PCR manual serum / plasma samples

miRCURY LNA™ Universal RTmicroRNA PCR

Instruction manual v6.2 #203301-203351 July 2016

Exosomes, serum/plasma and otherbiofl uid samples

EXIQON | miRCURY LNA™ Universal RT microRNA PCR serum/plasma/biofl uid | Instruction manual

Table of contents

Product summary 3I. Reagent kits 4II. Primer Sets and Panels 6Storage 10Additional required materials 10Recommended accompanying products 11

Product description 12 Control Assays 13

Profiling microRNA in blood serum/plasma samples 17

Before starting the experiment 18

Protocols 22 A. Individual assays using serum/plasma samples 22 B. Human and Mouse&Rat and Serum/plasma Focus microRNA PCR Panels using serum/plasma samples 31 C. Other Focus microRNA PCR Panels using serum/plasma samples 39 D. Pick-&-Mix microRNA PCR Panels using serum/plasma samples 47

Tips to protocol 57

Troubleshooting guide 65

FAQs 66

Related products 68

3


Product summary

The miRCURY LNA™ Universal RT microRNA PCR system is a microRNA-specifi c, LNA™-based system designed for sensitive and accurate detection of microRNA by quantitative real-time PCR using SYBR® Green. The method is based on universal reverse transcription (RT) followed by real-time PCR amplifi cation with LNA™ enhanced primers (for more details please see page 12). The miRCURY LNA™ Universal RT microRNA PCR portfolio is comprised of four types of reagent kits; including: • Universal cDNA synthesis kit II• RNA Spike-in kit • ExiLENT SYBR® Green master mix kit• microRNA primer sets available in pre-defi ned Human, Mouse&Rat and Focus PCR panels and customized Pick-&-Mix PCR panels as well as individual primer sets and reference genes. All PCR panels are Ready-to-Use delivered with one 10 μL PCR reaction per well.

Universal cDNA synthesis kit IIDescription page 4

RNA Spike-in kit (optional)

+microRNA and reference

gene primer sets

Description page 6,protocol page 22

Description page 9,protocol page 47

Description page 7,protocol page 31+39

Pick-&-Mix PCR panels(Ready-to-Use)

+ExiLENT SYBR® Green master mix

1, 2,5 or 20ml

Predefined PCR panelsHuman, Mouse&Rat, and Focus PCR pa

(Ready-to-Use)

Description page 5

Product overview

Figure 1.

4


I. Reagent kits

Universal cDNA synthesis kit II, 8-64 rxns (product # 203301)This kit contains all reagents required for fi rst-strand cDNA synthesis for 8-64 reactions1). The UniSp6 RNA Spike-in template can be used by itself or in combination with the cel-miR-39-3p template provided in the RNA Spike-in kit:

miRCURY LNA™ Universal RT microRNA PCR, Starter Kit (product # 203351)This kit contains all reagents required to perform 20 cDNA reactions of 10 μL volume and 100 PCR reactions. Kit includes primer set for the spike control UniSp6 (included), one candidate endogenous control primer set (miR-103a-3p) and two validated primer sets of your choice.

Contents Amount supplied

5× Reaction buffer2) 128 μL, 5x concentrated

Enzyme mix 64 μL, 10x concentrated

Nuclease free water 1 ml

UniSp6, RNA Spike-in template3) 12 fmol, dried down

Table 1.


5x Reaction buffer2) 128 μL, 5x concentrated

Enzyme mix 24 μL, 10x concentrated

UniSp6, RNA Spike-in template 12 fmol, dried down

UniSp6 RNA Spike-in control primer set v24), lyophilized 200 rxn

hsa-miR-103a-3p primer set (also works for mmu+rno), lyophilized

200 rxn

2 primer sets free of choice from stocked primers 2x 200 rxn

ExiLENT SYBR® Green master mix, 2x concentrated 500 μL

Nuclease free water 1.25 mL

Table 2.

1) Number of reactions is based on a standard reaction volume of 10 μL to 80 μL. Reaction volume depends on the application and number of assays to profi le. Please consult Figure 4 for details.

2) Includes universal reverse transcription primer.3) Used exclusively for the UniSp6 RNA Spike-in control primer set included in the ExiLENT SYBR® Green master mix kit.

5


ExiLENT SYBR® Green master mix

These kits contain all reagents required for PCR amplifi cation of microRNAs. In addition, a positive control assay, the UniSp6 RNA Spike-in control primer set, is provided with this kit for amplifi cation of the synthetic UniSp6 RNA spike-in provided in the Universal cDNA synthesis kit II.

ExiLENT SYBR® Green master mix, 1 ml (product # 203401). The master mix is provided in an amount suffi cient for 200 reactions of 10 μL:

ExiLENT SYBR® Green master mix, 2.5 ml (product # 203403). The master mix is provided in an amount suffi cient for 500 reactions of 10 μL:

ExiLENT SYBR® Green master mix, 20 ml (product # 203421). The master mix is provided in an amount suffi cient for 4000 reactions of 10 μL:

1) Used exclusively for detection of the UniSp6 RNA spike-in provided with the Universal cDNA synthesis kit II.


ExiLENT SYBR® Green master mix, 2x concentrated 2x 0.5 ml, 2x concentrated

Nuclease free water 1x 1.25 ml


Table 3.


ExiLENT SYBR® Green master mix, 2x concentrated 2x 1.25 ml, 2x concentrated

Nuclease free water 2x 1.25 ml


Table 4.


ExiLENT SYBR® Green master mix, 2x concentrated 2x 10 ml, 2x concentrated

Nuclease free water 1x 20 ml


Table 5.

Note:

UniSp6 primer set now supplied

lyophilized - Re-suspend in 220μL

nuclease free water before use!

6


II. Primer Sets and Panels

MicroRNA LNA™ PCR primer set (product # 204000-206xxx and 2100000-21xxxxx)LNA™ PCR primer sets are designed for optimal performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR® Green master mix. The performance of LNA™ primer sets will be aff ected if used in combination with less than optimal reagents. The primer sets are supplied in suffi cient amounts for 200 reactions of 10 μL.

Reference gene primer set (product # varies)The Reference gene primer sets are designed for use with the microRNA primers sets above as reference genes for normalization. The primer mix is supplied in suffi cient amount for 200 reactions of 10 μL.

Contents

LNA™ PCR Primer set (dried down)

Table 6.

Contents

LNA™ PCR Primer set (dried down)

Table 7.

7


Pre-defi ned microRNA PCR Panels (product # varies)The Human, Mouse&Rat and Focus microRNA PCR panels Ready-to-Use consist of either 96-well or 384-well PCR plates containing dried down LNA™ primer sets for one 10 μL real-time PCR reaction per well. The LNA™ primer sets are designed for optimal performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR® Green master mix. The performance of the LNA™ primer sets will be aff ected if they are used in combination with less than optimal reagents.

Contents Human miRNome panel I and II, V4384-well PCR plates supplied with LNA™ primer sets dried down, one 10 μL reaction per well:

Contents Mouse&Rat miRNome panel I and II, V4384-well PCR plates supplied with LNA™ primer sets dried down, one 10 μL reaction per well:

Panel I Panel II

372 LNA™ primer sets for theamplification of human microRNAs1)

380 LNA™ primer sets for theamplification of human microRNAs1)

3 inter-plate calibrators 3 inter-plate calibrators

3 primer sets for reference genes2) 1 blank well

5 RNA Spike-in control primer sets3)

1 blank well

Table 8.

Panel I Panel II372 LNA™ primer sets for theamplification of mouse and rat microRNAs1)

380 LNA™ primer sets for theamplification of mouse and rat microRNAs1)

3 inter-plate calibrators 3 inter-plate calibrators

3 primer sets for reference genes2) 1 blank well

5 RNA Spike-in control primer sets3)

1 blank well

Table 9.

8


Contents Serum/Plasma Focus microRNA PCR panel, V4PCR plates compatible with various real-time PCR instruments are available and supplied with LNA™ primer sets dried down, one 10 μL reaction per well:

Contents Cancer Focus microRNA PCR panel, V4PCR plates compatible with various real-time PCR instruments are available and supplied with LNA™ primer sets dried down, one 10 μL reaction per well:

1) Please go to www.exiqon.com/mirna-pcr to download plate layout fi les.2) Human Panels and Cancer Focus Panel: Three snRNAs (U6snRNA, SNORD38B, SNORD49A). Mouse&Rat Panels: Three snRNAs

(U6snRNA, RNU5G, RNU1A1). Serum/plasma Focus Panel: miR-103a-3p, miR-191-5p, miR-423-5p, miR-16-5p, miR-425-5p,miR-93-5p, miR-451a are regarded reference gene candidates.

3) The RNA Spike-in control primer sets targets the UniSp6 RNA spike-in supplied in the Universal cDNA synthesis kit II and the 4 RNA spike-ins contained in the RNA Spike-in kit (UniSp2, UniSp4, UniSp5, and cel-miR-39-3p).

96-well (1 plates) 384-well plate (4 panels per plate)84 LNA™ primer sets for the amplification of human microRNAs1)

4x84 LNA™ primer sets for the amplificationof human microRNAs1)

3 Primer sets for potential reference genes2)

4x3 Primer sets for potential reference genes2)

3 Inter-plate calibrators 4x3 Inter-plate calibrators

5 RNA Spike-in control primer sets3) 4x5 RNA Spike-in control primer sets3)

1 blank well 2x1 blank well

Table 11.

96-well (2 plates) 384-well plate (2 panels per plate)179 LNA™ primer sets for the amplificationof human microRNAs1+2)

2x179 LNA™ primer sets for the amplificationof human microRNAs1+2)

2x3 Inter-plate calibrators 2x6 Inter-plate calibrators

5 RNA Spike-in control primer sets3) 2x5 RNA Spike-in control primer sets3)

2 blank wells - 1 in each plate 2x2 blank wells

Table 10.

9


Pick-&-Mix microRNA PCR Panel, (product # 203893-203897 and 203818-203819)The Pick-&-Mix microRNA PCR Panels consist of either 96-well PCR plates or 384-well PCR plates containing custom selections of dried down microRNA LNA™ PCR primer sets for one 10 μL real-time PCR reaction per well, ready-to-use. PCR plates compatible with various real-time PCR instruments are available. The LNA™ primer sets are designed for optimal performance with the Universal cDNA Synthesis Kit II and the ExiLENT SYBR® Green master mix kit. The performance of the LNA™ primer sets will be aff ected if they are used in combination with less than optimal reagents.

Contents PCR plates supplied with customer defi ned LNA™ primer sets, reference gene primer sets, and RNA Spike-in control primer sets, dried down, one 10 μL reaction per well1):

1) Please go to www.exiqon.com/pick-and-mix to confi gure a Pick-&-Mix microRNA PCR Panel.

