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Applied Biosystems 7500 Real-Time PCR System之原理與應用介紹趙乃蓁 (Nai-Chen Chao)Field Application ScientistThe world leader in serving science

Agenda Real-time PCR Principle and Chemistry Assay Design Pre-designed Assay Custom Assay Primer Express Software Reverse Transcription (RT) and Real-time PCR Reaction One Step and Two Step Workflow Master mix Gene Expression Analysis Absolute Quantitation Relative Quantitation2

Agenda Data Analysis and Quality Control Applications SNP Genotyping miRNA analysis using TaqMan assay Introduction of ABI 7500 Real-Time PCR system Overview Software operation Create Study3

Real-time PCR Principle and Chemistry4The world leader in serving science

Traditional PCRIn traditional PCR, the product is detected only after amplification iscompleted, using methods such as gel electrophoresisCycle 15Cycle 2Cycle 3gel electrophoresis

Principle of Real-Time PCRlensNormalised reporter fluorescence (Rn)0.90capPCR vessel0.80Sample0.700.60 RnLower expression level0.500.40Threshold0.300.20No Template control(NTC)Baseline region0.100.00051015CT 2025PCR cycle number303540Exponential PhaseThermal blocksample6CT (Threshold Cycle): Calculated fractional cycle number at whichPCR amplification curve crosses the threshold of detection

Real-time PCR Signal Detection: Exponential Phase101PlateauLinear100ExponentialThresholdof detectionFluorescent 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7

Real-Time PCR ChemistriesTaqMan 5’-Nuclease Assay8SYBR GreenBinds double-strandedDNA

TaqMan Assay: 5’-nuclease AssayRforward primerprobeQ3'5'3'5'3'5'5'reverseprimer1. PolymerizationQ3'5'3'5'3'5'5'R2. Strand displacementQ3'5'3'5'5'3'5'3. CleavageR5'3'5'5'3'3'5'R Reporter (FAM, VIC, etc.)Q Quencher (NFQ/MGB, etc.)FRET: Fluorescence Resonance Energy Transfer9Q4. Polymerization completed

TaqMan Assay: TaqMan MGB probes Non-fluorescent Quencher (NFQ) “Dark” quencher acts as energy transfer acceptor that doesn’t emit a detectable fluorescentsignal Minor Groove Binder (MGB) Small molecule that fits snugly into minor groove of duplex DNA Stabilizes probe annealing Shorter probe length (13-25-mers)NFQ Higher specificity(non-fluorescent quencher)QRAGGCCTTGAGAGATATMGB(minor groove binder)RQAGGCCTTGAGAGATAT GCTACACAGTCCGGAACTCTCTATAGCATCACAC10

SYBR Green I Dye: Fluorescent DNA Binding Dye A ‘minor groove’-binding molecule specific to the minor groove of doublestranded DNA Fluoresces at an increased intensity when boundMajorGrooveMinorGroove11

SYBR Green I Dye: Melting Curve AnalysisPCRPolymerizationNormalized SYBR Green tive First Derivative

Assay DesignAssay primers Probe13The world leader in serving science

Applied Biosystems 提供Primers/Probe設計的全方位解決方案 Pre-Designed TaqMan Assay (ready-to-use)7 TaqMan Gene Expression Assays �劑組 提供所有相關生物資訊 (23 species) TaqMan microRNA and primary microRNA Assays TaqMan SNP Genotyping Assays TaqMan Copy Number Assays TaqMan 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14Custom TaqMan AssaysService

Pre-Designed TaqMan 15

Pre-Designed TaqMan Assay16

Custom TaqMan Assay Design mic-products/tools/cadt/17

Primer Design: Bioinformatic Evaluation Bioinformatics BLAST tool: Sequence specificity (https://blast.ncbi.nlm.nih.gov/Blast.cgi) SNPmasker: N masked SNPs (http://bioinfo.ebc.ee/snpmasker/) RepeatMasker: N masked repeat sequence (http://www.repeatmasker.org) Software Primer Express Software18

Primer Express Software: Sequence2. Find Primer/Probe1. Add DNA file or Copy & Paste19

