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1The world leader in serving science
韓世芸
Senior Field Application Scientist
Introduction to Real-Time PCR & StepOne PlusTM System Operation
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Real Time PCR Amplification plot features
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0 5 10 15 20 25 30 35 40
PCR cycle number
Baseline region
Threshold
CT
No Template control
Sample
Norm
alise
d re
porte
r flu
ores
cenc
e (R n
)
Geometric Phase:(Exponential Phase)Y= N0 2n
Lower expression level
ΔRn
capPCR vessel
thermal block
sample
lens
CT = threshold cycle: the calculated fractional cycle number at whichthe PCR product crosses a threshold of detection
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Real-Time PCR detection takes place in the “exponential phase”, while traditional detection takes place at the “plateau” of the reaction.
Exponential
Linear
PlateauGel
Low Ct (high copy #)
High Ct (low copy #)
PCR cycles→fluo
resc
ent s
igna
l →101
100
10-1
Threshold of detection
Y= N0 2n , CT 與起始濃度之對數值成反比
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Generate Real-Time Signal Using Fluorescent
1. TaqMan® probe or TaqMan® MGB probe chemistry: 5′ Nuclease assay
2. SYBR® Green I dye chemistry
FQ
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SYBR® Green I Dye
• A ‘minor groove’-binding molecule specific to the minor groove of double-stranded DNA
• It fluoresces at an increased intensity when bound
Major Groove
Minor Groove
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Polymerization Complete
Polymerization
SYBR Green 1®
- dsDNA minor-groove binding dye
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Check specificity of reactions using a dissociation curve (a.k.a. melt curve)
Fluorescence
Temperature60º 95º
One, clean peak = no apparentextraneous products
Temperature
Fluorescence
Derivative view
Multiple peak1. Primer conc. Optimization2. Re-design primer
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TaqMan Chemistry- FRET Principal
energy transfer
hv
Excitation
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Fluorogenic 5' nuclease assay(TaqMan® chemistry)
R Qforward primer
reverse primer
3'
3'5'
5'3'
5'
5'
1. Polymerisation
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. Polymerisation completed
QR
R = ReporterQ = Quencher
3'
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Minor Groove Binder (MGB)Small molecule that fits snugly into the minor groove of duplex DNAStabilizes probe annealing
Non Fluorescent Quencher (NFQ)“Dark” quencherActs as energy transfer acceptor that does not emit a detectable fluorescent signal
MGB probe design uses a special algorithm in Primer Express® Software.All probes will be short (13-25-mers)
TaqMan® MGB/NFQ Probes
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定性研究
• 基因型研究
-- SNP與疾病關聯性
• 病原菌偵測
• 基因體拷貝數變異
(Copy Number Variants)
• High Resolution Melt
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)
• Somatic Mutation檢測
同步定量PCR之應用
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Real Time PCR Applications
Absolute Quantitation vs. Relative Quantitation
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Absolute Quantitation-- measure viral copy number in samples
To determine the actual number of copies of a target nucleic acid within a sample with statistical confidence.
• needs a standard whose concentration is known absolutely
• identical amount of sample must be assayed for all unknowns
• unnecessary for most studies
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Log copy number
CT
valu
esCTs
0 1 2 3 4 5 6 7
CT is directly proportional to log of amount of input template
Absolute Quantitation
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Relative Quantitation
Relative— “n-fold” difference relative to the calibrator (no units)
-- 1. △△Ct analysis (**UB 2)-- 2. relative standard curve
♦ Standard: any stock RNA or DNA containing the appropriate target♦ Endogenous control normalized RNA input measurement and RT-efficiency difference♦(ex. 18S rRNA, GAPDH, HPRT, β-actin……..)
♦ The most powerful and widely used methodEx. Time Course (Treatment)
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Comparative Ct Method
step 2: Normalization to calibrator sample
ΔCt Sample – ΔCt Calibrator = ΔΔCt
step 1: Normalization to endogenous control
Ct Target gene – Ct Endogenous control = ΔCt
step 3: use the formula
2-ΔΔCtA calibrator sample is a sample to which unknown samples are compared (ex. untreated sample or control).