96-well PCR plates1) 384-well PCR plates1)

10 primer sets in 8 replicates 22 primer sets in 16 replicates

22 primer sets in 4 replicates 46 primer sets in 8 replicates

92 primer sets in 1 replicate 94 primer sets in 4 replicates

96-well flexible layout 380 primer sets in 1 replicate

384-well flexible layout

Table 12.

10


Storage

All microRNA PCR Panels, LNA™ primer sets and Reference gene primer setsThe PCR panels and primer sets are shipped dried down at room temperature. The primers can be stored between +4°C and -20°C. Under these conditions, all components are stable for at least 12 months. A� er resuspension, it is recommended to store LNA™ primer sets and Reference gene primer sets in aliquots at -20°C to avoid repeated freeze-thaw cycles.

Universal cDNA synthesis kit II and ExiLENT SYBR® Green master mixThese kits are shipped on dry ice in polystyrene containers and should be stored at -15°C to -25°C. Do not store in a frost-free freezer. Under these conditions, all components are stable until the expiry date on the package or vial. It is recommended that the RNA spike-in be stored in aliquots at -20°C a� er re-suspension to avoid repeated freeze-thaw cycles.

Additional required materials

Reagents not supplied• ROX or other passive reference dye (required on some PCR cyclers) see Tip 8 on page 59 for more.

Materials and Equipment not supplied• Nuclease-free PCR tubes or plates for use with individual assays • Nuclease-free, aerosol barrier pipette tips• Nuclease-free, low nucleic acid binding microcentrifuge tubes (e.g. Eppendorf DNA LoBind tubes and original 50 ml NUNC vials) • Sealing foils for PCR plates• Micro-centrifuge and plate centrifuge• Heating block, thermal cycler or other incubators• Real-time PCR instrument

Optional but recommended product• miRCURY LNA™ Universal RT microRNA PCR, RNA Spike-in kit (product# 203203)

See tip 8

11


Recommended accompanying products

Exiqon recommends the Exiqon GenEx qPCR so� ware for comprehensive and convenient data analysis. GenEx includes a wizard for import of Exiqon miRCURY™ Universal RT microRNA PCR data and off ers advanced methods to analyze real-time qPCR data in a few simple steps. The so� ware includes tools from selection and validation of reference genes, data pre-processing and comprehensive statistical analyses. For more information and to download a free trial, please go to www.exiqon.com/qpcr-so� ware. The following Exiqon GenEx products are available: Exiqon GenEx6 Industrial - Exiqon version of GenEx, qPCR analysis so� ware, industrial license

Exiqon GenEx6 Academic - Exiqon version of GenEx, qPCR analysis so� ware, academic license

Exiqon recommends the miRCURY™ RNA Isolation kits for purifi cation of total RNA or small RNA fraction. RNA purifi ed using the miRCURY™ RNA Isolation kits is fully compatible with the miRCURY LNA™ Universal RT microRNA PCR System. The following kits are available:

miRCURY™ RNA Isolation Kit – Cell & PlantProvides a rapid method for purifi cation of total RNA from cultured animalcells, small tissue samples, blood, bacteria, yeast, fungi and plants.

miRCURY™ RNA Isolation Kit – TissueSpecifi cally designed for purifi cation of total RNA from tissue samples.

miRCURY™ RNA Isolation Kit - Biofl uidsKit for purifi cation of low abundant small RNAs from samples such as serum, plasma, urine and CSF. See Tip 2 See tip 2

12


Product description

A unique system for microRNA profi lingmiRCURY LNA™ Universal RT microRNA PCR off ers the best available combination of performance and ease-of-use on the microRNA real-time PCR market because it unites two important features (Figure 2):

1. Universal RT – One fi rst-strand cDNA synthesis reaction provides template for all microRNA real-time PCR assays. This saves precious sample, reduces technical variation, requires use of less reagents, and saves time in the laboratory.

2. LNA™ PCR amplifi cation – Both PCR amplifi cation primers (forward and reverse) are microRNA specifi c and optimized with LNA™. The result is: 1) exceptional sensitivity as well as extremely low background enabling accurate quantifi cation of very low microRNA levels and 2) highly specifi c assays that allow discrimination between closely related microRNA sequences.

miRCURY LNA™ Universal RT microRNA PCR off ers solutions both for high-throughput microRNA expression profi ling and for quantifi cation of individual microRNAs.

Step 1: First-strand synthesis (RT)

Step 2: Real-time PCR amplification

Mature microRNAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAA

A)

B)

A)

B)

3’ degenerate anchor

miR-specific forward primer

miR-specific reverse primer

TTTTTTTTTTTTTTT

TTTTTTTTTTTTTTT

5’ universal tag

LNA LNA LNA

LNA LNA LNA

Schematic outline of the miRCURY LNA™ Universal RT microRNA PCR System.

Figure 2. A poly-A tail is added to the mature microRNA template (step 1A). cDNA is synthesized using a poly-T primer with a 3’ degenerate anchor and a 5’ universal tag (step 1B). The cDNA template is then amplified using microRNA-specific and LNA™-enhanced forward and reverse primers (step 2A). SYBR® Green is used for detection (step 2B).

13


Control Assays

There are 3 diff erent types of control assays available in the miRCURY LNA™ Universal RT microRNA PCR system: • Reference assays and reference candidates• Inter-plate calibrators• RNA spike-in assays

All of these control assays are available in the microRNA PCR panels. One RNA spike-in template is provided with the Universal cDNA Synthesis Kit II, while the assay that will detect this RNA spike-in is available in the ExiLENT SYBR® Green master mix. Additionally 4 RNA spike-in templates are available as a spike-in kit. The assays for detection of these 4 templates as well as the reference assays are available as individual primer sets.

Reference assays and reference candidatesThese assays detect small non-coding RNAs - either small nuclear RNA, small nucleolar RNA or microRNA – which frequently are found to be stably expressed across diff erent cells or tissues. Reference assays may therefore be candidate assays for normalization in a profi ling study with several samples. Though this is a good and recommended approach, great caution should be taken in the selection of reference genes. The danger of using endogenous reference genes lies in the assumption that a specifi c gene is expressed at the exact same level in all sample types. This is rarely true. The selection of reference genes should therefore be made with care, and should be specifi c to the sample set you are working with. The actual selection of reference genes to be used for normalization should always be based on a determination of the most stably expressed gene(s) which may be done using either GeNorm or NormFinder – both tools that are integrated within Exiqon’s GenEx data analysis so� ware. When applicable, we recommend using microRNA rather than small nuclear RNA or small nucleolar RNA for normalization. Firstly, small nuclear and nucleolar RNAs are longer RNA species than microRNA and may purify diff erently from microRNA. Moreover, small nuclear and nucleolar RNAs have entirely diff erent functions as well as sub-cellular locations, and fi nally certain samples like blood plasma does not contain the small nuclear and nucleolar RNAs. Global mean normalization is a preferred alternative to using reference genes for normalization when working with panels and samples where many microRNAs are screened per sample and where many microRNA are called (detected) in all samples. Exiqon’s GenEx will easily perform global mean normalization. To read more on normalization, see Tip 11. See tip 11

14


Inter-plate calibrators Three wells within the pre-defi ned Human, Mouse&Rat, and Focus PCR Panels contain the inter-plate calibrator assay (annotated as UniSp3 IPC in the plate layout fi les). Depending on the plate layout, the Pick-&-Mix Panels contain at least three inter-plate calibrators. Each of these wells, contain a pre-aliquoted primer pair and a DNA template and therefore the variation is very minimal from well-to-well and from plate-to-plate of these assays. The inter-plate calibrators are used for calibration between PCR plate runs which is very useful on some instruments that apply the cycle threshold method for Cq determination such as the ABI7900 PCR cycler. Since the inter-plate calibrators are independent of cDNA quality in order to give a signal (but may be aff ected by PCR inhibitors in the sample) they may be used to quality control each plate run.

Inter-plate calibration (IPC) can easily be performed in the data analysis so� ware Exiqon’s GenEx. Alternatively, IPC may be performed manually by using the IPC assay replicates as follows. For each plate, verify that the replicates have Cq standard deviation within 0.5. If this is not the case, eliminate the outlier if this can be identifi ed. Calculate the average of the replicates for each plate, the overall average (average of IPC values from all plates). The calibration factor is calculated as the diff erence between plate average and overall average for each plate (calibration factor = IPCplate-IPCoverall). An example is shown in Table 1. Finally, calibrate each plate by subtracting the calibration factor from all Cq values in the plate.

Plate 1 Plate 2 Plate 3

let-7c 21.12 20.93 21.34

IPC plate average 19.72 19.70 20.00

IPC overall average 19.81 19.81 19.81

Calibration factor -0.09 -0.11 0.19

let-7c calibrated 21.21 21.04 21.15

Table 13. Example of inter-plate calibration (IPC)

15


RNA spike-ins (synthetic control templates)The primary purpose of the RNA spike-ins and the matching primer pairs for detection of these is to provide controls for the quality of the RNA isolation, the cDNA synthesis reaction and the PCR. RNA isolations may vary in yield, purity and integrity. Some sample types may contain compounds that inhibit the cDNA synthesis or the PCR even though the RNA has been purifi ed using the best standard procedures. This may result in diff erent effi ciencies of the reverse transcription or PCR between compared samples. One way to control for diff erences in effi ciencies at each experimental level (isolation, cDNA synthesis, and PCR) is by adding known RNA spike-ins to the sample prior to isolation and cDNA synthesis. Use of the RNA spike-ins may also reveal if nucleases are present. A� er conducting the PCR but before initiating the data analysis, wells detecting RNA spike-ins are compared and outlier samples may be identifi ed and considered for exclusion in the further data analysis.

We have designed a fl ight of RNA spike-ins for this purpose. The UniSp6 RNA Spike-in template is provided with the Universal cDNA synthesis kit II. Additionally four RNA spike-in templates can be obtained with the separate RNA Spike-in kit. Here, a vial of three RNA spike-in templates, UniSp2, UniSp4 and UniSp5, mixed at diff erent concentrations can be used during RNA isolation. The cel-miR-39-3p RNA template provided in a separate vial in the RNA Spike-in kit can be mixed with the UniSp6 template from the Universal cDNA synthesis kit II to obtain two diff erent template concentrations. This combination can be added during the cDNA synthesis. Five wells in pre-defi ned PCR panel plates contain the matching primer sets. The selection of RNA Spike-in control primer set in the Pick-&-Mix PCR Panel can be customized to the specifi c need. A UniSp6 control primer set is also provided with the ExiLENT SYBR® Green master mix kit, which is to be used with our non-plate based PCR primer set products.The RNA spike-ins are shipped dried down and must be re-suspended before use.

If the RNA Spike-in kit with multiple RNA spike-ins is used, follow the protocol accompanying that product.

If UniSp6 is to be used alone:1. Re-suspend the UniSp6 RNA spike-in by adding 80 μl nuclease free water to the tube.2. Mix by vortexing and spin down. Leave for 20-30 min. on ice to fully dissolve RNA spike-in. Mix by vortexing and spin down. Store in aliquots at -20°C3. Prior to the RT reaction, add 1 μl synthetic spike-in (108 copies/μl) per 20 ng sample RNA.

