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Introduction to Real-Time PCR &StepOne PlusTM System Operation韓世芸Senior Field Application ScientistThe world leader in serving science1
Normalised reporter fluorescence (Rn)Real Time PCR Amplification plot featureslens0.900.800.70SampleLower expression level0.600.500.40ΔRnThreshold0.300.20No Template controlBaseline region0.10capPCR vesselthermal block0.0005101520CT25PCR cycle numbersample303540Geometric Phase:(Exponential Phase)Y N0 2nCT threshold cycle: the calculated fractional cycle number at whichthe PCR product crosses a threshold of detection2
Real-Time PCR detection takes place in the “exponential phase”, whiletraditional detection takes place at the “plateau” of the reaction.101GelPlateau100LinearThresholdof detectionExponentialfluorescent signal 10-1PCR cycles Low Ct(high copy #)High Ct(low copy #)Y N0 2n , CT 與起始濃度之對數值成反比3
Generate Real-Time Signal Using Fluorescent1. TaqMan probe or TaqMan MGB probechemistry: 5′ Nuclease assayFQ2. SYBR Green I dye chemistry4
SYBR Green I Dye A ‘minor groove’-binding molecule specific to the minorgroove of double-stranded DNA It fluoresces at an increased intensity when boundMajorGrooveMinor Groove5
SYBR Green 1 - dsDNA minor-groove binding dyePolymerizationPolymerization Complete6
Check specificity of reactions using a dissociationcurve (a.k.a. melt curve)Derivative viewFluorescenceFluorescence60ºTemperatureOne, clean peak no apparentextraneousproducts95ºMultiple peak1. Primer conc. OptimizationTemperature2. Re-design primer7
TaqMan Chemistry- FRET PrincipalhvExcitationenergy transfer8
Fluorogenic 5' nuclease assay(TaqMan chemistry) Rforward primerprobeQ3'5'3'5'3'5'5'Qreverseprimer1. Polymerisation3'5'3'5'3'5'5'2. Strand displacementRQ5'3'3'5'5'R3'5'3. CleavageQ5'3'5'5'3'3'5'4. Polymerisation completedR ReporterQ Quencher9
TaqMan MGB/NFQ ProbesMinor Groove Binder (MGB)Small molecule that fits snugly into the minor groove of duplex DNAStabilizes probe annealingNon Fluorescent Quencher (NFQ)“Dark” quencherActs as energy transfer acceptor that does not emit a detectable fluorescent signalMGB probe design uses a special algorithm inPrimer Express Software.All probes will be short (13-25-mers)10
同步定量PCR之應用基因定量 病毒定量: HBV, HCV,HPV 疾病相關基因之定量定性研究 基因型研究-- SNP與疾病關聯性 miRNA基因調控研究 病原菌偵測 siRNA knock down validation 基因體拷貝數變異 檢測基因轉殖食品(GMO) Somatic Mutation檢測(Copy Number Variants) High Resolution Melt11
Real Time PCR ApplicationsAbsolute Quantitation vs. Relative Quantitation12
Absolute Quantitation-- measure viral copy number in samplesTo determine the actual number of copies of a targetnucleic 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 studies13
CTsCT valuesAbsolute Quantitation0CT is directly proportional to log ofamount of input template1234567Log copy number14
Relative QuantitationRelative— “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 targetEndogenous control normalized RNA input measurement and RT-efficiency difference (ex. 18S rRNA, GAPDH, HPRT, β-actin .) The most powerful and widely used method Ex. Time Course (Treatment)15
Comparative Ct Methodstep 1: Normalization to endogenous controlCt Target gene – Ct Endogenous control ΔCtstep 2: Normalization to calibrator sampleΔCt Sample – ΔCt Calibrator ΔΔCtstep 3: use the formula-ΔΔCt2A calibrator sample is a sample to which unknown samplesare compared (ex. untreated sample or control).16
Comparative Ct MethodComparison of the c-myc expression levelin T 0, T 12, T 24, T 48 time course studyCalibratort 0timet 12t 24t 48total RNAtotal RNAtotal RNAtotal RNAcDNAcDNAcDNAcDNAC-myc GAPDHC-myc GAPDHC-myc GAPDHSpectrophotometer measure RNA quantityReverse Transcription: Ex. 5 ug RNA/ 50 uL 100 ng/uLC-myc GAPDHReal Time PCRUnknown samples( 50 ng): T 0, T 12, T 24, T 48Ct 30.5 Ct 23.6Ct 27 Ct 22.617
ΔΔCt Calculations (Comparative Ct)c-Myc GAPDH ΔCtΔΔCt2-ΔΔCtT 0 (calibrator)T 12hrT 24hr2524231010111514120-1-31.02.08.0T 48hr28101830.1Relative Quantityof Expression108.08t 06t 12 h41.02t 24 h2.00.1t 48 h018
時間自行運算結果!結果則可在 Analysis’: GeneExpression呈現基因表現趨勢只要在 Setup’ 設定好19
Relative Quantitation SoftwareIntegrated Software for data from multi-plate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StepOne Plus run to run variation 簡化分析流程, 快速分析大量樣品20