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Comparative Ct MethodComparison 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 PCRUnknown samples( 50 ng): T=0, T=12, T=24, T=48
Calibrator
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
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c-Myc GAPDH ΔCt ΔΔCt 2-ΔΔCt
T=0 (calibrator) 25 10 15 0 1.0T=12hr 24 10 14 -1 2.0T=24hr 23 11 12 -3 8.0T=48hr 28 10 18 3 0.1
ΔΔCt Calculations (Comparative Ct)
t = 0t = 12 ht = 24 ht = 48 h
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Rel
ativ
e Q
uant
ityof
Exp
ress
ion
2.0
8.0
1.0 0.1
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隨即得到基因相對定量趨勢!!不需再花時間自行運算結果!
結果則可在`Analysis’: Gene Expression呈現基因表現趨勢
只要在`Setup’ 設定好
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Relative Quantitation Software
快速運算△△ Ct =△ Ct (Unknown) -△ Ct (Calibrator), 2 -△△Ct
自動畫出相對定量比較圖, 完全不須經 Excel運算
Integrated Software for data from multi-plate
• run to run variation ↓
• 簡化分析流程, 快速分析大量樣品
StepOne Plus
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如何製備Real Time PCR的反應
TaqMan Chemistry SYBR Chemistry
2x TaqMan Master Mix 1x 10 ul(2x TaqMan Fast Master Mix)20x Probe/primer Assay Mix 1 ulWater NAcDNA 10-100 ng 5-10 ul
20 ul
2x Power SYBR Master Mix 1x 10 ul(2x Fast SYBR Master Mix)F Primer optimized NAR Primer optimized NAWater NAcDNA 10-100 ng 5-10 ul
20 ul
Real Time PCR:
PCR condition: 50℃, 2min95 ℃, 10 min95 ℃, 15 sec 40 cycles60 ℃, 1min
Standard modePCR condition: 95 ℃, 20 sec95 ℃, 1 sec 40 cycles60 ℃, 20 sec
Fast mode*Probe/ Primer Self-design:
Primer final conc. 300 nMProbe final conc. 200 nM
Reverse Transcription : High Capacity RNA-to-cDNA Kit2x RT Buffer 10 uL
20x RT Enzyme mix 1 uLRNA (up to 2ug) (up to) 9 uL
Total 20 uL
SYBR Green : + Melt curve Program
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Applied Biosystems Primers/Probe design total solution
• Option 1: Pre-Designed TaqMan® Assay (ready-to-use)• > 1.3 million TaqMan Gene Expression assays (for 23 species)• > 4.5 million TaqMan SNP assays• > 1.6 million TaqMan CNV assays for human• > 12,000 TaqMan microRNA assays (miRBase release 20, 206 listed species)• > 778 TaqMan Mutation Detection assays in 46 cancer genes• TaqMan Non-coding RNA assays, TaqMan pri-miRNA assays, TaqMan DME
assays ………
• Option 2: Custom Assay-- 代客設計 for all TaqMan assays• All-in One tube TaqMan-based Assay (20X mixture)
• Option 3: Primer Express® software• Software for designing real-time assays• 統一條件設計, 不同基因可同時上機, 不需測試PCR 條件
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How to search AB TaqMan assay on line?
http://www.lifetechnologies.com/tw/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays.html
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TaqMan® Gene Expression Array Cards and Plates
http://www.lifetechnologies.com/tw/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/taqman-gene-expression/real-time-
pcr-taqman-arrays.html
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3.定量PCR Primers/ Probe設計軟體
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清楚明確的 TaqMan Probe & Primer 設計規範
200 bp amplicon 500 bp amplicon
3. 定量PCR Primers/ Probe設計軟體
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Primer Express 3.0 Operation
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2. Find Primer/Probe
1. Add DNA file or Copy & Paste
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Design Parameter
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Result
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SYBR Green experiment procedure
1. Primer conc. Optimization• Primer Final conc. 100-300 nM• No primer dimer or
non-specific product involved
2. PCR Primer Efficiency Validation• Sample serial dilution to run standard curve for target gene and
endogenous control gene
3. Real sample run for each gene
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ΔΔCt Validation
Log of Input
Ct v
alue
GAPDH
C-Myc
∆ Ct
Y=0.049X+3.0179slope + 0.1ΔCt allowed
∆ Ct
Log of Input RNA
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簡易三步驟!!