16


An alternative application of the UniSp6 RNA spike-in is as inter-plate calibrator. This is only relevant when using individual LNA™ primer sets and Reference gene primer sets in a multi-plate set-up. The microRNA PCR panels already contain an inter-plate calibrator. Add 1 μl synthetic spike-in (108 copies/μl) to 20 ng of a complex RNA sample (e.g. total RNA from MS2, yeast, or a cell line; not provided with the kit). Proceed with fi rst strand synthesis and subsequently real-time PCR as described in the protocols of the current instruction manual. At least one spike-in amplifi cation reaction per PCR plate is used for inter-plate calibration (see Tip 9).

Experimental designBefore starting the experiment, it is essential to consider the experimental setup and consider the number of replicates needed for obtaining signifi cant results – replicates being technical as well as biological. The number of biological replicates required varies from experiment to experiment depending on the variation within and between the groups. We recommend that a No Template Control (NTC) is included in the study every time a new experiment is set up, to set the background level. The most optimal NTC is a mock up sample preparation including only carrier RNA as sample. The NTC should be run on all assays included in the study. We defi ne the background of an assay as 5 Cq values below the NTC level. Furthermore we recommend including spike-ins found in the Spike-in kit to provide full quality control over all steps in the profi ling (see fi gure 3). Finally it is necessary to include a number of candidate reference microRNAs, which are expected to be constitutively expressed across the diff erent experimental conditions, for data normalization.

See tip 9

UniSp2

Sample n

Sample n+1 Sample preparation evaluation

Sold as separate Spike-in kit (203203)

cDNA synthesis evaluation

Comes with Universal cDNAsynthesis kit II

Included in all panel products

Inter plate Calibration

NTC

UniSp4 UniSp5 cel-miR-39-3p UniSp6 UniSp3

Assays available asindividual assays or inready-to-use panels

Sample profiling

miR-A miR-X

Experimental design

Figure 3.

17


Profiling microRNA in blood serum/plasma samplesThe current instruction manual provides protocols and procedures for profi ling microRNA in blood plasma and serum samples as well as other biofl uid samples.

Plasma and serum are essentially cell free liquid samples. This means that only circulating RNA is extracted from these sample types, resulting in low total RNA concentrations, even if the microRNA fraction is readily detectable. The result of this is that measuring correct RNA concentrations is diffi cult, and that there is a high risk of increased loss during extraction. For this reason, we recommend adding carrier RNA during the extraction procedure, and subsequently using RNA amounts based on starting volume rather than RNA quantity. Please fi nd our recommended kit for RNA extraction on www.exiqon.com/mirna-isolation-biofl uids. In addition to these protocols we also recommend reading our guidelines for profi ling microRNA in blood serum and plasma: www.exiqon.com/serum-plasma-guidelines

Another challenge when working with plasma or serum is the high content of RT and/or PCR inhibitors in this type of sample. The presence of inhibitors means increasing the amount of sample input in order to obtain better sensitivity is not possible. The amount of inhibitors le� a� er extraction may vary greatly between diff erent extraction methods, but also from sample to sample. It is therefore highly recommended to test varying total RNA sample input amounts in the cDNA synthesis reaction (e.g. 2 μl, 4 μl and 6 μl per 20 μL cDNA synthesis reaction) on a number of individual assays or alternatively on a set of full panels: This will provide an idea about the microRNA level and possible presence of inhibitors in your particular serum/plasma sample.

18


Before starting the experiment

Before setting up a real-time PCR experiment, there are a number of practical experimental design parameters that should be considered:

RNA input - Blood serum, plasma and other biofl uids are particular sample types that require special RNA purifi cation procedures and the amount of RNA present in the samples can usually not be accurately determined. Due to the low levels of microRNAs and potentially high levels of inhibitors in samples derived from serum, plasma and other biofl uid samples, specifi c recommendations for how to set up experiments using these types of sample can be found in the previous chapter “Profi ling microRNA in blood serum/plasma samples”, page 17.

Information on how to extract and handle RNA can be found in the Tips section. In short, total RNA should be prepared using a method that preserves small RNA species. DNase treatment may be necessary. When using commercially available kits, please ensure that the total RNA preparation is guaranteed to contain microRNAs.

Important note

RNA work requires specifi c handling and precautions to prevent RNase contamination of the reagents and degradation of the RNA sample. Find information on how to handle RNA in the tips section starting on page 57. The tips section also provides simple guidelines for good laboratory practice to ensure optimal performance of PCR experiments.

19


Normalization – when running individual assays or when confi guring a Pick-&-Mix microRNA PCR panel it is important to consider how the data will be normalized. For tips and recommendations on choosing the correct reference genes and how to test and validate reference genes, please see section on reference assays (page 13) and the Tip 11 (page 63).

Excess volumes required for pipetting – Liquid handling with pipettes or pipetting robots require excess volumes of reagents due to loss during pipetting. The loss depends on the available pipetting system but losses in the range from 10% -25% are not uncommon. All protocols in the current instruction manual refl ect the required reaction volumes and pipetting volumes should be adjusted according to accommodate the pipetting loss of the available pipetting system.

ROX – ROX is a passive reference dye used by some PCR cycles to obtain a robust read over the entire array of wells in a 96- or 384- well PCR plate. The requirement for ROX is instrument dependent and we recommend to follow the instrument manufactures guidelines on this.

ABI instruments – the default settings on ABI real-time PCR cyclers are not suitable for running miRCURY LNA™ Universal RT microRNA PCR. Settings need to be changed from automatic to manual background and threshold settings to obtain valid PCR data (see also Tip 10). Furthermore, if the dataset is to be analyzed using the GenEx so� ware, it is important that the experiment is set up as an AQ experiment, not RQ. To ensure correct settings, download the instrument settings fi le at www.exiqon.com/sds.

RNA spike-ins – consider how the RNA spike-ins should be applied in the planned study. Please consult the section on RNA spike-ins on page 15.

See tip 11

See tip 8

See tip 10

20


Below are some general guidelines that can be used to perform microRNA expression profi ling on human and mouse serum and plasma samples, however, the exact protocol needed may vary according to the particular samples or organism involved.

1. RNA isolation Start with around 200μl human serum or 50 μl mouse /rat serum and follow the

recommended RNA extraction protocol including carrier RNA (e.g.MS2 bacteriophage total RNA, Roche Applied Sciences cat.# 10165948001). Elute purifi ed RNA in 50 μl. Addition of RNA Spike-in templates as quality controls for the RNA isolation using the RNA Spike-in kit is optional. Please see page 15 and the RNA Spike-in kit Instruction Manual for protocol details (exiqon.com/RNA-spike-in-kit).

2. First strand cDNA synthesis We recommend performing cDNA synthesis in a larger reaction volume than for normal

samples. In our experience, 8 μl of eluted RNA in a 40 μl RT reaction generally gives a good signal. One 40μl RT reaction gives enough cDNA for one 384-well plate. Note: The equivalent of 32 μL original serum/plasma is used per plate.

3. Real-Time PCR amplifi cation Due to the large RT reaction volume, the dilution factor of the cDNA is reduced to 50x

compared to 100x for standard samples. To avoid low concentrations of cDNA from adhering to tube surface the following procedure is recommended: Combine and mix gently 2x PCR Master mix and water, then add undiluted cDNA and mix.

21


Protocols for the fi rst-strand cDNA synthesis and real-time amplication follows on the next pages:

A. Individual assays using serum/plasma samples, please go to page 22 B. Human, Mouse&Rat, Serum/plasma Focus microRNA PCR Panels, please go to page 31 C. Other Focus microRNA PCR Panels, please go to page 39 D. Pick-&-Mix microRNA PCR Panels, please go to page 47

Figure 4 gives an overview and helps identifying which protocol to follow as well as the recommended cDNA reaction volume needed for a given sample and assay type.

Protocol overview

Figure 4.

Serum plasma or other biofluids

Cells or tissue

Sample type

Panels or primer sets:

Universal cDNA reactions per kit

Universal cDNA reaction volume

miRNome panel I

miRNome panel I+II

1-96 97-192

Individual assays(<96)

16 8 64 32 64

40 µL

50x

80 µL 10 µL 20 µL 10 µL

40xDilution of cDNAin ExiLENT Master Mix

Use biofluids manual from www.exiqon.com/pcr-manual-biofluids

Use standard printed m

anual or download from

w

ww

.exiqon.com/pcr-m

anual

Pick-&-Mix/Focus

22


Protocol A. Individual assays using serum/plasma samplesThis protocol is used for conducting the fi rst-strand cDNA synthesis and real time PCR, using the individual assays for human or mouse and rat (product numbers 204000-206XXX).

The levels of total RNA found in serum and plasma are very low and it is therefore RNA carriers should be applied to ensure robust RNA isolation. This makes it impossible to determine the concentration of RNA in a given sample a� er RNA extraction. It is therefore recommended to use the amount of starting material as a measure of input amount.

RNA inhibitors in serum/plasma will prevent appropriate cDNA synthesis and amplifi cation of microRNAs. The amount of inhibitors in a sample varies with samples and the RNA extraction method. It is therefore recommended to investigate how much extracted RNA to use in the RT reaction without experiencing inhibition, before starting the experiment. This can be done by running a few individual assays with diff erent amounts of RNA input volume e.g. 2 μl, 4 μl and 6 μl per 20 μL cDNA synthesis reaction (equivalent to 8 μL, 16 μL and 24 μL of serum/plasma, respectively). By increasing the RNA input the Cq values are reduced to a point where inhibition sets in. For additional information, please also read the section in the manual (page 17) describing general guidelines when conduction microRNA expression profi ling in serum and plasma samples the section about using RNA Spike-ins for quality control of the RNA isolation (page 15).

Before initiating PCR setup:Before using the LNA™ PCR primer set, the UniSp6 RNA spike-in control primer mix, or the Reference gene primer set for the fi rst time, the primers must be re-suspended and mixed.• Re-suspend the primer set by adding 220 μl nuclease free water to the tube. Mix by vortexing and spin down. Leave on ice for 20-30 minutes.

Prot

ocol

A. I

ndiv

idua

l ass

ays

usin

g se

rum

/pla

sma

sam

ples

23


Additional required materials:• 96- or 384-well plate real time PCR cycler• Thermocycler for fi rst-strand cDNA synthesis• 96/384 well plates or tube strips compatible with available real time PCR cycler• Micro centrifuge• Swing bucket centrifuge for 96/384 well plates

Checklist:• Have you considered excess volumes required for using liquid handling robotics?

– see page 19• Did you consider how to use the RNA spike-ins? – please see page 15• ROX: The ExiLENT SYBR® Green master mix, does not include the ROX passive reference dye.