如何製備Real Time PCR的反應Reverse Transcription : High Capacity RNA-to-cDNA Kit2x RT Buffer20x RT Enzyme mixRNA (up to 2ug)Total10 uL1 uL(up to) 9 uL20 uLReal Time PCR:TaqMan Chemistry2x TaqMan Master Mix1x(2x TaqMan Fast Master Mix)20x Probe/primer Assay MixWatercDNA10-100 ngSYBR Chemistry10 ul1 ulNA5-10 ul20 ulStandard mode*Probe/ Primer Self-design:Primer final conc. 300 nMProbe final conc. 200 nMPCR condition:50 , 2min95 , 10 min95 , 15 sec 40 cycles60 , 1min2x Power SYBR Master Mix1x10 ul(2x Fast SYBR Master Mix)F PrimeroptimizedNAR PrimeroptimizedNAWaterNAcDNA10-100 ng5-10 ul20 ulFast modePCR condition:95 , 20 sec95 , 1 sec 40 cycles60 , 20 secSYBR Green : Melt curve Program21
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 DMEassays 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22
How to search AB TaqMan assay on tml23
TaqMan Gene Expression Array Cards and html24
3.定量PCR Primers/ Probe設計軟體25
3. 定量PCR Primers/ Probe設計軟體清楚明確的 TaqMan Probe & Primer 設計規範200 bp amplicon500 bp amplicon26
Primer Express 3.0 Operation27
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2. Find Primer/Probe1. Add DNA file or Copy & Paste29
Design Parameter30
Result31
SYBR Green experiment procedure1. Primer conc. Optimization Primer Final conc. 100-300 nM No primer dimer ornon-specific product involved2. PCR Primer Efficiency Validation Sample serial dilution to run standard curve for target gene andendogenous control gene3. Real sample run for each gene32
ΔΔCt ValidationCt value Ct CtLog of Input RNAC-MycGAPDHLog of InputY 0.049X 3.0179slope 0.1ΔCt allowed33
The StepOnePlus Real-Time PCR System簡易三步驟!!34
StepOnePlus Real-Time PCR System: The Basics 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 volume435
StepOnePlus Real-Time PCR System: The Basics 4-color instrument FAM /SYBR Green dyes VIC /JOE dyes NED /TAMRA dye ROX dye36
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 PC3. Analyze your data37
ColocatedDirect control of instrument via computer1. Setup: You can design the experiment on the PCconnected to the StepOne System2. Run: Start the run from the PC connected to the StepOneSystem and Real-Time data collection3. Analyze the results on the PC connected to the StepOneSystem38
StepOne只能暫存一個檔案39
StepOnePlus Compatible Consumables 樣品量多時– P/N 4346907MicroAmp Fast 96-Well Reaction Plate (0.1 mL) -10 plates– P/N 4360954MicroAmp Optical Adhesive Film - 25 films 樣品量少時– P/N 4358293MicroAmp Fast 8-Tube Strip (0.1 mL) - 125 strips– P/N 4323032MicroAmp Optical 8-Cap Strip - 300 strips Place the tray containing the tube, Loadat least 16 tube40
The flat edge of an applicator is rubbed back-and-forth along the length ofthe plate with a significant downward pressure to form a complete seal ontop the wellsNote: Pressure is required to activate the adhesive on the optical cover41
Operation Notes Directly load fast optical 96-well plate into the instrument9 If using fast 8-tube stripes, load the tubes with fast 96well tray Do not mark any labels on the consumables9 This may increase the background signal Avoid bubbles when pipetting into each well9 Centrifuge samples42
StepOne Software v2.1 OperationThe world leader in serving science43
1. Run: QuickStartQuick Start 先上機 !44
2. Setup: Experiment Propertiesa. Experiment Name 及檔案儲存位置b.選擇 Experiment Typec.選擇使用螢光系統d.選擇Ramp Speede.選擇實驗樣品種類45
3. Setup: Run Method4.46
5. Setup: Plate Setup 定義基因和樣品名稱47
6-1. Setup: Plate Setup決定基因和樣品位置 (for ddCt)48
6-2. Setup: Plate Setup決定基因和樣品位置 (for Standard curve)49
Automatic Standard Curve Setup50
7. AnalyzeAnalysis : Amplification Plot3. Analyze or Re-analyze1.2. Auto or Manual4. Check Threshold51
How to Set the Threshold?Auto or Manual Threshold?Manual threshold could be used to set fixedthreshold when doing run to run comparison52
Analysis Report53
Analysis : Gene Expression54
Analysis : Standard Curve55
Analysis : Multicomponent Plot56
Analysis : Raw Data Plot57
QC Summary Help Your Troubleshooting58
Analysis : Melt Curve (SYBR Green)59
Analysis : Allelic Discrimination60
Data exportExport to Excel, PowerPoint or save as jpeg61
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 VeritiThermal Cycler62
Red linesto delineatezones63
Setup: Run Method64
Useful rs/real-time-pcr-experimental-configuration.html65
Useful ome.html66
On-Line Support 7
Thanks for your �品應用支援服務專線: 0800-251-326 #3E-mail: Support.TW@lifetech.comThe world leader in serving science68
2x Power SYBR Master Mix 1x 10 ul (2x Fast SYBR Master Mix) F Primer optimized NA. R Primer optimized NA. Water NA. cDNA 10-100 ng 5-10 ul. 20 ul. Real Time PCR: PCR condition: 50 , 2min. 95 , 10 min. 95 , 15 sec 40 cycles. 60 , 1min . Standard mode. PCR condition: 95 , 20 sec