The StepOnePlus™ Real-Time PCR System
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StepOnePlus™ Real-Time PCR System: The Basics
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● 96-Well Block- One block, 2 speeds–Fast cycling: 40 cycles in under 40 minutes–Standard cycling: 40 cycles in under 2 hours–10-30µl reaction volume
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StepOnePlus™ Real-Time PCR System: The Basics
• 4-color instrument• FAM™/SYBR® Green dyes• VIC®/JOE™ dyes• NED™/TAMRA™ dye• ROX™ dye
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Standalone (PC-Free)
只需輕按觸控螢幕 不需電腦連線也能上機!!
1. Start the run from the touchscreen2. After run, download the file (.eds)
to your PC
3. Analyze your data
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Colocated
Direct control of instrument via computer
1. Setup: You can design the experiment on the PC connected to the StepOne System
2. Run: Start the run from the PC connected to the StepOneSystem and Real-Time data collection
3. Analyze the results on the PC connected to the StepOneSystem
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StepOne只能暫存一個檔案
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● 樣品量多時– P/N 4346907
MicroAmp® Fast 96-Well Reaction Plate (0.1 mL) -10 plates– P/N 4360954
MicroAmp® Optical Adhesive Film - 25 films
StepOnePlus™ Compatible Consumables
★ Place the tray containing the tube, Load at least 16 tube
● 樣品量少時– P/N 4358293
MicroAmp® Fast 8-Tube Strip (0.1 mL) - 125 strips– P/N 4323032
MicroAmp® Optical 8-Cap Strip - 300 strips
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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
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• Directly load fast optical 96-well plate into the instrument
If using fast 8-tube stripes, load the tubes with fast 96-well tray
• Do not mark any labels on the consumables
This may increase the background signal
• Avoid bubbles when pipetting into each well
Centrifuge samples
Operation Notes
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StepOne Software v2.1 Operation
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Quick Start 先上機 !
1. Run: QuickStart
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2. Setup: Experiment Properties
a. Experiment Name 及檔案儲存位置
b.選擇 Experiment Type
c.選擇使用螢光系統
d.選擇Ramp Speed
e.選擇實驗樣品種類
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3. Setup: Run Method4.
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5. Setup: Plate Setup 定義基因和樣品名稱
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6-1. Setup: Plate Setup 決定基因和樣品位置 (for ddCt)
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6-2. Setup: Plate Setup 決定基因和樣品位置 (for Standard curve)
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Automatic Standard Curve Setup
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7. Analyze
3. Analyze or Re-analyze
1.
2. Auto or Manual
4. Check Threshold
Analysis : Amplification Plot
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How to Set the Threshold?Auto or Manual Threshold?
Manual threshold could be used to set fixed threshold when doing run to run comparison
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Analysis Report
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Analysis : Gene Expression
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Analysis : Standard Curve
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Analysis : Multicomponent Plot
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Analysis : Raw Data Plot
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QC Summary Help Your Troubleshooting
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Analysis : Melt Curve (SYBR Green)
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Analysis : Allelic Discrimination
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Data export
Export to Excel, PowerPoint or save as jpeg
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StepOnePlus™ Real-Time PCR System: The Basics
● Veriflex™ Block-One block, Six Zones-The same “Better than gradient” feature from Veriti™ 96-well Thermal Cycler
*Image from Veriti Thermal Cycler
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Red linesto delineate zones
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Setup: Run Method
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Useful information--中文線上課程
http://www.lifetechnologies.com/tw/zt/home/taiwan/real-time-pcr-webinars/real-time-pcr-experimental-configuration.html
http://www.lifetechnologies.com/tw/zt/home/taiwan/real-time-pcr-webinars/real-time-pcr-experimental-configuration.htmlhttp://www.lifetechnologies.com/tw/zt/home/taiwan/real-time-pcr-webinars/real-time-pcr-experimental-configuration.html
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Useful information
http://www.lifetechnologies.com/tw/zt/home.html
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On-Line Support Center
https://www.lifetechnologies.com/tw/en/home/technical-resources/technical-reference-library/real-time-digital-PCR-
instruments-support-center.html
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68The world leader in serving science
www.lifetechnologies.com.tw產品應用支援服務專線: 0800-251-326 #3E-mail: [email protected]
Thanks for your attendance!
