The world leader in serving science 謝瑋珍 (Sophia Hsieh, Ph.D.) Field Application Scientist Applied Biosystems® QuantStudio™ 7 Flex Real- Time PCR System之原理與應用介紹
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The world leader in serving science
謝瑋珍 (Sophia Hsieh, Ph.D.)
Field Application Scientist
Applied Biosystems® QuantStudio™ 7 Flex Real-Time PCR System之原理與應用介紹
2
• Principle of Real-time PCR
- TaqMan® V.S. SYBR® Green
- Reverse Transcription and Real-time PCR Reaction
• Real-time PCR Quantitation Methods
• Application
• Assay design
- Predesigned Assay Search
- Primer Express
• QuantStudio™ 7 Flex Real-Time PCR System
• Thermo Fisher Cloud
Agenda
3
Polymerase Chain Reaction (PCR)
PCR is a method to amplify specific genetic sequences.
Cycle 1 Cycle 2 Cycle 3
4
Polymerase Chain Reaction (PCR)
5
Traditional PCR
In traditional PCR, the product is detected only after amplification
is completed, using methods such as gel electrophoresis
6
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
0 5 10 15 20 25 30 35 40
PCR cycle number
Baseline region
Threshold
CT
No Template control
Sample
No
rmali
sed
rep
ort
er
flu
ore
scen
ce (
Rn)
Exponential Phase
Rn
cap
PCR vessel
thermal block
sample
lens
CT = threshold cycle: calculated fractional cycle number at which
PCR amplification curve crosses the threshold of
detection
Principle of Real-time PCR
Lower expression level
7
Real-time PCR Signal Detection: Exponential Phase
Exponential
Linear
PlateauGel
Low Ct
(high copy #)
High Ct
(low copy #)
PCR cycles→
flu
ore
sc
en
t
sig
na
l→101
100
10-1
Threshold
of detection
Y= N0 2n , CT 與起始濃度之對數值成反比
8
Real-time PCR Chemistries
SYBR® Green I dye
Fluorogenic 5’ Nuclease
Assay
TaqMan® and TaqMan® MGB
Binds Double-
stranded DNA
9
R Qforward primer
reverse
primer
3'
3'
5'
5'3'
5'
5'
1. Polymerization
probe
Q3'
3'
5'
5'3'
5'
5'
2. Strand displacement
Q3'
3'
5'
5'3'
5'
5'
3. Cleavage
R
3'
5'
5'3'
5'
5'
4. Polymerization completed
QR
R = Reporter (FAM, VIC, etc.)
Q = Quencher (NFQ/MGB, etc.)
3'
TaqMan® Assay: Fluorogenic 5‘-nuclease Assay
10
• Minor Groove Binder (MGB)
• Small molecule that fits snugly into minor groove of duplex DNA
• Stabilizes probe annealing
• Non-fluorescent Quencher (NFQ)
• “Dark” quencher acts as energy transfer acceptor that doesn’t emit a detectable
fluorescent signal
• Shorter probe length (13-25-mers)
TaqMan® Probe: TaqMan® MGB/NFQ Probes
AGGCCTTGAGAGATAT
GCTACACAGTCCGGAACTCTCTATAGCATCACAC
R
||||||||||||||||
Q
AGGCCTTGAGAGATAT
RQ
(non-fluorescent quencher) NFQ
Q
MGB(minor groove binder)
11
• A ‘minor groove’-binding molecule specific to the minor groove of double-
stranded DNA
• Fluoresces at an increased intensity when bound
Real-time PCR Chemistries: SYBR® Green I Dye
Major
Groove
Minor
Groove
12
SYBR® Green I Dye: Melting Curve Analysis
Polymerization
CompletePolymerization
PCR
Denaturation
melt
Normalized SYBR ® Green Signal Negative First Derivative
13
single target amplification
Temperature
Derivative R
eport
er
(-R
n)
• Use NTC to check whether non-specific product is primer dimer
• If the non-specific product is primer-dimer:• Optimize primer concentration
• Re-design primer pair
Temperature
Non-target
Temperature
Non-target
(NTC: No Template Control)
SYBR® Green I Dye: Melting Curve Analysis
14
Real-time PCR Chemistries
TaqMan® Assay SYBR® Green I Dye
Specificity More specific Less specific
Probe hybridization
Sensitivity Very high Very high
Flexibility Multiplex PCR No probe required
SNP detection Screening tool