Please follow instrument manufactures recommendations• ABI instruments the use of manual background and threshold settings is necessary for

obtaining correct PCR data. Make sure to have the optimal settings by downloading the instrument settings fi le at www.exiqon.com/sds. Furthermore, if the data is to be analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ

• Have you optimized the input amount to the RT reaction in order to avoid inhibition?

Protocol A. Individual assays

using serum/plasm

a samples

24


Workfl ow for individual primer sets (per sample)

Phase I: Prepare RNA sampleSee page 19 for recommendations

Phase II: cDNA synthesisSee protocol page 13.

Phase III: real-time PCR amplificationSee protocol page 15.- Prepare adequate amount of pre-mixed primer - Mix with ExiLENT SYBR® Green Master mix and distribute into wells- Add cDNA to all primer sets to be analyzed

Phase IV: Data analysisSee data analysis guide online- Export data for further analysis- Data pre-processing, normalization and statistical analysis

4

3

2

1

0

-1Normal Tumor Tumor

stroma

Tumor Total

• miR-21

• let-7a

Rel

ativ

e ex

pres

sion

(log

2)

LNATM primer sets

Prot

ocol

A. I

ndiv

idua

l ass

ays

usin

g se

rum

/pla

sma

sam

ples

25


ProtocolThe miRCURY LNA™ Universal RT microRNA PCR protocol is a two-part protocol consisting of:1. First-strand cDNA synthesis (Step 1-5)2. Real-time PCR amplifi cation (Step 6-11)

Important: Keep reagents and reactions on ice (or at 4˚C) at all times.

First strand synthesis:

Step 1RNA input volume

Since the RNA concentration in extractions from serum and plasma cannot be determined with accurately we recommend using the amount of starting material as a measure for input amount.

Step 2Prepare reagents

Gently thaw the 5x Reaction buffer and nuclease-free water, and immediately place on ice. Mix by vortexing. Re-suspend the RNA spike-in(s) according to the appropriate RNA Spike-in protocol (see page 15), leave on ice for 15-20 minutes. Immediately before use, remove the Enzyme mix from the freezer, mix by flicking the tubes and place on ice. Spin down all reagents.


using serum/plasm

a samples

26


1) The equivalent of 8 μl original serum/plasma is used per 10 μL reverse transcription reaction, respectively. Optimal volumes should be determined on individual assays prior to panel experiments.

2) The protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20˚C for up to 5 weeks (optional stoe at 4˚C for up to 4 days). It is recommended that synthesized cDNA is stored in “low-nucleic acid binding” tubes or plates.

Step 4Mix and spin reagents

Mix the reaction by very gentle vortexing or pipetting to ensure that all reagents are thoroughly mixed. After mixing, spin down.

Step 5Incubate and heat inactivate2)

• Incubate for 60 min at 42˚C.• Heat-inactivate the reverse transcriptase

for 5 min at 95˚C. • Immediately cool to 4°C.• Store at 4°C or freeze.

Step 3 Combine reagents according to Table 14

Note: remember to calculate necessary excess volume for pipetting and robotic dead volume

If performing first-strand cDNA synthesis on multiple RNA samples, it is recommended to prepare an RT working solution of the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the proportion indicated in the first four lines of Table 14).The following procedure is recommended:1. Prepare the required amount of RT working solution and place it on ice.2. Dispense RT working solution into nuclease free tubes.3. Dispense template RNA in each tube.

Reagent Volume (μL), RT reaction

5x Reaction buffer 2

Nuclease-free water 4.5

Enzyme mix 1

Synthetic RNA spike ins, optional replace with H2O if omitted

0.5

Template total RNA1) 2

Total volume 10

Table 14. Reverse transcription reaction setup

Prot

ocol

A. I

ndiv

idua

l ass

ays

usin

g se

rum

/pla

sma

sam

ples

27


Step 7Dilute cDNA template 40x in nuclease free water3)

Immediately before use, dilute only the amount of cDNA template needed for the planned real-time PCR reactions 40x in nuclease free water (e.g. add 390 μl nuclease free water to each 10 μl of reaction). It is recommended to use “low-nucleic acid binding” tubes or plates. It is not recommended to store the 1:40 dilution of cDNA.

Recommendation: Include a passive reference dye in the cDNA dilution if advised by instrument manufacturer. Please note that the PCR Master mix does not include ROX. The amount of ROX required is instrument dependent and it is important to refer to the manufacturer’s recommendations when deciding how much ROX to use.

See tip 8

3) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.

Step 6Prepare reagents for real-time PCR

Place cDNA (from Step 5), nuclease free water and PCR Master mix on ice and thaw for 15-20 min. Protect the PCR Master mix vials from light. Immediately before use, mix the PCR Master mix by pipetting up and down. The rest of the reagents are mixed by vortexing and spun down.

qPCR protocol:


using serum/plasm

a samples

28


4) If using a 96-well cycler with a minimum recommended volume of 20 μL (as some ABI instruments), then use 10μL reaction volume and set the instrument settings at 20μL.

5) The PCR primer set must be dissolved prior to real-time PCR set-up, see page 22.

Step 8Combine PCR Master mix, PCR primer set and cDNA according to Table 15

Mix thoroughly


When multiple real-time PCR reactions are performed with the same microRNA primer set, it is recommended to prepare a primer:master mix working-solution of the PCR primers and the PCR Master mix (in the proportion indicated in Table 15). The following procedure is recommended:1. Prepare the required amount of PCR primer:master mix working-

solution (see Table 3) and place it on ice. It is recommended to include 10% excess of all reagents in the master mix to compensate for pipetting excess material.

2. Place the relevant volume of primer:master mix working-solution in PCR tubes/wells (see Table 3) and spin tubes/plate briefly in a centrifuge (1500g for 1 minute), to avoid air bubbles.

3. Add cDNA template to each tube/well.

ReagentVolume (μL), 96/384-well plate, tubes or strips

PCR Master mix 5

PCR primer set5) 1

Diluted cDNA template 4

Total volume 10

Table 15. Real-time PCR reaction, pr. 10 μL reaction4)


Mix the reaction by gentle pipetting to ensure that all reagents are mixed thoroughly. After mixing cap tubes or strips or seal the plate with optical sealing as recommended by the manufacturer. Spin down in a centrifuge (1500g for 1 minute). The experiment can be paused at this point. Store the reactions protected from light at 4°C for up to 24 hours.

Prot

ocol

A. I

ndiv

idua

l ass

ays

usin

g se

rum

/pla

sma

sam

ples

29


6) Five additional amplifi cation cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40.7) The ramp-rate of cooling from 95˚C to 60˚C should be set to 1.6˚C/s. This is equivalent to 100% under standard cycling conditions on the

ABI 7500, 7900 and Viia7 instruments. If the ramp rate of cooling is too rapid, performance may be compromised.8) Melting curve analysis of the PCR product(s) is recommended to verify specifi city and identity of the amplifi cation reaction. Melting curve

analysis is an analysis step built into the so� ware of instruments. Please follow the instructions provided by the supplier. Note: The Tm of a PCR product depends on buff er composition, salt concentration and the PCR instrument.

ABI instrument user?Apply manual baseline and threshold settings! Go to www.exiqon.com/sds to download settings file

Step 10Real-time PCR amplification

Perform real-time PCR amplification followed by melting curve analysis according to Table 16.

Process stepSettings, LC480 instrument6)

Settings, other instruments4)

Polymerase Activation/Denaturation

95˚C, 10 min 95˚C, 10 min

Amplification 45 amplification cycles at 95˚C, 10 s60˚C, 1 min, ramp-rate 1.6̊ C/s7)

Optical read

40 amplification cycles at95˚C, 10 s60˚C, 1 min, ramp-rate 1.6̊ C/s7)

Optical readMelting curve analysis8) Yes Yes

Table 16. Real-time PCR cycle conditions


using serum/plasm

a samples

30


See tip 11

Step 11Analyze data

Perform initial data analysis using the software supplied with the real-time PCR instrument to obtain raw Cq values (Cp or Ct, depending on PCR instrument). If you are using an ABI instrument, please note that it is not recommended to use auto Ct settings. For a guide on how to set manual baseline and threshold, refer to Tip 10, page 61 in the Tips section. Furthermore, if the data is to be analyzed using Exiqon GenEx, the experiment must be set up as an AQ experiment, not RQ. If you are using a Roche LC480 instrument, we recommend analysis using the 2nd derivative method.

For tips on normalization, please see Tip 11, page 63. We recommend performing normalization and further data analysis with the Exiqon GenEx qPCR analysis software (www.exiqon.com/mirna-pcr-analysis). Please refer to our data analysis guide for recommendations.

Prot

ocol

A. I

ndiv

idua

l ass

ays

usin

g se

rum

/pla

sma

sam

ples

31


Protocol B. Human and Mouse&Rat and Serum/plasma Focus microRNA PCR Panels using serum/plasma samplesThis protocol is used for conducting the fi rst-strand cDNA synthesis and real time PCR, using serum or plasma in combination with the following products:• Human miRNome PCR Panel (product numbers 203611 to 203618)• Mouse&Rat miRNome PCR Panel (product numbers 203709 to 203716)• Serum/plasma Focus microRNA PCR Panel The levels of total RNA found in serum and plasma are very low and it is therefore RNA carriers should be applied to ensure robust RNA isolation. This makes it impossible to determine the concentration of RNA in a given sample a� er RNA extraction. It is therefore recommended to use the amount of starting material as a measure of input amount.

RNA inhibitors in serum/plasma will prevent appropriate cDNA synthesis and amplifi cation of microRNAs. The amount of inhibitors in a sample varies with samples and the RNA extraction method. It is therefore recommended to investigate how much extracted RNA to use in the RT reaction without experiencing inhibition, before starting the experiment. This can be done by running a few individual assays with diff erent amounts of RNA input volume e.g. 2 μl, 4 μl and 6 μl per 20 μL cDNA synthesis reaction (equivalent to 8 μL, 16 μL and 24 μL of serum/plasma, respectively). By increasing the RNA input the Cq values are reduced to a point where inhibition sets in. For additional information, please also read the section in the manual (page 17) describing general guidelines when conduction microRNA expression profi ling in serum and plasma samples and the section about using RNA Spike-ins for quality control of the RNA isolation (page 15).

Protocol B. H

uman and M

ouse&R

at and Serum/plasm

aFocus m

icroRN

A PCR

Panels using serum/plasm

a samples

32


Additional required materials:• 96- or 384-well plate real time PCR cycler• Thermocycler for fi rst-strand cDNA synthesis• Micro centrifuge• Sealing foils for PCR plates• Swing bucket centrifuge for 96/384 well plates• Recommended: Liquid handling robot for pipetting

Checklist:• Have you considered excess volumes required for using liquid handling robotics? – see page 19• Did you consider how to use the RNA spike-ins? – please see page 15• ROX: The ExiLENT SYBR® Green master mix, does not include the ROX passive reference dye. Please follow instrument manufactures recommendations.• ABI instruments: The use of manual background and threshold settings is necessary for obtaining correct PCR data. Make sure to have the optimal settings by downloading the instrument settings fi le at www.exiqon.com/sds. Furthermore, if the data is to be analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ.• Have you optimized the input amount to the RT reaction in order to avoid inhibition?