Questions?
Introduction to Real-Time PCR & StepOne PlusTM System Operation投影片編號 2投影片編號 3Generate Real-Time Signal Using FluorescentSYBR® Green I Dye投影片編號 6Check specificity of reactions using a dissociation curve (a.k.a. melt curve)TaqMan Chemistry- FRET PrincipalFluorogenic 5' nuclease assay�(TaqMan chemistry)TaqMan® MGB/NFQ Probes投影片編號 11Real Time PCR Applications��Absolute Quantitation vs. Relative QuantitationAbsolute Quantitation�-- measure viral copy number in samples投影片編號 14投影片編號 15Comparative Ct Method投影片編號 17ΔΔCt Calculations (Comparative Ct)隨即得到基因相對定量趨勢!!�不需再花時間自行運算結果!Relative Quantitation Software 如何製備Real Time PCR的反應Applied Biosystems Primers/Probe design total solutionHow to search AB TaqMan assay on line?TaqMan® Gene Expression Array Cards and Plates投影片編號 25清楚明確的 TaqMan Probe & Primer 設計規範Primer Express 3.0 Operation投影片編號 28投影片編號 29Design ParameterResultSYBR Green experiment procedureDDCt Validation投影片編號 34StepOnePlus™ Real-Time PCR System: The BasicsStepOnePlus™ Real-Time PCR System: The BasicsStandalone (PC-Free)� �只需輕按觸控螢幕 不需電腦連線也能上機!! Colocated投影片編號 39StepOnePlus™ Compatible Consumables投影片編號 41Operation NotesStepOne Software v2.1 Operation1. Run: QuickStart 2. Setup: Experiment Properties3. Setup: Run Method5. Setup: Plate Setup 定義基因和樣品名稱投影片編號 48投影片編號 49 Automatic Standard Curve Setup7. Analyze投影片編號 52Analysis ReportAnalysis : Gene ExpressionAnalysis : Standard Curve投影片編號 56投影片編號 57投影片編號 58投影片編號 59投影片編號 60Data exportStepOnePlus™ Real-Time PCR System: The Basics投影片編號 63Setup: Run MethodUseful information--中文線上課程Useful informationOn-Line Support CenterThanks for your attendance!�Questions?What is HRM?High Resolution Melt Analysis (HRM)Reading a High Resolution Melt CurveReading a High Resolution Melt CurveApplicationsMutation ScanningSNP Genotyping: Class 1 SNP data WorkflowHRM reaction preparationContinuous Melt CurveHRM Software v3.0: Easy NavigationHRM Software v3.0: Plate Layout ViewHRM Software v3.0: Advanced Assay Settings Library & Expected # of ClustersHRM Software v3.0: Separately analyze multiple assays on a single plateHRM Data數據和圖形簡易輸出! 超easy~�Export to Excel, PowerPoint or save as jpegUser Guide and Quick SheetHRM Handbook is Now Available!投影片編號 87How to search AB TaqMan assay on line?miRNA Quantitation by Real-Time PCR� Individual TaqMan MicroRNA AssaysIndividual TaqMan MicroRNA Assays WorkflowWhat is Sickle-cell Anemia?What are SNPs?Single Nucleotide PolymorphismTaqMan® SNP Genotyping Assay OverviewAfter each cycle . . .Genotyping Assay 如何製備SNP Genotyping 的反應投影片編號 99TaqMan® Copy Number Assays workflow How to determine DNA copy number (an example)CopyCaller® Software for Data AnalysisBrowse/New Experiments: NewSelect Experiment : Save投影片編號 105投影片編號 106Basic Operation試劑多樣性 符合你所有需求!試劑多樣性 符合你所有需求!