+/- application
Optimization Ready to use 20x primer/probe
mix - no need to optimize
Need to optimize PCR program
Gold standard for MAQC Need to check primer-dimer info
PCR efficiency 100±10% Need to check PCR efficiency
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Reverse Transcription and Real-time PCR Reaction
16
One-step vs Two-step Workflows
• One-step Technology
• RT and PCR are performed in
single buffer system
• √ One tube, one step
• √ Reduce chance of cross-
contamination
• √ Easy for high throughput workflow
• √ Cost effective when few
targets/sample analyzed
• √ Uses gene-specific primers
• X cDNA cannot be stored
• Two-step Technology
• RT and PCR are performed
in two separate reactions
• √ Cost advantaged when
interrogating multiple targets
• √ cDNA can be stored and
used for further experiments
• √ Best choice if RNA is
limiting
• X Multiple steps, longer time
to result
17
One-step Workflow: Real-time PCR Reactions
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• TaqMan® Fast Virus 1-Step Master Mix (PN 4444434)
• 4X master mix to amplify both RNA and DNA
• Formulated to handle common RT-PCR inhibitors found in blood, stool, and
other difficult samples
• Up to triplex (ROX as passive reference)
• TaqPath™ 1-Step Multiplex Master Mix (PN A28526)
• 4X master mix to amplify both RNA and DNA
• Tolerant to common RT-PCR inhibitors
• Manufactured in an ISO 13485 certified facility
• Up to quadruplex (Mustang Purple as passive reference): Only in QS5
One-step Workflow: Master Mixes
19
Two-step Workflow: Real-time PCR Reactions
Reverse Transcription : High Capacity RNA-to-cDNA Kit
TaqMan Chemistry SYBR Chemistry
2x TaqMan Master Mix 1x 10μl
20x Probe/primer Assay Mix 1x 1μl
Water NA
cDNA 1-100 ng 5-10μl
20μl
2x Power SYBR Master Mix 1x 10μl
F Primer optimized NA
R Primer optimized NA
Water NA
cDNA 1-100 ng 5-10μl
20μl
Real-time PCR:
PCR condition:
50℃, 2min
95 ℃, 10 min
95 ℃, 15 sec 40 cycles
60 ℃, 1min
Standard mode
2X RT Buffer 10μl
20X RT Enzyme Mix 1μl
Sample (up to 2μg) Up to 9μl
Nuclease-Free water To 20μl
SYBR Green:
- Check Primer Concentration
- Add Melt Curve Program
PCR condition:
95 ℃, 20 sec
95 ℃, 1 sec 40 cycles
60 ℃, 20 sec
Fast mode
20
Two-step Workflow: Master Mixes
Standard Mode
• TaqMan® Chemistry
• TaqMan® Universal Master Mix II
(PN 4440038)
• TaqMan® Gene Expression
Master Mix (PN 4369016)
• SYBR® Green Chemistry
• Power SYBR® Green PCR Master
Mix (PN 4367659)
Fast Mode
• TaqMan® Chemistry
• TaqMan® Fast Universal Master
Mix (PN 4366072)
• TaqMan® Fast Advanced Master
Mix (PN 4444557)
• SYBR® Green Chemistry
• Fast SYBR® Green Master Mix
(PN 4385612)
• PowerUp™ SYBR® Green Master
Mix (PN A25742)
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Gene expression assay: Quantification Method
22
• Absolute Quantitation vs. Relative Quantitation
Real-time PCR Quantitation Methods
23
Log copy number
CT
valu
es
CTs
0 1 2 3 4 5 6 7
CT is directly proportional to log of
amount of input template
➢主要應用於病毒量及病原菌偵測➢To determine the actual number of copies
of a target nucleic acid within a sample with
statistical confidence.
絕對定量 (Absolute Quantitation)
24
• To determine fold differences of a target nucleic acid in a
starting material with statistical confidence.
• ΔΔCt analysis (most common)
• Relative standard curve
• Need endogenous gene normalizes the amount of sample
added
• e.g. GAPDH, β-actin, etc
• Need at least 1 reference sample
• e.g. untreated, WT, time=0, etc.
