WIXLIB

Introduction to Real-Time PCR &StepOne PlusTM Operation林有啓 (Eugene Lin, Ph.D.)Field Application ScientistThe world leader in serving science1

Normalised reporter fluorescence (Rn)Real Time PCR Amplification plot featureslens0.900.800.70SampleLower expression level0.600.500.40 RnThreshold0.300.20No Template controlBaseline region0.10capPCR vesselthermal block0.0005101520CT25PCR cycle numbersample303540Geometric Phase:(Exponential Phase)Y N0(1 E) x 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

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 Melt4

Generate Real-Time Signal Using Fluorescent1. TaqMan probe or TaqMan MGB probechemistry: 5 Nuclease assayFQ2. SYBR Green I dye chemistry5

SYBR Green Dye A ‘minor groove’-binding molecule specific to the minorgroove of double-stranded DNA It fluoresces at an increased intensity when boundMajorGrooveMinor Groove6

SYBR Green - dsDNA minor-groove binding dyePolymerizationPolymerization Complete7

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:1. Optimize primer concentration2. Re-design primer pair8

TaqMan Chemistry- FRET PrincipalhvExcitationenergy transfer9

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 Quencher10

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)11

Real-time PCR ChemistriesSpecificity Highly specific Probe Hybridization Less specificSensitivity Very High Very HighFlexibility Multiplex PCR SNP detection /- application No Probe is required Screening toolOptimization Ready to use 20x primer/probe mix - Need to optimize PCR programno need to optimize Need to check primer-dimer info Gold standard for MAQC Need to check PCR efficiency PCR efficiency 100% 10%12

2-step qRT-PCRHigh CapacitycDNA RT Kit(P/N 4368814)High CapacityRNA-to-cDNA (P/N 4387406)Step1:Convert RNA to cDNA(random primers/oligo dT)Multipletubes2hr RT0.5- 1hr RTStep 2:Amplify and quantify cDNA(gene-specific primers)13

Preparing 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 , 1min10μl1μlNA5-10μl2x Power SYBR Master Mix 1x10μlF PrimeroptimizedNAR PrimeroptimizedNAWaterNAcDNA1-100 ng 5-10μl20μlFast modeSYBR Green:20μl- Check Primer Concentration:PCR condition: 100-300nM95 , 20 sec95 , 1 sec 40 cycles - Add Melt (Dissociation) Curve60 , 20 sec14

Real Time PCR ApplicationsAbsolute Quantitation vs. Relative Quantitation15

絕對定量 (Absolute Quantitation)CT values 主要應用於病毒量及病原菌偵測 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 template1234567Log copy number16

相對定量(Relative Quantitation)Relative— “n-fold” difference relative to the calibrator (no units)-- 1. Ct analysis (most common)-- 2. relative standard curveStandard: any stock RNA or DNA containing the appropriate targetEndogenous control normalized RNA input measurement and RT-efficiency difference(ex. 18S rRNA, GAPDH, HPRT, -actin .) The most powerful and widely used method Ex. Time Course (Treatment)17

Comparative Ct Methodstep 1: Normalization to endogenous controlCt Target gene – Ct Endogenous control Ctstep 2: Normalization to calibrator sample Ct Sample – Ct Calibrator Ctstep 3: use the formula- Ct2A calibrator sample is a sample to which unknown samplesare compared (ex. untreated sample or control).18

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 22.619

ΔΔCt Calculations (Comparative Ct)c-MycT 0 (calibrator)T 12hrT 24hrT 48hr25242328GAPDH10101110Relative Quantityof Expression10 Ct Ct2- Ct151412180-1-331.02.08.00.18.0t 08t 12 h6t 24 h41.022.0t 48 h0.1020

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 15,000 TaqMan microRNA assays (miRBase release 20, 206 listed species) TaqMan Mutation Detection assays TaqMan Non-Coding RNA assays Option 2: Custom / Custom Plus Assay All-in One tube TaqMan-based Assay (20X mixture) Option 3: Primer Express software Software for designing real-time assays 上機條件皆相同 不用再花時間測試primer溫度了21

How to search AB TaqMan assay on database/Gene name orRefSeq Accession .22

TaqMan Gene Expression Array s.html23

Pre-configured TaqMan Gene ExpressionArray Plate Pre-configured platesfor 135 differentdiseases, pathways,and biologicalprocesses 96-well Fast(0.1 ml) plate Species Human Mouse RatHuman Notch SignalingHuman Alzheimer's DiseaseHumanABC TransportersHuman AngiogenesisHuman p53 SignalingHuman ApoptosisHuman CMV & MAPK PathwaysHuman Immune ResponseHuman PDGF PathwayHuman InflammationHuman Cholera InfectionHuman PhosphodiesteraseHuman Phagocytosis of MicrobesHuman Protein Kinase PathwaysHuman Pathogenesis of ALSHuman Stem Cell PluripotencyHuman ABC TransportersMouse Alzheimer's DiseaseHuman p53 SignalingMouse Immune ResponseHuman CMV & MAPK PathwaysMouse Stem Cell PluripotencyHuman PDGF PathwayRat InflammationHuman Cholera InfectionRat PhosphodiesteraseHuman Phagocytosis of MicrobesHuman AndrogensHuman Pathogenesis of ALSHuman ChemokinesHuman TNF Superfamily PathwayHuman CYP450 and other OxygenasesHuman TNFR1 PathwayHuman DiabetesHuman TNFR2 PathwayHuman EstrogensHuman Toll Comparative PathwayHuman Heat Shock ProteinsHuman Toll-Like Receptors PathwayHuman Hedgehog PathwayHuman Transcription of mRNAHuman HypoxiaHuman Transcription of rRNAHuman LDL PathwaysHuman Transcription of tRNAHuman Ligand Gated Ion ChannelsMouse lipid regulated genesHuman MAP Kinase PathwaysRat Endogenous ControlsHuman NeurotransmittersMouse Endogenous ControlsHuman AndrogensHuman ChemokinesHuman DiabetesHuman EstrogensHuman Growth FactorsHuman HematopoisisHuman NGF PathwayHuman NFkB PathwayHuman OsteogenesisHuman TGFB PathwayHuman Tumor MetastasisHuman TNFR1 PathwayHuman TNFR2 PathwayHuman WNT PathwayHuman Hox GenesHuman BMP PathwayHuman IL-2 PathwayHuman IL-9 PathwayHuman iNOS SignalingHuman IL-1 PathwayHuman IL-10 PathwayHuman Growth FactorsHuman HematopoisisHuman EGF PathwayHuman FGF Pathway24