Prot

ocol

B. H

uman

and

Mou

se&

Rat

and

Ser

um/p

lasm

aFo

cus

mic

roR

NA

PCR

Pan

els

usin

g se

rum

/pla

sma

sam

ples

33




Phase III: real-time PCR amplificationSee protocol page 36.- Mix cDNAs with PCR Master mix- Add cDNA: PCR Master mix to PCR plates


4

3

2

1

0


stroma

Tumor Total

miR-21 let-7a

Rel

ativ

e ex

pres

sion

(log

2)

Workfl ow for Human, Mouse&Rat and Serum/plasma Focus microRNA PCR Panels (per sample)

Protocol B. H

uman and M

ouse&R

at and Serum/plasm

aFocus m

icroRN

A PCR


a samples

34


ProtocolThe miRCURY LNA™ Universal RT microRNA PCR protocol is a two-step protocol consisting of:1. First-strand cDNA synthesis (Step 1-5)2. Real-time PCR amplifi cation (Step 6-9)




Since the RNA concentration in extractions from serum and plasma cannot be determined accurately we recommend using the amount of starting material as a measure for input amount.

Step 2 Prepare reagents


Prot

ocol

B. H

uman

and

Mou

se&

Rat

and

Ser

um/p

lasm

aFo

cus

mic

roR

NA

PCR

Pan

els

usin

g se

rum

/pla

sma

sam

ples

35


Step 3Combine reagentsaccording to Table 17


If performing first-strand cDNA synthesis on multiple RNA samples, it is recommended to prepare an RT working solution of the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the proportions indicated in the first four lines of Table 17).The following procedure is recommended:1. Prepare the required amount of RT working solution and place it on

ice.2. Dispense RT working solution into nuclease free tubes.3. Dispense template RNA in each tube.

The 96 well protocol provide sufficient cDNA for one full Serum/plasma panel whether located in two 96-well plates or distributed within 192 wells of a 384 well plate. The 384 well protocol is enough for one ready-to-use 384 well plate (panel I or II).




• Incubate for 60 min at 42°C.• Heat-inactivate the reverse transcriptase

for 5 min at 95°C. • Immediately cool to 4°C.• Store at 4°C or freeze.

Reagent

96 well panel Volume (μL)

384 well panel Volume (μL)

5x Reaction buffer 4 8

Nuclease-free water 9 18

Enzyme mix 2 4


1 2

Template total RNA1) 4 8

Total volume 20 40


Protocol B. H

uman and M

ouse&R

at and Serum/plasm

aFocus m

icroRN

A PCR


a samples

36


See tip 8

Step 7Combine PCR Master mix, water and cDNA and add to PCR plates2)

Mix thoroughly

Dilute cDNA template 50x in nuclease free water and mix 1:1 with 2x PCR Master mix. The following procedure is recommended to avoid low concentrations of cDNA from adhering to tube surface:1. Before removing the plate seal, briefly spin down the plate(s) in a plate

centrifuge.2. Combine 2x PCR Master mix and water

• Serum/plasma Focus panel: 1000 μl 2x master mix and 980 μl water• Human, Mouse&Rat panels: 2000 μl 2x master mix and 1960 μl water

3. Mix gently and spin down.4. Add 20 μL cDNA (Serum/plasma Focus panel) or 40 μL cDNA (Human

and Mouse&Rat panels) and mix3).5. Add 10 μL PCR Master mix:cDNA mix to each well2).6. Seal the plate with optical sealing as recommended by the instrument

manufacturer.7. Spin plate briefly in a plate centrifuge (1500g for 1 minute), to collect

the sample.

The experiment can be paused at this point. Store the reactions protected from light at 4°C for up to 24 hours.


qPCR protocol:



Prot

ocol

B. H

uman

and

Mou

se&

Rat

and

Ser

um/p

lasm

aFo

cus

mic

roR

NA

PCR

Pan

els

usin

g se

rum

/pla

sma

sam

ples

37


1) The equivalent of 16 or 32μl original serum/plasma is used per 20 or 40 μL reverse transcription reaction, respectively. Optimal volumes should be determined on individual assays prior to panel experiments.

2) The protocol can be interrupted at this stage. The undiluted cDNA may be kept at -20˚C for up to 5 weeks (optional store at 4˚C for up to 4 days). It is recommended that synthesized cDNA is stored in “low-nucleic acid binding” tubes or plates.

3) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting. 4) If using a 96-well cycler with a minimum recommended volume of 20 μL (like some ABI instruments), then use 10μL reaction volume

and set the instrument settings at 20μL. 5) Five additional amplifi cation cycles are required when using the LC480 instrument to allow collection of assay data with Cp-values up to 40.6) The ramp-rate of cooling from 95˚C to 60˚C should be set to 1.6˚C/s. This is equivalent to 100% under standard cycling conditions on the









95˚C, 10 min 95˚C, 10 min


Optical read


Optical read

Melting curve analysis6) Yes Yes


Protocol B. H

uman and M

ouse&R

at and Serum/plasm

aFocus m

icroRN

A PCR


a samples

38


See tip 11

Step 9Analyze data

Perform initial data analysis using the software supplied with the real-time PCR instrument to obtain raw Cq values (Cp or Ct, depending on PCR instrument). If you are using an ABI instrument, please note that it is not recommended to use auto Ct settings. For a guide on how to set manual baseline and threshold, refer to Tip 10, page 61 in the Tips section. Alternatively, use ABI settings files available fromwww.exiqon.com/sds. Furthermore, if the data is to be analyzed using Exiqon GenEx, the experiment must be set up as an AQ experiment, not RQ. If you are using a Roche LC480 instrument, we recommend analysis using the 2nd derivative method.


Prot

ocol

B. H

uman

and

Mou

se&

Rat

and

Ser

um/p

lasm

aFo

cus

mic

roR

NA

PCR

Pan

els

usin

g se

rum

/pla

sma

sam

ples

39


Protocol C. Other Focus microRNA PCR Panels using serum/plasma samplesThis protocol is used for conducting the fi rst-strand cDNA synthesis and real time PCR, using the Focus microRNA PCR panels (96 assays pr. sample in either 96 well or 384 well plates).

The levels of total RNA found in serum and plasma are very low and it is therefore necessary to apply RNA carriers to ensure robust RNA isolation. The low concentrations make it impossible to determine the concentration of RNA in a given sample a� er RNA extraction. It is therefore recommended to use the amount of starting material as a measure of input amount.

RNA inhibitors in serum/plasma will prevent appropriate cDNA synthesis and amplifi cation of microRNAs. The amount of inhibitors in a sample varies with samples and the RNA extraction method. It is therefore recommended to investigate how much extracted RNA to use in the RT reaction without experiencing inhibition, before starting the experiment. This can be done by running a few individual assays with diff erent amounts of RNA input volume e.g. 2 μl, 4 μl and 6 μl per 20 μL cDNA synthesis reaction (equivalent to 8 μL, 16 μL and 24 μL of serum/plasma, respectively. By increasing the RNA input the Cq values are reduced to a point where inhibition sets in. For additional information, please also read the section in the manual (page 17) describing general guidelines when conduction microRNA expression profi ling in serum and plasma samples and the section about using RNA spike-ins for quality control of the RNA isolation (page 15).

Protocol C. Other Focus microR

NA

PCR


a samples

40


Additional required materials:• 96- or 384-well plate real time PCR cycler• Thermocycler for fi rst strand synthesis• Micro centrifuge• Sealing foils for PCR plates• Swing bucket centrifuge for 96/384 well plates• Recommended: Liquid handling robot for pipetting

Checklist:• Have you considered excess volumes required for using liquid handling robotics? – see page 19• Did you consider how to use the RNA spike-ins? – please see page 15• ROX: The ExiLENT SYBR® Green master mix, does not include the ROX passive reference dye. Please follow instrument manufactures recommendations.• ABI instruments: The use of manual background and threshold settings is necessary for obtaining correct PCR data. Make sure to have the optimal settings by downloading the instrument settings fi le at www.exiqon.com/sds. Furthermore, if the data is to be analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ• Have you optimized the input amount to the RT reaction in order to avoid inhibition?

Prot

ocol

C. O

ther

Foc

us m

icro

RN

A P

CR

Pane

ls u

sing

ser

um/p

lasm

a sa

mpl

es

41




Phase III: real-time PCR amplificationSee protocol page 44.- Mix cDNAs with PCR Master mix- Add cDNA: PCR Master mix to PCR plates


4

3

2

1

0


stroma

Tumor Total

miR-21 let-7a

Rel

ativ

e ex

pres

sion

(log

2)

Workfl ow for Focus microRNA PCR Panels (per sample)


NA

PCR


a samples

42









Prot

ocol

C. O

ther

Foc

us m

icro

RN

A P

CR

Pane

ls u

sing

ser

um/p

lasm

a sa

mpl

es

43


1) The equivalent of 8μl original serum/plasma is used per 10 μL reverse transcription reaction, respectively. Optimal volumes should be determined on individual assays prior to panel experiments.


Step 3Combine reagentsaccording to Table 19


When performing first-strand cDNA synthesis on multiple RNA samples, it is recommended to prepare an RT working solution of the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the proportions indicated in the first four lines of Table 19).The following procedure is recommended:1. Prepare the required amount of RT working solution and place it

on ice.2. Dispense RT working solution into nuclease free tubes.3. Dispense template RNA in each tube.

Reagent96 well panel Volume (μL)

5x Reaction buffer 2

Nuclease-free water 4.5

Enzyme mix 1


0.5

Template total RNA1) 2

Total volume 10








NA

PCR


a samples

44


See tip 8

Step 7Combine cDNA, water and PCR Master mix and add to PCR plates3)

Mix thoroughly

The following procedure is recommended:1. Before removing the plate seal, briefly spin down the plate(s) in a

plate centrifuge.2. Combine 2x PCR Master mix and water: 500 μL 2x master mix and

495 μL water.3. Mix gently and spin down.4. Add 10 μL cDNA5. Add 10 μL PCR Master mix: cDNA mix to each well4).6. Seal the plate with optical sealing as recommended by the

instrument manufacturer.



qPCR protocol:

3) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.4) If using a 96-well cycler with a minimum recommended volume of 20 μL (like some ABI instruments), then use 10μL reaction volume

and set the instrument settings at 20μL.



Prot

ocol

C. O

ther

Foc

us m

icro

RN

A P

CR

Pane

ls u

sing

ser

um/p

lasm

a sa

mpl

es

45


Step 8Spin plate

Spin plate briefly in a plate centrifuge (1500g for 1 minute), to collect the sample.







