Tissue to C T : Actual qRT-PCR Results from LCM Samples Joel L. “Jock” Moore, Jr. June 24, 2009.

Tissue to CT: Actual qRT-PCR Results from LCM Samples

Joel L. “Jock” Moore, Jr.

June 24, 2009

Tissue Preparation

• M303 was an infarct model that also received an intramyocardial injection of hMSCs.

• Mouse hearts were harvested, embedded in OCT, and snap-frozen in liquid N2.

• Hearts were stored > 2 years at -80 °C

M222 & M224 were mice with normal hearts. Each received intramyocardial injections of hMSCs.

Slide Preparation

• PALM PEN-Membrane slides were sprayed with RNaseZap (Ambion), dipped in DEPC-treated H2O 2X, then allowed to dry.

Prior to cutting tissue on the cryostat, all slides were UV-treated for ≥ 30 min. This not only sterilizes the slides but also allows tissue to adhere more easily to the PEN membrane (UV light alters its hydrophobic nature).

Slide Preparation• For each animal, multiple 10 μm sections were

cut with an RNase-free cryostat blade and placed on the membrane slides.

• A few sections were also placed on RNase-free glass slides to serve as pre-staining RNA quality control samples.– Cost saving

– More difficult

• Slides were placed back-to-back in a Falcon 50ml conical tube, then stored at -80 °C for a few days.

• Overall, the dryer and colder your tissue sections are, the less likely ribonuclease activity will degrade your RNA

• OCT must be removed before microdissection

• Keep all reagents ICE-cold

• Limit aqueous phase staining steps

• End your staining protocol with a dehydration step e.g., 100% EtOH (NOT xylenes). This will allow you to work longer on the LCM system with minimal RNA degradation

Staining Protocol: General Tips

Example H&E Staining Protocol

• Retrieved Falcon tube from freezer and let thaw (~ 5 min).

• “Fixed” membrane slide in 70% EtOH, 2 ½ min. In the meantime, dipped the glass slide in DEPC-treated H2O then scraped tissue directly into a microfuge tube containing lysis buffer.

• Hematoxylin QS (Vector Labs), 30 sec.• Dip in DEPC-treated H2O• Eosin-Y, few seconds.• Quick increasing EtOH series (70%, 96%,

100%).• Removed excess and let dry on Robomover

stage.• While drying, used the 5X objective to scan

the entire slide.

PALMRobo Software Settings

• These settings will vary dramatically by objective, embedding material, tissue type, even staining protocol.

• My settings using a 10X objective– RoboLPC joint = 16 μm

– Cut speed = 50 μm/sec

– Cut/LPC Focus Δ = - 3 (Cut: 87, LPC: 84)

– Cut/LPC Energy Δ = +40 (Cut: 52, LPC: 92)

• Fine-tune & save your settings before performing experiments on precious tissue samples.

M224 Injection Site

M224: Injection Site, Adhesive Cap

M303 Infarct Site

M303: Infarct Site, Adhesive Cap

M303: Normal Myocardium

M303: Normal Myocardium, Adhesive Cap

Examples of LPC Elements

•M303

•Infarct site: 20 Circular elements @ ~ 38,000 μm2 each (~ 760,000 μm2 total)

•Normal myocardium: 9 circular elements @ ~ 67,000 μm2 each (~ 600,000 μm2 total)

•M224

•Injection site: 4 elements of varying size (~ 128,000 μm2 total)

•Normal myocardium: 6 circular elements @ ~ 37,000 μm2 each (~ 225,000 μm2 total)

5X Scan, M303 Post-LMPC

* Unsuccessful LPC-elements

RNA Preservation & Extraction

• I used QIAGEN’s RNeasy Plus Micro Kit (Cat # 74034)– Small elution volume of 12 μl

• Ambion’s RNAqueous-Micro Kit (Cat # AM1931) is another popular choice.

• Because ß-mercaptoethanol permanently denatures RNases and lysis buffers are designed to stabilize RNA, you can relax once your sample is submerged in this solution!

RNA Characterization

• Agilent 2100 Bioanalyzer (RNA Pico 6000 Lab-on-a-Chip Kit)

• This system uses microfluidics technology to perform an electrophoretic analysis of RNA using only 1 µL of an RNA sample.

