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Applied Biosystems StepOnePlus Real-timePCR System之原理與應用介紹蔡如芸 (Judy Tsai, Ph.D.)Field Application ScientistThe world leader in serving science

lenscapPCR vesselNormalised reporter fluorescence (Rn)Principle of Real-time PCRthermal blocksample0.900.800.70SampleLower expression level0.600.500.40 RnThreshold0.30No Template control0.20Baseline region0.100.0005101520CT25PCR cycle number303540Exponential PhaseCT threshold cycle: calculated fractional cycle number at whichPCR amplification curve crosses the threshold ofdetection2

Real-time PCR Signal Detection: Exponential Phase101100GelPlateauLinearThresholdof detectionExponentialfluorescentsignal 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

Real-time PCR ChemistriesTaqMan and TaqMan MGBFluorogenic 5’ NucleaseAssay4SYBR Green I dyeBinds Doublestranded DNA

TaqMan Assay: Fluorogenic 5‘-nuclease AssayRforward primerprobeQ3'5'3'5'3'5'5'reverseprimer1. PolymerizationQ3'5'3'5'3'5'5'2. Strand displacementRQ5'3'3'5'3. Cleavage5'R3'5'Q5'3'5'5'3'3'5'4. Polymerization completedR Reporter (FAM, VIC, etc.)Q Quencher (NFQ/MGB, etc.)5

TaqMan Probe: TaqMan MGB/NFQ Probes 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 detectablefluorescent signal MGB probe design uses a special algorithm in Primer Express Software Shorter probe length (13-25-mers)(non-fluorescent quencher)QRAGGCCTTGAGAGATATRNFQQMGB(minor groove binder)QAGGCCTTGAGAGATAT GCTACACAGTCCGGAACTCTCTATAGCATCACAC6

Real-time PCR Chemistries: SYBR Green I Dye A ‘minor groove’-binding molecule specific to the minor groove of doublestranded DNA Fluoresces at an increased intensity when boundMajorGrooveMinorGroove7

SYBR Green I Dye: Melting Curve aturation8

SYBR Green I Dye: Melting Curve Analysis 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 pair9

Real-time PCR ChemistriesSpecificityTaqMan AssaySYBR Green I DyeMore specificLess specificProbe hybridizationSensitivityVery highVery highFlexibilityMultiplex PCRNo probe requiredSNP detectionScreening tool /- applicationOptimization10Ready to use 20xprimer/probe mix - noneed to optimizeNeed to optimize PCRprogramGold standard for MAQCNeed to check primerdimer infoPCR efficiency 100 10%Need to check PCRefficiency

Reverse Transcription and Real-timePCR Reaction11The world leader in serving science

One-step vs Two-step Workflows One-step Technology RT and PCR are performed insingle buffer system RT and PCR are performedin two separate reactions One tube, one step Cost advantaged wheninterrogating multiple targets Reduce chance of crosscontamination cDNA can be stored andused for further experiments Easy for high throughput workflow Best choice if RNA islimiting Cost effective when fewtargets/sample analyzed Uses gene-specific primers X cDNA can not be stored12 Two-step Technology X Multiple steps, longer timeto result

One-step Workflow: Real-time PCR Reactions13

Two-step Workflow: Real-time PCR ReactionsReverse Transcription : High Capacity RNA-to-cDNA Kit2X RT Buffer10μl20X RT Enzyme Mix1μlSample (up to 2μg)Up to 9μlNuclease-Free waterTo 20μlReal-time PCR:TaqMan ChemistrySYBR Chemistry2x TaqMan Master Mix1x20x Probe/primer Assay Mix 1xWatercDNA1-100 ngStandard modePCR condition:50 , 2min95 , 10 min95 , 15 sec 40 cycles60 , 1min1410μl1μlNA5-10μl2x Power SYBR Master Mix 1x10μlF PrimeroptimizedNAR PrimeroptimizedNAWaterNAcDNA1-100 ng 5-10μl20μl20μlFast modePCR condition:95 , 20 sec95 , 1 sec 40 cycles60 , 20 secSYBR Green:- Check Primer Concentration- Add Melt Curve Program