95˚C, 10 min 95˚C, 10 min


Optical read


Optical read




NA

PCR


a samples

46


Step 10Analyze data

Perform initial data analysis using the software supplied with the real-time PCR instrument to obtain raw Cq values (Cp or Ct, depending on PCR instrument). If you are using an ABI instrument, please note that it is not recommended to use auto Ct settings. For a guide on how to set manual baseline and threshold, refer to Tip 10, page 61 in the Tips section. Alternatively, use ABI settings files available from www.exiqon.com/sds. Furthermore, if the data is to be analyzed using Exiqon GenEx, the experiment must be set up as an AQ experiment, not RQ. If you are using a Roche LC480 instrument, we recommend analysis using the 2nd derivative method.





See tip 11

Prot

ocol

C. O

ther

Foc

us m

icro

RN

A P

CR

Pane

ls u

sing

ser

um/p

lasm

a sa

mpl

es

47


Protocol D. Pick-&-Mix microRNA PCR Panels using serum/plasma samplesThis protocol is used for conducting the fi rst-strand cDNA synthesis and real time PCR, using the Pick-&-Mix microRNA PCR Panel, 96-well plates (product number 203893-203897) and 384-well plates (product number 203818-203819).

The levels of total RNA found in serum and plasma are very low and it is therefore RNA carriers should be applied to ensure robust RNA isolation. This makes it impossible to determine the concentration of RNA in a given sample a� er RNA extraction. It is therefore recommended to use the amount of starting material as a measure of input amount.

RNA inhibitors in serum/plasma will prevent appropriate cDNA synthesis and amplifi cation of microRNAs. The amount of inhibitors in a sample varies with samples and the RNA extraction method. It is therefore recommended to investigate how much extracted RNA to use in the RT reaction without experiencing inhibition, before starting the experiment. This can be done by running a few individual assays with diff erent amounts of RNA input volume e.g. 2 μl, 4 μl and 6μl per 20 μL cDNA synthesis reaction (equivalent to 8 μL, 16 μL and 24 μL of serum/plasma, respectively). By increasing the RNA input the Cq values are reduced to a point where inhibition sets in. For additional information, please also read the section in the manual (page 17) describing general guidelines when conduction microRNA expression profi ling in serum and plasma samples and the section about using RNA Spike-ins for quality control of the RNA isolation (page 15).

Protocol D. Pick-&

-Mix m

icroRN

A PCR


a samplesPCR

Panels

48


Additional required materials:• 96- or 384-well plate real time PCR cycler• Thermocycler for fi rst-strand cDNA synthesis• Micro centrifuge• Recommended: Liquid handling robot for pipetting• Sealing foils for PCR plates• Recommended: Liquid handling robot for pipetting

Checklist:• Have you considered excess volumes required for using liquid handling robotics? – see

page 19• Did you consider how to use the RNA spike-ins? – please see page 15• ROX: The ExiLENT SYBR® Green master mix, does not include the ROX passive reference

dye. Please follow instrument manufactures recommendations• ABI instruments: The use of manual background and threshold settings is necessary

for obtaining correct PCR data. Make sure to have the optimal settings by downloading the instrument settings fi le at www.exiqon.com/sds. Furthermore, if the data is to be analyzed using GenEx, the experiment must be set up as an AQ experiment, not RQ.

• Have you optimized the input amount to the RT reaction in order to avoid inhibition?

Prot

ocol

D. P

ick-

&-M

ix m

icro

RN

A P

CRPa

nels

usi

ng s

erum

/pla

sma

sam

ples

PCR

Pan

els

49




Phase III: Real-time PCR amplificationSee protocol page 53.- Mix cDNAs with PCR Master mix- Add cDNA: PCR Master mix to primer sets replicates (indicated by colored frames)


4

3

2

1

0


stroma

Tumor Total

miR-21 let-7a

Rel

ativ

e ex

pres

sion

(log

2)

Workfl ow for Pick-&-Mix microRNA PCR Panels (per sample)

Protocol D. Pick-&

-Mix m

icroRN

A PCR


a samplesPCR

Panels

50


Pick-&-Mix microRNA PCR Panel layoutsOverview of the pre-defi ned plate layouts available for 96-well and 384-well plates of the Pick-&-Mix Panel. Wells in dark gray and light gray are pre-occupied by interplate-calibrators (UniSp3 IPC) and RNA spike-in controls (UniSp6), respectively.

10 microRNAs x 8 samples

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC

1 2 3 4 5 6 7 8 9 10 CP IPC


A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 IPC

13 14 15 16 17 18 19 20 21 22 23

13 14 15 16 17 18 19 20 21 22 23

13 14 15 16 17 18 19 20 21 22 23

13 14 15 16 17 18 19 20 21 22 23

CP

1 2 3 4 5 6 7 8 9 10 11 IPC

CP

1 2 3 4 5 6 7 8 9 10 11 IPC

CP

1 2 3 4 5 6 7 8 9 10 11 IPC

CP

92 microRNAs x 1 sample

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 121 2 3 4 5 6 7 8 9 10 11 IPC

13 14 15 16 17 18 19 20 21 22 23

25 26 27 28 29 30 31 32 33 34 35

37 38 39 40 41 42 43 44 45 46 47

49 50 51 52 53 54 55 56 57 58 59

61 62 63 64 65 66 67 68 69 70 71

73 74 75 76 77 78 79 80 81 82 83

85 86 87 88 89 90 91 92 93 94 95

CP

IPC

IPC

60

72

84

96


A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

CP IPC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22


A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 CP

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

IPC

CP

IPC

CP

IPC

CP

IPC

CP

IPC

CP

IPC

CP

IPC

CP

IPC


A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 1 22 33 44 55 66 77 88 99 1010 1111 1212

1 1 22 33 44 55 66 77 88 99 1010 1111 1212

13 13 1414 1515 1616 1717 1818 1919 2020 2121 2222 2323 2424

13 13 1414 1515 1616 1717 1818 1919 2020 2121 2222 2323 2424

25 25 2626 2727 2828 2929 3030 3131 3232 3333 3434 CPCP 3636

25 25 2626 2727 2828 2929 3030 3131 3232 3333 3434 CPCP 3636

37 37 3838 3939 4040 4141 4242 4343 4444 4545 4646 4747 4848

37 37 3838 3939 4040 4141 4242 4343 4444 4545 4646 4747 4848

49 49 5050 5151 5252 5353 5454 5555 5656 5757 5858 5959 6060

49 49 5050 5151 5252 5353 5454 5555 5656 5757 5858 5959 6060

61 61 6262 6363 6464 6565 6666 6767 6868 6969 7070 IPCIPC 7272

61 61 6262 6363 6464 6565 6766 6767 6868 6969 7070 IPCIPC 7272

73 73 7474 7575 7676 7777 7878 7979 8080 8181 8282 8383 8484

73 73 7474 7575 7676 7777 7878 7979 8080 8181 8282 8383 8484

85 85 8686 8787 8888 8989 9090 9191 9292 9393 9494 9595 9696

85 85 8686 8787 8888 8989 9090 9191 9292 9393 9494 9595 9696

380 microRNAs x 1 sample

A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 2 43 65 87 109 1211 1413 1615 1817 2019 2221 2423

25 26 2827 3029 3231 3433 3635 3837 4039 4241 4443 4645 4847

5049 51 5352 5554 5756 5958 6160 6362 6564 6766 6968 7170 72

73 74 7675 7877 8079 8281 8483 8685 8887 9089 9291 9493 IPC95

97 98 10099 102101 104103 106105 108107 110109 112111 114113 116115 118117 120119

121 122 124123 126125 128127 130129 132131 134133 136135 138137 140139 142141 144143

145 146 148147 150149 152151 154153 156155 158157 160159 162161 164163 166165 168167

169 170 172171 174173 176175 178177 180179 182181 184183 186185 188187 190189 IPC191

193 194 196195 198197 200199 202201 204203 206205 208207 210209 212211 214213 216215

217 218 220219 222221 224223 226225 228227 230229 232231 234233 236235 238237 240239

241 242 244243 246245 248247 250249 252251 254253 256255 258257 260259 262261 264263

265 266 268267 270269 272271 274273 276275 278277 280279 282281 284283 286285 CP287

289 290 292291 294293 296295 298297 300299 302301 304303 306305 308307 310309 312311

313 314 316315 318317 320319 322321 324323 326325 328327 330329 332331 334333 336335

337 338 340339 342341 344343 346345 348347 350349 352351 354353 356355 358357 360359

361 362 364363 366365 368367 370369 372371 374373 376375 378377 380379 382381 IPC383Prot

ocol

D. P

ick-

&-M

ix m

icro

RN

A P

CRPa

nels

usi

ng s

erum

/pla

sma

sam

ples

PCR

Pan

els

51









Protocol D. Pick-&

-Mix m

icroRN

A PCR


a samplesPCR

Panels

52


1) The amount of cDNA required per sample depends on the amount microRNA analyzed per sample. The volumes suggested here provides excess amount of cDNA, which ensures the highest possible reproducibility because these volumes can be pipetted with great accuracy.

2) The equivalent of 16 or 32μl original serum/plasma is used per 20 or 40 μL reverse transcription reaction, respectively. Optimal volumes should be determined on individual assays prior to panel experiments.

Step 3Combine reagents according to Table 21

When performing first-strand cDNA synthesis on multiple RNA samples, it is recommended to prepare an RT working solution of the 5x Reaction buffer, water, Enzyme mix and RNA spike ins (in the proportions indicated in the first four lines of Table 21).The following procedure is recommended:1. Prepare the required amount of RT working solution and place it on ice.2. Dispense RT working solution into nuclease free tubes.3. Dispense template RNA in each tube.

The amount of 50x fold diluted cDNA needed for the different Pick-&-Mix layouts can be seen in Table 22 – Step 7.

Reagent

Volume (μL) < 100 microRNA analyzed per sample

Volume (μL)> 100 microRNA analyzed per sample

5x Reaction buffer 2 4

Nuclease-free water 4.5 9

Enzyme mix 1 2


0.5 1

Template total RNA2) 2 4

Total volume 10 20

Table 21. Reverse transcription reaction setup per sample1)



Prot

ocol

D. P

ick-

&-M

ix m

icro

RN

A P

CRPa

nels

usi

ng s

erum

/pla

sma

sam

ples

PCR

Pan

els

53


qPCR protocol:







Protocol D. Pick-&

-Mix m

icroRN

A PCR


a samplesPCR

Panels

54


See tip 8

4) Adjust volumes to accommodate your in-house liquid handling system and the losses these have when pipetting.

Step 7Dilute cDNA template 50x in nuclease free water4)

Dilute the cDNA from the RT reactions to give a final 50x dilution. Suggested cDNA dilution procedures is shown in Table 22 along with required volumes of diluted cDNA for the different Pick-&-Mix pre-defined layouts. For fully customized layouts dilutions may be adjusted to the specific replicate scheme. It is recommended to use “low-nucleic acid binding” tubes or plates. It is not recommended to store the 1:50 dilution of cDNA.