• Its software generates an electropherogram, a virtual gel-image, and a quantitation estimate.

• It is qualitatively accurate at [RNA] between 50 – 5000 pg/μl, but its quantitation accuracy is reported as 30% CV (Coefficient of Variance = SD/mean).

• The RNA integrity number (RIN) is a value automatically assigned to total RNA samples by the Bioanalyzer’s software. Rather than using a ratio of ribosomal bands, the software uses the entire electrophoretic trace to identify the 28S &18S ribosomal peaks while also taking into account the presence or absence of degradation products. Agilent touts the system as the de facto standard for RNA integrity since the assigned RIN is independent of sample concentration, instrument and analyst.

Agilent 2100 Bioanalyzer

1 10

Maximum degradation

Lowest RNA quality

Minimum degradation

Highest RNA quality

The RIN Continuum

• What the RIN can do:– Obtain a numerical assessment of the integrity of RNA. – Directly compare RNA samples, e.g. before and after archiving,

or compare integrity of the same tissue across different labs. – Ensure repeatability of experiments, e.g. if RIN shows a given

value and is suitable for microarray experiments, then the RIN of the same value can always be used for similar experiments given that the same organism/tissue/extraction method is used.

• What the RIN cannot do:– Tell a scientist ahead of time whether an experiment will work or

not if no prior validation was done (e.g. RIN of 5 might not work for microarray experiments, but might work well for an appropriate RT-PCR experiment. Also, a RIN that might be good for a 3' amplification might not work for a 5' amplification)

Agilent 2100 Bioanalyzer

*Directly from Agilent’s website

M222 RNA Degradation: ~40 min post thaw

RIN: 8.4RIN: 8.4

RIN: 7.6RIN: 7.6

We could not locate M222’s injection site on this particular membrane slide. The normal myocardium LCM sample, however, looks to

be of sufficient RNA quality.

RIN: 8.3RIN: 8.3

M224 RNA Degradation: ~40 min post thaw

RIN: 8.8RIN: 8.8

RIN: 6.9RIN: 6.9

RIN: 9.5RIN: 9.5

M224’s Possible Injection Site LCM sample yielded high quality RNA

M224’s Normal Myocardium LCM sample also showed promise.

RIN: 7.9RIN: 7.9

M303 RNA Degradation: ~40 min post thaw

RIN: 8.9RIN: 8.9

RIN: 6.8RIN: 6.8

RIN: 8.6RIN: 8.6

M303 Normal Myocardium LCM sample should produce qRT-PCR results, but…

The M303 Infarct LCM sample is not looking good.

RIN: 1.0RIN: 1.0

RNA Quantitation• Agilent

• Nanodrop

SampleSample [RNA] in pg/[RNA] in pg/μμll

M222 Normal Myocardium 527

M224 Possible I.S. 93

M224 Normal Myocardium 15

M303 Normal Myocardium 123

SampleSample [RNA] in pg/[RNA] in pg/μμll

M222 Normal Myocardium 2,100

M224 Possible I.S. 1,600

M224 Normal Myocardium 3,000

M303 Normal Myocardium 1,760

Nanodrop Spectra

qRT-PCR

• cDNA was synthesized using Invitrogen’s SuperScript First Strand Synthesis system

• Made 10 ng cDNA based on Nanodrop figures

• Used Taqman Probes to search for Human GAPDH and Mouse GAPDH. This will answer the question, “Are these human or mouse cells?”

qRT-PCR Results

Probe Sample CT #1 CT #2 CT #3 Avg. CT SD

Human GAPDHM222 Normal Myocardium - - - - -

Human GAPDHM224 Possible I.S. 28.879 29.015 28.925 29.01 0.07

Human GAPDH M224 Normal Myocardium - - - - -

Human GAPDH M303 Normal Myocardium - - - - -

*CT cut-off set at 40 cycles

Changes for future experiments

Quantitation of RNA?

Amplification of RNA?

Pre-amplification of cDNA?

Quantitation of cDNA? (Standard curves, Picogreen fluorometric assay, etc.)

http://www.ecu.edu/cs-dhs/internalmed/ec/lasercapture.cfm

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