• Check primer PCR efficiency if using SYBR Green Dye
相對定量 (Relative Quantitation)
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• 2μg RNA in 20μl RT
• Gene name: C-Myc and GAPDH
• cDNA 4-fold serial dilution: 10μl cDNA + 30μl H2O (25ng/μl)• 1. 25ng/μl
• 2. 6.25 ng/μl
• 3. 1.56 ng/μl
• 4. 0.39ng/μl
• 5. NTC (duplicate for each sample)
•每個濃度點各做二重複
• Prepare a Premix for each gene
• Aliquot 15μl of Premix to each well
• Add 5μl of RT product to the well
• Real-time PCR reaction
相對定量 (Relative Quantitation): PCR Efficiency Validation
30μl H2O 30μl H2O 30μl H2O 30μl H2O
25ng/μl 6.25 ng/μl 1.56 ng/μl 0.39 ng/μl
10μl 10μl 10μl
26
90 ≦ Eff% ≦ 110 → ∆∆ Ct
Eff% < 90 → Relative standard curve
相對定量 (Relative Quantitation): PCR Efficiency Validation
27
相對定量 (Relative Quantitation):Comparative Ct Method (ΔΔCt)
Comparison of the c-myc expression level
in T=0, T=12, T=24, T=48 time course study
Reverse Transcription: Ex. 5 ug RNA/ 50 uL =100 ng/uL
Spectrophotometer measure RNA quantity
Real Time PCR
Unknown samples( 50 ng): T=0, T=12, T=24, T=48
Reference Sample
t=0 t=12 t=24 t=48time
total RNA
cDNA
total RNA
cDNA
total RNA
cDNA
total RNA
cDNA
C-myc GAPDH C-myc GAPDH C-myc GAPDH C-myc GAPDH
Ct=30.5 Ct=23.6 Ct=27 Ct=22.6
28
相對定量 (Relative Quantitation):Comparative Ct Method (ΔΔCt)
step 2: Normalization to reference sample
Ct sample – Ct reference sample = Ct
step 1: Normalization to endogenous control
Sample: Ct c-Myc – Ct GAPDH = Ct sample
Reference: Ct c-Myc – Ct GAPDH = Ct reference sample
step 3: use the formula
2-Ct
A reference sample is a sample to which unknown samples
are compared (e.g. untreated sample or control).
29
相對定量 (Relative Quantitation):Comparative Ct Method (ΔΔCt)
30
Data Analysis- 4 check points
1. 完整的擴增曲線
2. 每個target gene的Threshold
設置在各自正確的位置
3. Melt curve plot (for SYBR Green only)
4. 技術性重複(Technical Replicate)
31 The world leader in serving science
Application
32
Real-time PCR Applications
定性研究
•基因型研究
-- SNP與疾病關聯性
•基因體拷貝數變異(Copy Number Variants)
• Somatic Mutation檢測
基因定量
•病毒定量: HBV, HCV,HPV…
•疾病相關基因之定量
• miRNA基因調控研究
• siRNA knock down validation
•檢測基因轉殖食品(GMO)
33
• Single Nucleotide Polymorphisms (SNPs) are single base changes in
genomic DNA.
• SNPs may cause disease, alter metabolisms or act as markers.
• SNPs in diploid loci commonly have 3 genotypes.
SNP Genotyping
A
A
A
G
G
G
Homozygous Heterozygous Homozygous
34
TaqMan® SNP Genotyping Assay Overview
35
Allelic Discrimination (SNP) Data
G
A
Homozygous GG
Heterozygous GAHomozygous AA
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Assay Design
37
Applied Biosystems提供Primers/Probe設計的全方位解決方案
Custom TaqMan Assays Service
• Custom TaqMan Assays
▪ All-in One tube TaqMan-based Assay
• Primer Express Software
▪ 上機條件皆相同~不用再花時間測試primer溫度了
7• Pre-Designed TaqMan® Assay (ready-to-use)
▪ TaqMan Gene Expression Assays
• > 1,800,000 assays
• 提供所有相關生物資訊 (23 species)
▪ TaqMan microRNA and primary microRNA
Assays
▪ TaqMan SNP Genotyping Assays
▪ TaqMan Copy Number Assays
▪ TaqMan Mutation Detection Assays
38
Finding the Right Assay for Your Research
• Search for the assay you need quickly and easily
http://www.thermofisher.com/tw/en/home/life-science/pcr/real-time-pcr/real-
time-pcr-assays.html
• Powerful search engine
• Streamlined search interface
• Flexibility to search by gene name, gene alias or assay ID
39
Finding the Right Assay for Your Research
40
TaqMan Array Plates
Ready-to use plates with preloaded
TaqMan Assays!
41
TaqMan Array Plates_Powerful data analysis
Powerful data analysis!!
