WIXLIB

Applied Biosystems QuantStudio 3 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

Polymerase Chain Reaction (PCR)2

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 ofdetection3

Real-time PCR Signal Detection: Exponential Phase101GelPlateauLinear100ExponentialThresholdof detectionfluorescentsignal 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4

Real-time PCR ChemistriesTaqMan and TaqMan MGBFluorogenic 5’ NucleaseAssay5SYBR 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.)6

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 GCTACACAGTCCGGAACTCTCTATAGCATCACAC7

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 boundMajorGrooveMinorGroove8

SYBR Green I Dye: Melting Curve aturation9

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 pair10

Real-time PCR ChemistriesSpecificityTaqMan AssaySYBR Green I DyeMore specificLess specificProbe hybridizationSensitivityVery highVery highFlexibilityMultiplex PCRNo probe requiredSNP detectionScreening tool /- applicationOptimization11Ready 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 Reaction12The 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 stored13 Two-step Technology X Multiple steps, longer timeto result

1-step qRT-PCR: Real-time PCR Reactions14

1-step qRT-PCR: Master Mixes TaqMan Fast Virus 1-Step Master Mix (PN 4444434) 4X master mix to amplify both RNA and DNA Formulated to handle common RT-PCR inhibitors found in blood, stool, andother difficult samples Up to triplex (ROX as passive reference) TaqPath 1-Step Multiplex Master Mix (PN A28522) 4X master mix to amplify both RNA and DNA Tolerant to common RT-PCR inhibitors Manufactured in an ISO 13485 certified facility Up to quadruplex (does not include passive reference)15

2-step qRT-PCR: 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 , 1min1610μl1μlNA5-10μl2x 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

2-step qRT-PCR: 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)17

Real-time PCR Quantification Methods Absolute Quantification vs. Relative Quantification18

絕對定量 (Absolute Quantification)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 template1912345Log copy number67

相對定量 (Relative Quantification) 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 Dye20

相對定量 (Relative Quantification): 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 21Prepare 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 Quantification): PCR Efficiency Validation90 Eff% 110 CtEff% 90 Relative standard curve22

相對定量 (Relative Quantification): Comparative Ct (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 23.623Ct 27 Ct 22.6

相對定量 (Relative Quantification): Comparative Ct (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).24

相對定量 (Relative Quantification): Comparative Ct (ΔΔCt)25

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 assaysMaturemiRNA26 SKU A25576, Size S, 250 reactions TaqMan advanced miRNA cDNAsynthesis kit

TaqMan Advanced miRNA Assays: How it Works27

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

Copy Number Variation (CNV)Copy Number Variation (CNV)29 A structural genomic variant involving copy numberchanges in comparison to a reference genome Deletion or duplication events involving 1 kb ofDNA. Most are 10 Kb; some rare CNVs 1 MbCNVs are found in normal individuals and have also beenassociated with disease and other phenotypes

Workflow of TaqMan Copy Number Variation Assays 1.6M Pre-Designed TaqMan Copy Number Assays availableFAM -labeled1CopyCaller TEST ASSAYStandard TaqMan protocolVIC -labeledCONTROL ASSAY2(i.e.: RNase P)gDNA31 ng / µL PCR rxnqPCRTaqMan Genotyping MasterMix3044 replicates per gDNAsample

Determination of DNA Copy Number31

CopyCaller Software-輕鬆獲得CNV結果32 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

What are SNPs?MomDad Diploid organisms 2 sets of chromosomes Each person has 2 copies or 2 alleles of each gene – 1 alleleon each chromosome. Each person receives 1 allele from each parent. If both alleles are the same, the person is homozygous forthat gene. If the alleles differ, then the person is heterozygous for thatgene.33

TaqMan SNP Genotyping Assay Overview34

Allelic Discrimination (SNP) DataHomozygous AAHeterozygous GAAHomozygous GGG35

Types of Cancer Mutations Germline mutations Inherited mutations Present in all cells Heterozygote (50%) orhomozygote (100%) profile Single gene to multi-genes SNP to large chromosomerearrangement36 Somatic mutations Mutations associated with thecancer itself Present in some somatic cells (i.e.CTC) Require sensitive methods todetect minor allelic frequency Single gene to multi-gene SNP to large chromosomerearrangementExample: BRCA1Example: PI3KBreast CancerBreast, Colon Cancer

TaqMan Mutation Detection Assays (TMDA) Somatic Mutation Detection by castPCR Technology TMDA Product Line Summary Assays for 778 key mutations from 46 cancer genes Corresponding gene reference assays Wild-type assays for a subset of mutation targets Internal Positive Control Reagents (IPC kit) Mutation Detector Software Somatic mutations reported in the importantgenes related to biological pathways such asEGFR, Ras-Raf, KIT, FLT3, and PDGFRA High sensitivity (0.1-1%) for use with FFPEsamples and biopsies High specificity for generating accurate results37 Gene List 1 JAK2KRASKITMPLNPM1NRASPDGFRAPIK3CAPTENTP53VHL

TaqMan Mutation Detection Assays (TMDA) Superior Sensitivity – 0.1 % High Specificity Simple and scalable workflow – 3 hrs from sample to resultsCompetitive Allele-Specific TaqMan PCR - castPCR38

Mutation Detector Software39

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40

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.html41

TaqMan Gene Expression Array ml42

Targets and Pathway Information43

Plate Layout and Assay ID44

定量PCR Primers/ Probe設計軟體45

清楚明確的 TaqMan Probe and Primer 設計規範200 bp amplicon46500 bp amplicon

47

Sequence2. Find Primer/Probe1. Add DNA file or Copy & Paste48

Design Parameter49

Results50

Check Tm of Primers51