Two-step Workflow: Master MixesStandard Mode TaqMan ChemistryFast Mode TaqMan Chemistry TaqMan Universal Master Mix II(PN 4440038) TaqMan Fast Universal MasterMix (PN 4366072) TaqMan Gene ExpressionMaster Mix (PN 4369016) TaqMan Fast Advanced MasterMix (PN 4444557) SYBR Green Chemistry Power SYBR Green PCR MasterMix (PN 4367659) SYBR Green Chemistry Fast SYBR Green Master Mix(PN 4385612) PowerUp SYBR Green MasterMix (PN A25742)15

Real-time PCR Quantitation Methods Absolute Quantitation vs. Relative Quantitation16

絕對定量 (Absolute Quantitation)CTsCT values 主要應用於病毒量及病原菌偵測 To determine the actual number of copiesof a target nucleic acid within a sample withstatistical confidence.0CT is directly proportional to log ofamount of input template1712345Log copy number67

相對定量 (Relative Quantitation) To determine fold differences of a target nucleic acid in a startingmaterial with statistical confidence. ΔΔCt analysis (most common) Relative standard curve Need endogenous gene normalizes the amount of sample added Endogenous control (e.g. GAPDH, β-actin, etc.) Most powerful and widely used method Check primer PCR efficiency if using SYBR Green Dye18

相對定量 (Relative Quantitation): PCR Efficiency Validation 2μg RNA in 20μl RT 100ng cDNA/μl Gene name: C-Myc and GAPDH cDNA 4-fold serial dilution: 10μl cDNA 30μl H2O (25ng/μl) 1.2.3.4.5.25ng/μl6.25 ng/μl1.56 ng/μl0.39ng/μlNTC (duplicate for each sample)10μl25ng/μl10μl10μl6.25 ng/μl1.56 ng/μl0.39 ng/μl30μl H2O30μl H2O30μl H2O 每個濃度點各做二重複30μl H2O 19Prepare a Premix for each geneAliquot 15μl of Premix to each wellAdd 5μl of RT product to the wellReal-time PCR reaction

相對定量 (Relative Quantitation): PCR Efficiency Validation90 Eff% 110 CtEff% 90 Relative standard curve20

相對定量 (Relative Quantitation):Comparative Ct Method (ΔΔCt)Comparison of the c-myc expression levelin T 0, T 12, T 24, T 48 time course studyReference Samplet 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 23.621Ct 27 Ct 22.6

相對定量 (Relative Quantitation):Comparative Ct Method (ΔΔCt)step 1: Normalization to endogenous controlSample: Ct c-Myc – Ct GAPDH Ct sampleReference: Ct c-Myc – Ct GAPDH Ct reference samplestep 2: Normalization to reference sample Ct sample – Ct reference sample Ctstep 3: use the formula- Ct2A reference sample is a sample to which unknown samplesare compared (e.g. untreated sample or control).22

相對定量 (Relative Quantitation):Comparative Ct Method (ΔΔCt)23

Introducing TaqMan Advanced miRNA Assays SKU A28007, 50 reactions Excellent sensitivity in biological samples(serum/plasma, tissue) Low input amount (2µl) saves precioussamples; no need to run multiple RTs andsplit sample Universal cDNA can be used for anymiRNA assay cDNA can be archived for future miRNAstudies TaqMan advanced miRNA assaysMaturemiRNA24 SKU A25576, Size S, 250 reactions TaqMan advanced miRNA cDNAsynthesis kit

TaqMan Advanced miRNA Assays: How it Works25

TaqMan Advanced miRNA Assays: High SpecificitymiRNA UGGUGGUmiRNA SequenceAGU AGG UUG UAU AGU UAGU AGG UUG UGU GGU UAGU AGG UUG UAU GGU UAGU AGG UUG CAU AGU UAGG AGG UUG UAU AGU UAGU AGA UUG UAU AGU UAGU AGU UUG UAC AGU UAGU AGU UUG UGC UGU U***** *Let-7 miRNA family:differences as smallas single basemismatchesSynthetic TemplateTaqManAdvancedmiRNA %0%100%0%Let7i0%0%0%0%0%0%4%100%Extremely lowcross-reactivity,usually 1% or lower