42
Primer Design: Bioinformatic Evaluation
•Bioinformatics
• BLAST tool: Sequence specificity
(https://blast.ncbi.nlm.nih.gov/Blast.cgi)
• SNPmasker: N masked SNPs (http://bioinfo.ebc.ee/snpmasker/)
• RepeatMasker: N masked repeat sequence
(http://www.repeatmasker.org)
•Software
• Primer Express® Software
43
44
Sequence
2. Find Primer/Probe
1. Add DNA file or Copy & Paste
45
清楚明確的 TaqMan Probe and Primer 設計規範
200 bp amplicon 500 bp amplicon
46
Design Parameter
47
Results
48
Check Tm of Primers
49
SYBR Experiment Note
•PCR Efficiency Validation
• Serially-diluted sample to generate standard curve for each primer set
• At least 5 dilution point in triplicate
• Check the R square value and PCR efficiency (90% ~ 110%)
•Primer Optimization
• Use NTC to check whether non-specific product is primer dimer (NTC is
important!)
• If the non-specific product is primer-dimer:
• Optimize primer concentration
• Re-design primer pair
•Test with samples that are comparable to real experiment for each
gene
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Applied Biosystems QuantStudio™ 7 Real-Time PCR System
51
• Responsive touch screen interface
• Start a run from touch screen (stand-
alone) or attached PC (tower/laptop)
• Onboard memory stores >100 runs
• 4 Interchangeable Blocks
− (384-well, TaqMan Array Cards, 96-
well and Fast 96-well)
• OptiFlex® System
• Intuitive software with ReadiApp®
Application and QuickStart functions
QuantStudio™ 7 Flex Real-Time PCR System
52
QuantStudio™ 7: OptiFlex™ System
• Decoupled excitation and emission filters
• Select up to 21 color combination
Channel Dye Examples Excitation Filter Emission Filter
1
FAM™, SYBR®, SYTO®9 (MeltDoctor™), Fluorescein,
SYPRO® Orange 470 ± 15nm 520 ± 15nm
2 VIC®, JOE™, TET™, HEX™ 520 ± 10nm 558 ± 12nm
3 TAMRA™, NED™, BODIPY® TMR-X 550 ± 10nm 586 ± 10nm
4 ROX™, Texas Red® 580 ± 10nm 623 ± 14nm
5 LIZ® 640 ± 10nm 682 ± 14nm
6 Alexa Fluor®, Joda-4 662 ± 10nm 711 ± 12nm
Emission
Excitation
53
Block Change
• Block change typically in less than 1 min
• Heated Cover (96 vs. 384)
• Plate Adaptor (96 vs. 384 vs. TAC)
• 384-well and TAC Blocks
• 96-well and Fast 96-well
Validated Programmable
96 10-100uL 1-200uL
Fast 96 5-30uL 1-100uL
Validated Programmable
384 5-20uL 1-30uL
TAC 1uL 1uL
54
QuantStudio™ 7: Adaptor Change
The door opens, and plate holder swings out. Pull out the knob on the plate holder, shown here, you can take out plate adaptor.
55
• For 96-Well (0.2mL) sample block
-- MicroAmp® Optical 96-Well Reaction Plate (0.2mL)
(P/N N8010560)
-- MicroAmp® Optical Adhesive Film
(P/N 4360954)
-- MicroAmp® Optical 8-Tube Strip(0.2mL)
(P/N 4316567)
-- MicroAmp® Optical 8-Cap Strip
(P/N 4323032)
QuantStudio™ 7 Flex Supported Consumables
* Need to use MicroAmp®
96-Well Tray/Retainer Set
(for 0.2mL)
★ Load at least 16 tubes
56
• For 96-Well (0.1mL) sample block
-- MicroAmp® Fast Optical 96-Well Reaction Plate(0.1mL)
(P/N 4346907)
-- MicroAmp® Optical Adhesive Film
(P/N 4360954)
-- MicroAmp® Fast 8-Tube Strip(0.1mL)
(P/N 4358293)
-- MicroAmp® Optical 8-Cap Strip
(P/N 4323032)
QuantStudio™ 7 Flex Supported Consumables
* Need to use MicroAmp®
96-Well Tray ( for 0.1mL)
★ Load at least 16 tubes
57
Sealing the Plate
Note: Pressure is required to activate
the adhesive on the optical cover
The flat edge of an applicator is rubbed back-and-forth along the length of the plate
with a significant downward pressure to form a complete seal on top the wells
Downward pressure applied
in back-and-forth motions
across the top of the plate
Downward pressure applied
in back-and-forth motions
across the top of the plate
58
Sealing the Plate
The end of an applicator is rubbed around all the outside edges of the plate with a
significant downward pressure to form a complete seal around the outside wells
Downward pressure
applied in small
back - and - forth motions
along all the edges
Downward pressure
applied in small
back - and - forth motions
along all the edges
Note: Pressure is required to activate the adhesive on the optical cover