清楚明確的 TaqMan Probe and Primer 設計規範TaqMan CR產物大小建議在50-150 70oC (Quatification assay)65-67oC (Allelic Discrimination assay)Tm值：58-60oCProbe長度：13-25 bases (TaqMan MGB probe)13-30 bases (TaqMan probe)Primer長度：20 bases �前5個序列裡不能超過2個C dye在5’端第二個序列也不能為G)200 bp amplicon500 bp GG-MGB-3’ or �或以上的CG di-nucleotidesba. 針對TaqMan MGB probeb. 參數可選擇設定20200 bp amplification500 bp amplification

SYBR Experiment Note PCR Efficiency Validation Serially-diluted sample to generate standard curve for each primer set At least 5 dilution point in triplicate Check the R square value and PCR efficiency (90% 110%) Primer Optimization Use NTC to check whether non-specific product is primer dimer (NTC is important!) If the non-specific product is primer-dimer: Optimize primer concentration Re-design primer pair Test with samples that are comparable to real experiment for each gene21

Reverse Transcription (RT) andReal-time PCR Reaction22The world leader in serving science

One-step vs Two-step WorkflowsOne-step TechnologyTwo-step Technology RT and PCR are performed insingle buffer system RT and PCR are performed intwo separate reactions One tube, one step Reduce chance of crosscontamination Cost advantaged wheninterrogating multiple targets cDNA can be stored and usedfor further experiments Easy for high throughput workflow Best choice if RNA is limiting Cost effective when fewtargets/sample analyzed Uses gene-specific primersX cDNA cannot be stored23X Multiple steps, longer time toresult

One-step Workflow: Real-time PCR ReactionsComponentPCR machine program24

One-step Workflow: Master MixesStandard ModeFast Mode TaqMan Chemistry TaqMan Chemistry RNA-to-Ct 1-Step Kit (PN# 4392653) SYBR Green Chemistry TaqMan Fast Virus 1-Step MasterMix (PN# 4444432) Power SYBR Green RNA-to-CT 1Step Kit (PN# 4391178)Tolerant to PCR Inhibitors!25

Two-step Workflow: Real-time PCR ReactionsReverse Transcription : High Capacity RNA-to-cDNA Kit2X RT Buffer10 μl20X RT Enzyme Mix1 μlSample (up to 2 μg)Up to 9 μlNuclease-Free waterTo 20 μlStep 1Step 2Step 3Temp. (oC)37954Time605 Real-time PCR:SYBR ChemistryTaqMan Chemistry2x TaqMan Master Mix1x10 μl20x Probe/primer Assay Mix1x1 μlNAWatercDNA1-100 ngStandard modePCR condition:50 , 2 min95 , 10 min95 , 15 sec40 cycles60 , 1min262x Power SYBR Master Mix1x10 μlF PrimeroptimizedNAR PrimeroptimizedNA5-10 μlWater20μlcDNANA1-100 ng5-10 μl20μlFast modePCR condition:95 , 20 sec95 , 1 sec60 , 20 sec 40 cyclesSYBR Green:- Check Primer Concentration- Add Melt Curve Program

Two-step Workflow: Master MixesStandard ModeFast Mode TaqMan Chemistry TaqMan Chemistry TaqMan Universal Master Mix II(PN 4440038) TaqMan Fast Universal Master Mix(PN 4366072) TaqMan Gene Expression MasterMix (PN 4369016) TaqMan Fast Advanced Master Mix(PN 4444557) SYBR Green Chemistry Power SYBR Green PCR Master Mix(PN 4367659) SYBR Green Chemistry Fast SYBR Green Master Mix (PN4385612) PowerUp SYBR Green Master Mix(PN A25742)27

Gene Expression Assay28The world leader in serving science

絕對定量 (Absolute Quantitation) 主要應用於病毒量及病原菌偵測CT values To determine the actual number ofcopies of a target nucleic acid within asample with statistical confidence.CTs0CT is directly proportional to log ofamount of input template2912345Log copy number67

相對定量 (Relative Quantitation) To determine fold differences of a target nucleic acid in a starting materialwith 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.) Need at least 1 reference sample Reference group (e.g. untreated, time 0, etc.) Check primer PCR efficiency if using SYBR Green Dye30