TaqMan SNP Genotyping Assay Overview27

Allelic Discrimination (SNP) DataHomozygous AAHeterozygous GAAHomozygous GGG28

Applied Biosystems 提供Primers/Probe設計的全方位解決方案 TaqMan Gene Expression Assays 1,300,000 個已設計及測試過的基因定量試劑組 提供所有相關生物資訊 (23 species)7Custom TaqMan AssaysService TaqMan microRNA and primary microRNA AssaysTaqMan SNP Genotyping AssaysTaqMan Copy Number AssaysTaqMan Mutation Detection Assays 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29

Finding the Right Assay for Your Research Search for the assay youneed quickly and easily Powerful search engine Streamlined search interface Flexibility to search by genename, gene alias or assay nce/pcr/real-time-pcr/realtime-pcr-assays.html30

TaqMan Gene Expression Array ml31

Targets and Pathway Information32

Plate Layout and Assay ID33

定量PCR Primers/ Probe設計軟體34

清楚明確的 TaqMan Probe and Primer 設計規範200 bp amplicon35500 bp amplicon

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Sequence2. Find Primer/Probe1. Add DNA file or Copy & Paste37

Design Parameter38

Results39

Check Tm of Primers40

SYBR Green Experiment Notes1. Primer Concentration Optimization Primer final concentration No primer dimer or non-specific product involved2. PCR Primer Efficiency Validation Serially-diluted sample to generate standard curve for target gene andendogenous control gene3. Test with samples that are comparable to real experiment for eachgene41

StepOnePlus Real-time PCR System簡易三步驟!42

StepOnePlus Real-time PCR System: the Basics Hardware Specifications Power switch in the back Automatically enters standby mode after 4hr During standby mode, the touchscreen can beactivated with a simple touch Entire system becomes activated by pressing the softblue button 96-Well Block One block, 2 speeds Fast cycling: 40 cycles in 40 minutes Standard cycling: 40 cycles in under 2 hours 10-30µl reaction volume43

StepOnePlus Real-time PCR System: the Basics 4-color instrument FAM /SYBR Green dyes VIC /JOE dyes ROX dye NED /TAMRA dye44

StepOnePlus : Veriflex Block Veriflex Block One block, Six Zones The same “Better than gradient” feature from Veriti 96-well Thermal Cycler*Image from VeritiThermal Cycler45

StepOnePlus : Consumables 樣品量多時 MicroAmp Fast 96-Well Reaction Plate(0.1 ml) -10 plates (P/N 4346907) MicroAmp Optical Adhesive Film - 25films (P/N 4360954) 樣品量少時 MicroAmp Fast 8-Tube Strip (0.1 ml) 125 strips (P/N 4358293) MicroAmp Optical 8-Cap Strip - 300strips (P/N 4323032) Place the tray containing the tube, Load at least 16 tube46

Sealing the PlateThe flat edge of an applicator is rubbed back-and-forth along the length of the platewith a significant downward pressure to form a complete seal on top the wellsDownward pressure appliedin back-and-forth motionsacross the top of the plateNote: Pressure is required to activatethe adhesive on the optical cover47

Sealing the PlateThe end of an applicator is rubbed around all the outside edges of the plate with asignificant downward pressure to form a complete seal around the outside wellsDownward pressureapplied in smallback - and - forth motionsalong all the edgesNote: Pressure is required to activate the adhesive on the optical cover48

StepOnePlus : Operation Notes Directly load fast optical 96-well plate into the instrument If using 8-tube strips, load strips with fast 96-well tray Save your data with USB device after each run (standalone) Do not label on the consumables This may increase the background signal Avoid bubbles when pipetting into each well Centrifuge samples49

Standalone (PC-free) Operation只需輕按觸控螢幕 不需電腦連線也能上機!!1. Start run from touch-screen2. After run, download the file (.eds)to your PC3. Analyze your data50

Browse/New Experiments: New51

Select Experiment : Save52

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Standalone: 只能暫存一個檔案 插入USB, 待 icon 出現在右下角, 即可點選 Collect Results檔案會自動存到USB 中55

StepOneTM v2.3 Software 一套軟體可以符合全方位的應用 (1280x1024 pixel resoltion) 絕對定量 Quantification - Standard Curve 56相對定量 Quantification – Comparative Ct ( Ct)相對定量 Quantification - Relative Standard CurveMelting Curve AnalysisGenotypingPresence/Absence

1. Run: QuickStart57