Targets and Pathway Information25

Plate Layout and Assay IDAssay ID123456789AHs99999901 s1Hs99999905 m1Hs99999909 m1Hs99999908 m1Hs00153836 m 1Hs00923299 m 1Hs00155658 m 1Hs00609603 m 1Hs00174915 m 1BHs00609638 m 1Hs00370078 m 1Hs00234930 m 1Hs00233470 m 1Hs00233476 m 1Hs00176144 m 1Hs00176148 m 1Hs00231733 m 1Hs00754870 s1CHs00157619 m 1Hs00357739 m 1Hs00211913 m 1Hs00193363 m 1Hs00193364 m 1Hs00192033 m 1Hs00174143 m 1Hs00174131 m 1Hs00171410 m 1DHs00386448 m 1Hs00166367 m 1Hs00221445 m 1Hs00195432 m 1Hs00183425 m 1Hs00232222 m 1Hs00232068 m 1Hs00195437 m 1Hs00178579 m 1EHs00427259 m 1Hs00602137 m 1Hs00177066 m 1Hs00385075 m 1Hs00765707 m 1Hs00180562 m 1Hs00178463 m 1Hs00403062 m 1Hs00234686 m 1FHs00955491 gHHs00608187 m 1Hs99999918 m 1Hs00234244 m 1Hs00234245 m 1Hs00745761 s1Hs00610319 m 1Hs00559661 m 1Hs00234257 m 1GHs01117001 m 1Hs00415315 m 1Hs00193764 m 1Hs00271352 s1Hs00245109 m 1Hs00188614 m 1Hs00178154 m 1Hs00171132 m 1Hs00220998 m 1HHs00246256 m 1Hs00764128 s1Hs00205566 m 1Hs00179759 m 1Hs00410929 m 1Hs00224203 m 1Hs00368884 g1Hs00369400 m 1Hs00377065 m 1Gene Sym URF2INHBEIL17FACVR1C26

Configure Custom TaqMan Array Plate TaqMan Array Plates Fixed and custom formats: 8, 16,32, 48, and 96 96-well “plates” Uses inventoried assays 10µl reaction volume forStepOnePlus 27

What are SNPs? 單核苷酸多型性（Single Nucleotide �生的單個核苷酸鹼基之間的變異 �因於SNP的基因差異。 診斷及預測致病風險評估 藥物基因體學及新藥的開發MomDad Each person has 2 copies or 2 alleles of each gene – 1 allele on eachchromosome. Each person receives 1 allele from each parent. If both alleles are the same, the person ishomozygous for that gene. If the alleles differ, then the person isheterozygous for that gene.28

TaqMan SNP Genotyping Assay Overview29

Allelic Discrimination (SNP) DataHomozygous AAHeterozygous GAAHomozygous GGG30

miRNA Quantitation by Real-Time PCRMaturemiRNAAssayforNorthern Blot ResultSynthetic miRNA Targetlet-7a let-7b let-7c let-7dlet-7a31

Individual TaqMan MicroRNA Assays Aligned with release 19 of the miRBase 12,000 TaqMan microRNA assays Assay include: Human,Mouse,Rat,Drosophila melanogaster,Caenorhabditis elegans,Arabidopsis thaliana 193 listed species 46 endogenous controls (small nuclear RNAs)U6 snRNA, RNU6B, RNU43, RNU44, RNU48, Z30, snoRNA202 . Each assay contain: 1 RT primer, 1 TaqMan Assay Additional Applied Biosystems Products required to run miRNA assays-- TaqMan MicroRNA RT kit (200 rxn, 4366596/ or 1000 rxn, 4366597)-- TaqMan Universal PCR Master Mix32

Copy Number Variation (CNV)-基因拷貝數變異 ��的一種重要分子機制. 存在操作繁瑣，解析度低等問 py Number Variation (CNV) A structural genomic variant involving copy number changes incomparison to a reference genome Deletion or duplication events involving 1 kb of DNA. Most are 10 Kb; some rare CNVs 1 Mb CNVs are found in normal individuals and have also beenassociated with disease and other phenotypes33

Workflow of TaqMan Copy Number Variation Assays 1.6M Pre-Designed TaqMan Copy Number Assays availableFAM -labeled1CopyCallerTEST ASSAYVIC -labeledCONTROL ASSAYStandard TaqMan protocol2(i.e.: RNase P)gDNA31 ng / µL PCR rxnqPCRTaqMan4Master Mix4 replicates per gDNAsample34

Determination of DNA Copy Number35

CopyCaller Software-輕鬆獲得CNV結果 Flexible 不需要已知拷貝數的 樣品當control Free 免費下載分析軟體 Easy to use �輕鬆了解判讀結果 Results with confidence value �的結果36