59
Operation Notes
• Make sure the correct heat cover, block and adaptor are installed
• Use a tray with the 96-well adaptor for 8-tube strips
• Do not mark on the consumables
• This may increase the background signal
• Avoid bubbles when pipetting into each well
• Centrifuge samples
60
TaqMan array card
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TaqMan Array Card diagram
Reaction volume: 1 ul/ well
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TaqMan® Array Card Parts
Micro Fluidic Card with up to 384 TaqMan® Assays and 8-Ports
080
180
PortsTaqMan®
Assay
63
1. Load
2. 2. Spin
1200 rpm, 2x 1min
3. Seal
4. Run
TaqMan Array Card workflow
64
QuantStudio™ 7: Sealing TaqMan® Array Card
65
QuantStudio™ 7: 單機操作
• Start the run from touchscreen
• After the run, transfer data file (.eds) to an USB
• Analyze data on your own computer
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• 一套軟體可以符合全方位的應用 (1280x1024 pixel resolution )
• 絕對定量 Quantification - Standard Curve
• 相對定量 Quantification – Comparative Ct (△△Ct)
• 相對定量 Quantification - Relative Standard Curve
• Melting Curve Analysis
• Genotyping
• Presence/Absence
• QuantStudio™ Software V1.3
• Data analysis for QuantStudio™ 6 and 7 Flex and ViiA™ 7 Real-Time PCR
Systems
• http://www.thermofisher.com/tw/en/home/technical-resources/software-
downloads/quantstudio-flex-real-time-pcr-system.html
Quantstudio™ Software
67
1. Experiment Setup
68
2. Setup: Experiment Properties
69
3. Setup: Run Method
70
4. Run: Start Run
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5-1. Setup: Assign 決定基因和樣品位置 (Standard Curve)
圈選樣品擺放位置, 再從左邊勾選樣品名稱與偵測的基因
72
Standard Curve Setup
73
5-2. Setup: Define 定義基因和樣品名稱 (△△Ct)
輸入偵測的基因及使用的螢光
輸入樣品名稱
74
5. Setup: Assign 決定基因和樣品位置
圈選樣品擺放位置, 再從左邊勾選樣品名稱與偵測的基因
75
6. Analysis : Amplification Plot 3. Analyze or Re-analyze
2. Auto or Manual
4. Check Threshold
1.
76
Analysis: View Well Table
77
Analysis: Gene Expression
78
Analysis: Standard Curve
79
Analysis: Derivative Melt Curve (SYBR Green)
80
Analysis: QC Summary Helps with Troubleshooting
81
數據和圖形簡易輸出! 超easy~
Export to Excel, PowerPoint, PDF or save as JPEG / Copy
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Real-time PCR 中文線上講座
http://www.thermofisher.com/tw/en/home/taiwan/real-time-pcr-webinars/real-time-pcr-
experimental-configuration.html
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Create your template or analyze your data on ThermoFisher Cloud
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Analysis Modules - Compatible Instrument Systems
Real-Time PCR SystemSupported software
version(s)
File
extension
Applied Biosystems™ 7900HT Fast Real-Time PCR System v2.4 or later *.sds
Applied Biosystems™ 7500 Real-Time PCR System
Applied Biosystems™ 7500 Fast Real-Time PCR System
v2.0.5 or later *.eds
Applied Biosystems™ StepOne™ and StepOnePlus™
Real-Time PCR Systems
v2.0.1, v2.1, or
later
Applied Biosystems™ ViiA™ 7 Real-Time PCR System v1.1 or later
Applied Biosystems™ QuantStudio™ 12K Flex Real-Time
PCR System
v1.1.1 or later
Applied Biosystems™ QuantStudio™ 6 Flex Real-Time
PCR System
v1.0 or later
Applied Biosystems™ QuantStudio™ 7 Flex Real-Time
PCR System
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ThermoFisher Cloud Account Registration
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In the Cloud - Home
Home
Quick access to
Files, Applications,
and Instruments
Manage Profile
Switch to Region
Get Notifications
Monitor your
favorite
instruments
Filter your
applications
by type
File manager
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In the Cloud - Home
Home
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In the Cloud - Apps
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Analyze Data
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Real-time PCR 中文線上講座
http://www.thermofisher.com/tw/en/home/taiwan/real-time-pcr-webinars/real-time-pcr-
experimental-configuration.html
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