Relative Quantitation: PCR Efficiency Validation Gene name: c-Myc and GAPDH 2 μg RNA in 20 μl RT 100 ng cDNA/μl cDNA 4-fold serial dilution: 10 μl cDNA 30 μl H2O (25 ng/μl)1. 25 ng/μl2. 6.25 ng/μl3. 1.56 ng/μl10 μl10 μl10 μl4. 0.39 ng/μl5. NTC (duplicate for each sample) Prepare a Premix for each gene Aliquot 15 μl of Premix to each well Add 5 μl of RT product to the well Real-time PCR reaction3125 ng/μl6.25 ng/μl30 μl H2O30 μl H2O1.56 ng/μl30 μl H2O0.39 ng/μl30 μl H2O

Relative Quantitation: PCR Efficiency Validation90 Eff% 110 CtEff% 90 Relative standard curve or re-design32

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 0t 12t 24t 48timeSpectrophotometermeasure RNA quantitytotal RNAcDNA33total RNAcDNAC-myc GAPDHC-myc GAPDHCt 30.5 Ct 23.6Ct 27 Ct 22.6total RNAcDNAC-myc GAPDHtotal RNAcDNAC-myc GAPDHReverse TranscriptionEx. 5 ug RNA / 50 uL 100 ng/uLReal-Time PCRUnknown samples (50 ng)

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 referencestep 2: Normalization to reference sample Ct (sample) – Ct (reference sample) Ctstep 3: use the formula- Ct2A reference sample is a sample to which unknown samples are compared(e.g. untreated sample or control)34

相對定量 – Comparative Ct Method 02.08.00.1T 0 hrT 12 hrT 24 hrT 48 hrRelative Quantity ofExpression97t 0t 12t 24t 4865432103588210.1

Data Analysis – 4 check points 完整的擴增曲線 Baseline Exponential phase Linear phase Plateau 每個target gene的Threshold設置在各自正確的位置 Exponential phase Melt curve plot (for SYBR Green only) Single peak no primer-dimer 技術性重複(Technical Replicate) 三重複為佳 排除(Omit)異常樣本36

ApplicationsSNP GenotypingmiRNA analysis using TaqMan assay37The world leader in serving science

TaqMan SNP Genotyping Assay Overview38

Allelic Discrimination (SNP) DataHomozygous AAHeterozygous GAAHomozygous GGG39

MicroRNA Analysis Using TaqMan AssaysTaqMan MicroRNA AssaysTaqMan Advanced MicroRNA Assays40

TaqMan miRNA AssayTaqMan technology goes small with bigbenefits for miRNA research Highly specific—quantitate only thebiologically active mature miRNAs, notprecursors Sensitive—requires only 1–10 ng of total RNAor equivalent to conserve limited samples Wide dynamic range—up to 7 logs—detecthigh and low expressors in a single experiment Custom assays available—you specify thesequence, and we will design an assay Fast, simple, and scalable—two-step realtime RTPCR assay quickly provides high-qualityresults41

TaqMan Advanced miRNA Assays Excellent sensitivity in biological samples (serum/plasma, tissue) Low input amount (2 μL) saves precious samples; no need to runmultiple RTs and split sample Universal cDNA can be used for any miRNA assay cDNA can be archived for future miRNA studies TaqMan advanced miRNA assays SKU A25576, 250 reactions TaqMan advanced miRNA cDNA synthesis kit SKU A28007, 50 reactions42

TaqMan Advanced miRNA Chemistry: How it Works43

TaqMan Advanced miRNA Assays: Extremely 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 small as single basemismatchesSynthetic TemplateTaqManAdvancedmiRNA %0%100%0%0%0%Extremely low 0%0%0%100%0%Let7i0%0%0%0%0%0%4%100%usually 1% or lower

Applied Biosystems 7500Real-Time PCR System45The world leader in serving science

Multiplexing CapabilitiesCustom dye must fluoresce within 520 to 650 nm spectral range and be calibrated before use46

ABI 7500 Real-Time PCR System: Consumables 樣品數量多時 MicroAmp Optical 96-Well Reaction Plate (0.2mL) - 10 plates (P/N N8010560) MicroAmp Optical Adhesive Film - 25 films (P/N4360954) 樣品數量少時 MicroAmp Optical 8-Tube Strip (0.2 mL) - 125strips (P/N 4316567) MicroAmp Optical 8-Cap Strip - 300 strips (P/N4323032) Load at least 2 strips with tray (on column 1 and12)47