Numbers in parenthesis designates total number of assays/sample, including controls and inter plate calibrators. Loss from pipetting is not included in the volumes listed in the right column (diluted cDNA needed) .


Pick-&-Mix plate configuration

Suggested cDNA dilution procedure

Volume (μL) of diluted cDNA needed for each Pick-&-Mix plate

8x10 (12) 4 + 196 60

4x22 (24) 4 + 196 120

1x92 (96) 10 + 490 480

16x22 (24) 4 + 196 120

8x46 (48) 6 + 294 240

4x94 (96) 10 + 490 480

1x380 (384) 40 + 1960 1920

Table 22. Amount of 50x diluted cDNA needed in Pick-&-Mix plates

Prot

ocol

D. P

ick-

&-M

ix m

icro

RN

A P

CRPa

nels

usi

ng s

erum

/pla

sma

sam

ples

PCR

Pan

els

55


Step 8Combine cDNA and PCR Master mix 1:1 and add to PCR plates

Mix thoroughly

The following procedure is recommended:1. Before removing the plate seal, briefly spin down the plate(s) in a

plate centrifuge.2. Combine 2x PCR Master mix and 50x diluted cDNA 1:1 (e.g. 500 μl 2x

master mix and 500 μl diluted cDNA).3. Mix gently by inverting the tube, spin down.4. Add 10 μL PCR Master mix:cDNA mix to each well.4)

5. Seal the plate with optical sealing as recommended by the instrument manufacturer.


Step 9Spin plate

Spin plate briefly in a plate centrifuge (1500g for 1 minute), to remove air bubbles.







95˚C, 10 min 95˚C, 10 min


Optical read


Optical read



Protocol D. Pick-&

-Mix m

icroRN

A PCR


a samplesPCR

Panels

56


Step 11Analyze data

Perform initial data analysis using the software supplied with the real-time PCR instrument to obtain raw Cq values (Cp or Ct, depending on PCR instrument). If you are using an ABI instrument, please note that it is not recommended to use auto Ct settings. For a guide on how to set manual baseline and threshold, refer to Tip 10, page 61 in the Tips section. Alternatively, use ABI settings files available from www.exiqon.com/sds. Furthermore, if the data is to be analyzed using Exiqon GenEx, the experiment must be set up as an AQ experiment, not RQ. If you are using a Roche LC480 instrument, we recommend analysis using the 2nd derivative method.


5) If using a 96-well cycler with a minimum recommended volume of 20 μL (like some ABI instruments), then use 10μL reaction volume and set the instrument settings at 20μL.




See tip 11

Prot

ocol

D. P

ick-

&-M

ix m

icro

RN

A P

CRPa

nels

usi

ng s

erum

/pla

sma

sam

ples

PCR

Pan

els

57


Tips to protocol

Tip 1. RNA extraction and template preparation

Purifi cation and preparation of total RNA that includes small RNAs (<200 nt) from a biological sample is the fi rst critical step for a successful expression profi ling analysis of microRNAs. Therefore, the method used for RNA sample preparation is critical to the success of the experiment. The following points should be considered before starting the experiment: • We recommend using the miRCURY™ RNA Isolation kit for isolation of total RNA that

contains the small RNA fraction. If not using one of the miRCURY™ RNA Isolation kits, it is important that the method used to isolate RNA from a sample should give a quantitative recovery of small RNAs and should not result in a substantial loss of the small RNA fraction. This also applies when using commercially available kits. Make sure the total RNA preparation is guaranteed to contain microRNA.

• If commercially available purifi ed RNA is used, it is important to make sure that the RNA is guaranteed to contain small RNAs and is representative of the microRNAs in the species and/or tissue from which it was isolated.

• The comparison of samples prepared using diff erent RNA isolation methods is not recommended. However, if this is necessary, it is recommended to include the measure of a reference small RNA which has a consistent and unvaried expression level in order to allow for comparison of microRNA levels in the diff erent sample preparations.

• The isolation of RNA and the reaction steps preceding real-time PCR should be performed in rooms separate from where real-time PCR experiments will take place in order to avoid contamination with amplicon.

• It is recommended that the integrity of isolated RNA be assessed before proceeding with quantitative real-time PCR experiments. This may be performed either on the Agilent 2100 Bioanalyzer or by denaturing gel electrophoresis.

• If necessary, treat RNA preparations with DNases to remove contaminating DNA that may interfere with the real-time PCR results.

• Purifi ed total RNA should be dissolved in nuclease-free water at a stock concentration of at least 1 μg/μl. See recommendations for storage conditions in Tip 3.

58


Tip 2. Guidelines for serum, plasma and biofl uid samples

Plasma and serum are essentially cell free liquid samples. This means that only circulating RNA is extracted from these sample types, resulting in low total RNA concentrations, even if the microRNA fraction is readily detectable. The result of this is that measuring correct RNA concentrations is diffi cult, and that there is a high risk of increased loss during extraction. For this reason, we recommend using RNA amounts based on starting volume rather than RNA quantity. Comprehensive guidelines for microRNA profi ling in blood serum/plasma can be downloaded at www.exiqon.com/serum-plasma-guidelines.

Tip 3. General guidelines for handling and storage of RNA

The following precautions should be taken to prevent RNase contamination and degradation of the RNA sample and reagents:• Always wear disposable gloves.• Use nuclease-free, low nucleic acid binding plasticware and fi lter barrier pipette tips.• Keep tubes capped when possible. Always spin tubes before opening.• For long-time storage, RNA may be stored at -80˚C. Avoid repeated freezing and thawing

cycles.

Tip 4. Good PCR laboratory practice

To reduce the risk of contaminating PCRs with “old” PCR amplicons, and consequently obtain false results: • Always wear a clean lab coat. Use separate lab coats for RNA sample preparation, cDNA

synthesis and when setting up PCR reactions or handling PCR products.• Change gloves o� en, especially whenever you suspect they may have been contaminated. • Establish and maintain designated areas for PCR set-up, PCR amplifi cation, and gel

electrophoresis of PCR products.• Never bring amplifi ed PCR products into the PCR set-up area.• Spin down all reaction and sample tubes before opening. Open and close all reagent and

sample tubes carefully, trying not to splash or spray PCR samples.• Keep reactions and components capped whenever possible.• Use fi lter barrier pipette tips to avoid aerosol-mediated contamination

of your pipetting device.• Clean laboratory benches and equipment regularly.

59


Tip 5. Keep reagents and reactions cool at all times

In order to ensure optimal performance of the miRCURY LNA™ Universal RT microRNA PCR system it is important that the reagents and reactions are kept on ice (or at 4˚C) as much as possible during the protocol (apart from reaction steps specifi cally involving raised temperatures).

Tip 6. First strand cDNA synthesis

Store cDNA samples in nuclease-free low nucleic acid-binding micro centrifuge tubes, e.g. Eppendorf DNA LoBind tubes.

Tip 7. Recommended controls

The following controls are recommended and should be included in the experimental set-up:• Reverse transcription/no enzyme controls, i.e. fi rst-strand cDNA synthesis reactions

performed without the Enzyme mix. If a PCR product is amplifi ed from this control reaction it indicates genomic DNA or PCR product contamination of the template RNA.

• Non-template controls in the real-time PCR amplifi cation, i.e. real-time PCR reactions performed without any cDNA template. This control will reveal PCR product contamination of the reaction.

• Blank purifi cation or carrier only, i.e. when purifying RNA in the presence of carrier RNA (such as for serum and plasma samples). This control will reveal any non-specifi c signals originating from the carrier RNA alone.

Tip 8. Passive reference dye (ROX)

Many real-time PCR instruments will only produce reliable results when a passive reference dye such as ROX is added to the PCR reaction. The reference dye is used to normalize signals from individual PCR wells in order to enable comparison of real-time PCR amplifi cation signals across an entire PCR-plate.

It is recommended to determine whether your real-time PCR instrument has this type of requirement. The amount of ROX to include in the PCR reaction depends on the requirements of the real-time PCR instrument and must be adjusted accordingly. Please follow the supplier’s instructions for preparation and concentrations of ROX solutions.

60


Typically, real-time PCR instruments that allow excitation at individual wavelengths for individual dyes (most fi lter wheel based instruments) require less ROX than instruments that use a single excitation wavelength for all fl uorophores (most laser based instruments use excitation at 488 nm).

It is important to note that excessive amounts of ROX may inhibit the PCR reaction. It may be recommended to perform a titration of ROX amounts in order to determine the optimal concentration for a particular instrument-system combination. It is possible to use the spike-in template (provided in the Universal cDNA synthesis kit II) and the Control primers (provided in the ExiLENT SYBR® Green master mix kit) to perform such titrations. Please contact us (exiqon.com/contact) for the most up-to-date information regarding passive reference dyes.

Recommended ROX concentrations are found here:

Tip 9. Inter-plate calibration

When setting up large scale experiments with several PCR plates involved, individual run diff erences may be observed. One way of avoiding these having an eff ect on the results is setting up the experiment in such a way that all samples for one gene are run on the same plate. However, this is not always feasible. When Cq values for one gene have to be compared across plates, it is recommended to employ an inter-plate calibrator.

Instrument ROX concentration

ABI 7900HT 300-500 nM

ABI 7900HT FAST 300-500 nM

ABI Viia7 30-50 nM

ABI Viia7 FAST 30-50 nM

ABI 7500 FAST 30-50 nM

ABI StepOnePlus 300-500 nM

ABI 7300 300-500 nM

ABI 7500 30-50 nM

ABI 7700 300-500 nM

ABI 7000 300-500 nM

Table 24.

61


An inter-plate calibrator is a template-assay combination which is the same in all plates, and always located in the same well across diff erent plates. This can then be used to calibrate all plates to give the same Cq value for the calibrator, thereby reducing run-to-run variance. Please note that all contain inter-plate calibrators.

Tip 10. Guidelines for real-time PCR data collection using ABI instruments

On cyclers using baseline and threshold values for Cq (Ct) calculations, such as ABI 7900HT, it is important that the proper settings are used. Use of the automatic function of the so� ware for these settings does not seem to produce optimal results for SYBR® Green based assays. O� en the baseline is set erroneously on non-detected assays, and this in turn gives false positives, therefore do not use automatic settings. Another issue to consider when using automatic settings is that the settings may diff er between plates resulting in data that cannot be compared directly. Inter-plate calibration may not fully resolve this issue, since each assay has a separately calculated baseline and threshold. Instead, both threshold and baseline should be set manually, applying the same settings for all assays on the plate.

The following principles should be applied to manual baseline and threshold settings:

Baseline: The baseline should be calculated in the cycle interval before the amplifi cation takes off (see Figure 3).

Threshold: The threshold should then be set with the Y-axis in log scale where all assays are in the log linear phase, and the threshold above background for all assays (see Figure 3).

Note: The optimal threshold value may vary between individual machines and experiments.

Important note

If ROX passive reference dye has not been used in the PCR reactions, make sure the SDS so� ware is set-up without reference dye correction.

