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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

0

2

4

6

8

10

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

66

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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www.lifetechnologies.com.tw產品應用支援服務專線: 0800-251-326 #3E-mail: Support.TW@lifetech.com

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!