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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
2
Real Time PCR Amplification plot features
0.00
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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).
17
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
18
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
20
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
21
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
22
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
23
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
24
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
27
Primer Express 3.0 Operation
28
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2. Find Primer/Probe
1. Add DNA file or Copy & Paste
30
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
33
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
34
簡易三步驟!!
The StepOnePlus™ Real-Time PCR System
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StepOnePlus™ Real-Time PCR System: The Basics
4
● 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
43The world leader in serving science
StepOne Software v2.1 Operation
44
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
52
How to Set the Threshold?Auto or Manual Threshold?
Manual threshold could be used to set fixed threshold when doing run to run comparison
53
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
58
QC Summary Help Your Troubleshooting
59
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
62
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
63
Red linesto delineate zones
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Setup: Run Method
65
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
67
On-Line Support Center
https://www.lifetechnologies.com/tw/en/home/technical-resources/technical-reference-library/real-time-digital-PCR-
instruments-support-center.html
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!