62


Baseline setting

Figure 5.

63


Tip 11. Quick guide to normalization of microRNA qPCR experiments

The purpose of normalization is to remove technical and biological variation between samples that is not related to the biological changes under investigation. Proper normalization is critical for the correct analysis and interpretation of results from real-time PCR experiments. The most commonly used option for normalization is to use stably expressed reference genes.

In general it is recommended to test several endogenous control candidates (reference genes) before setting up the actual microRNA expression analysis. These candidates should be chosen among genes that can be expected to be stably expressed over the whole range of samples being investigated. They can be stably expressed small non-coding RNA, or stably expressed microRNAs, and chosen based on literature or pre-existing data (e.g. microarray analysis or qPCR panel screening).

Exiqon off ers primer sets for a number of diff erent small RNAs which tend to be stably expressed, and are therefore o� en good candidates for reference genes. It’s important to keep in mind that in spite of being small non-coding RNAs, most of these are signifi cantly larger than microRNA and therefore may have diff erent extraction effi ciency and stability. U6 is one such reference gene which is o� en used. However, U6 is signifi cantly larger than microRNAs and has a diff erent sub-cellular distribution. The existence of several diff erent isoforms also makes it a suboptimal reference gene. 5S ribosomal RNA is another popular option, but this RNA has a much higher expression level than most microRNAs, and is o� en found as a PCR contaminant.

If working with blood serum or plasma, please note that only circulating RNA are present. In this case the small non-coding RNAs (5S, U6, SNORs etc) are not good candidates for reference genes, since they are most probably not present in the sample.

Using stably expressed microRNAs as reference genes off ers several advantages such as equal size, extraction effi ciency and stability, as well as having expression levels within a similar range of the target microRNAs.

64


Several candidates can be found in the literature, including miR-191-5p, miR-103a-3p,let-7a-5p, and miR-16-5p. Microarray or qPCR panel screening data may also be used when choosing candidate reference genes.

All reference gene candidates should be empirically validated for each study. A number of diff erent so� ware packages exist for evaluating the optimal nature and number of endogenous controls, and for applying multiple endogenous controls for normalizing target expression. One such option is the GenEx so� ware from MulitD, sold through Exiqon with a special application for Exiqon PCR panels. GenEx incorporates both GeNorm and Normfi nder for fi nding the optimal reference genes, and is easy and intuitive to use for the actual normalization.

An alternative option for normalization of data from panels (profi ling a high number of microRNAs) is to normalize against the global mean; that is, the average of all expressed microRNAs This can be a good option in samples with a high call-rate (expressed microRNAs), but should be used with caution in samples with low call-rates. It is also not a good option in samples for which the general microRNA expression level is changed.

For further information on normalization and references, we recommend our data-analysis guide available online as well as the guide to microRNA normalization fromwww.gene-quantifi cation.de.

Tip 12. Melting curves

Melting curves are a good tool for evaluating the specifi city of the assay. When setting up the melt curve temperature gradient on the instrument, it is recommended to start at 60˚C.

65


Troubleshooting guide

Problem Suggestion

PCR signal in samples amplified from first-strand synthesis reactions performed without reverse transcriptase

This typically indicates contamination of the template RNA with genomic DNA. Perform DNase treatment of the RNA sample. If this does not solve the problem RNA samples or other reagent may be contaminated with PCR products.

PCR signal in no-template PCR reaction

This typically indicates contamination of the cDNA template or PCR reagents with amplified PCR product. Exposing the reactions to elevated temperatures (i.e. room temperature) during any part of the protocol increases the risk of background signals. It is important that the reagents and assembled reactions are kept cool (on ice or 4̊ C) at all times (see Tip 4,page 58 for details).

Generated signalsare weak

• On some real-time PCR cyclers, gain-settings are adjustable. Make sure the gain settings of your real-time PCR cycler have been set to accommodate the signals generated from the specific assay.

• RNA samples may contain PCR inhibitors. Further purification or an alternative RNA extraction method may be necessary. Check positive controls.

No fluorescent signal is detected during the PCR

Confirm that you have a PCR product by running an aliquot of your PCR reaction on an agarose gel.

No fluorescent signal detected during the PCR, but a PCR amplicon can be detected by agarose gel electrophoresis

• Check that the filter in the real-time PCR cycler was set to either SYBR® Green or FAM/FITC.

• Check that the optical read is at the correct step of the real-time PCR cycles.

• Adjust the baseline in the real-time PCR cycler software.

66


FAQs

What kind of real-time PCR instruments is miRCURY LNA™ Universal RT microRNA PCR compatible with?miRCURY LNA™ Universal RT microRNA PCR is compatible with all instruments capable of reading green fl uorophores such as fl uorescein/FITC/FAM and SYBR® Green. The system has been tested and found to work on real-time PCR instruments from several leading suppliers of this type of instrument.

What kind of settings should I use on my real-time PCR instrument?If your real-time PCR instrument supports fl uorophores such as fl uorescein/FITC/FAM or SYBR® Green your instrument must be set to detect these fl uorophores.

Is miRCURY LNA™ Universal RT microRNA PCR compatible with other SYBR® Green master mixes?We do not recommend using other SYBR® Green master mixes for real-time PCR analysis with the LNA™ PCR primer sets or Ready-to-Use panels. The primer sets have been optimized and validated using the miRCURY LNA™ ExiLENT SYBR® Green master mix and the performance of the primer sets will be compromised by using a diff erent master mix (which may contain diff erent salt and/or enzyme concentrations).

My RNA is already enriched for microRNAs, how much should I use in the real-time PCR experiments?miRCURY LNA™ Universal RT microRNA PCR is developed for use on total RNA and we do not recommend enriching for small RNAs. Samples of enriched microRNAs are diffi cult to quantitate accurately making it very tricky to ensure the same amount of sample is added to each reaction. If necessary, a total RNA equivalent should be used for the enriched sample, e.g. use a proportional amount of enriched sample resulting from 20 ng of total RNA. It may be necessary to try a couple of diff erent amounts of enriched sample to ensure that the results fall within the linear range of the assay.

67


What is the recommended experimental set up for Universal RT microRNA real-time PCR?It is generally accepted that the reverse transcription (RT) reaction gives rise to more variation than the PCR reaction. It is therefore advisable to perform replicate RT reactions, ideally 3 separate reactions with 1-2 PCR reactions for each RT. It is further recommended to always include at least three biological replicates (separate RNA extractions) of each sample type in order to allow statistical analysis of the results. If small changes in microRNA expression are expected, it may be necessary to include more replicates to ensure a signifi cant result. In general it is recommended that replicates should be included at any stage during sample procurement, processing, RNA isolation, etc. that could give rise to variation between samples.

A tech note on guidelines for setting up microRNA qPCR experiments can be downloaded at www.exiqon.com/miRNA-qPCR-guidelines.

Additional information is available at www.exiqon.com/mirna-pcr

68


Related products

Exiqon off ers a broad variety of tools enabling new discoveries concerning the expression, function and spatial distribution of microRNAs:

miRCURY LNA™ microRNA Array, microarray kitPre-printed miRCURY LNA™ microRNA Array microarray slides, available for hsa, mmu & rno and other species. Kit includes hybridization and wash buff ers as well as synthetic spike-in microRNAs.

miRCURY LNA™ microRNA Power labeling kitFor fl uorescent labeling of microRNAs from total RNA samples ready for array hybridization.

miRCURY LNA™ microRNA Array, ready-to-spot probe setReady-to-spot oligos for direct printing of arrays, or coupling in bead-based applications.

miRCURY LNA™ microRNA Detection ProbesFor in situ hybridization and northern blotting of all annotated microRNAs.

miRCURY LNA™ microRNA ISH Optimization kit (FFPE)Complete kit with control probes and hybridization buff er for easy set up of microRNA in situ hybridization.

miRCURY LNA™ microRNA Inhibitors, Power Inhibitors and Family InhibitorsUnravel the function of microRNAs by microRNA inhibition. Sophisticated LNA™ design ensures potent inhibition of all microRNAs regardless of their GC content. Chemically modifi ed, highly stable Power Inhibitors for unrivalled potency. Available for in vitro and in vivo studies.

miRCURY LNA™ microRNA Inhibitor LibraryFor genome-wide high throughput screening of microRNA function.

miRCURY LNA™ Target Site BlockersFor inhibition of microRNA binding to a specifi c mRNA target.

69


Notice to purchaserExiqon, LNA and miRCURY are registered trademarks of Exiqon A/S, Vedbaek, Denmark. SYBR Green is a licensed trademark of Invitrogen. All other trademarks are the property of their respective owners.

Locked-nucleic Acids (LNAs™) are protected by US Pat No. 6,268,490, US Pat No. 6,770,748, US Pat No. 6,639,059, US Pat No. 6,734,291 and other applications and patents owned or licensed by Exiqon A/S. Products are provided to buyers for research use only. The products in their original or any modifi ed form may be used only for the buyer’s internal research purposes and not for commercial, diagnostic, therapeutic, or other use, including contract research. The buyer may not provide products to third parties in their original or any modifi ed form. The purchase of products does not include or carry an implied right or license for the buyer to use such products in their original or any modifi ed form in the provision of services to third parties, and a license must be obtained directly from Exiqon A/S for such uses.

Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056 and 6,171,785. The purchase of this product includes a limited, non transferable immunity from suit under the foregoing patent claims for using only this amount of product solely in Contract Research, including reporting results of purchaser’s activities for a fee or other commercial consideration, and also for the purchaser’s own internal research. No right under any other patent claim is conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

Furthermore, this product is provided under an agreement between Molecular Probes, Inc., a wholly owned subsidiary of Invitrogen Corporation, and EXIQON and the manufacture, use, sale or import of this product is subject to one or more U.S. Patents and corresponding international equivalents. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer, where such research does not include testing, analysis or screening services for any third party in return for compensation on a per test basis. The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, 29851 Willow Creek Road, Eugene, OR 97402. Tel: (541) 465-8300, Fax: (541) 335-0354.

Further, the purchase of this product includes a limited, non-transferable license under specifi c claims of U.S. Patent Nos. 6,174,670 and 6,569,627, owned by the University of Utah Research Foundation and licensed to Roche Diagnostics GmbH and Idaho Technology, Inc., to use only the enclosed amount of product according to the specifi ed protocols. No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670 and 6,569,627, other than for the amount of product contained herein.

For life science research use only. Not for use in diagnostic procedures

13-0

099

- 92

0004

- v

6.2

- 07

/201

6 -

SERU

M

Outside North AmericaExiqon A/S Skelstedet 16DK-2950 Vedbaek, DenmarkPhone +45 45 650 929Fax +45 45 661 888

North AmericaExiqon Inc. 12 Gill Street, Suite 1650Woburn, MA 01801, United StatesPhone (781) 376 4150Fax (781) 376 4152

exiqon.com

PCR manual serum / plasma samples

Documents