相對定量實(shí)驗(yàn)設(shè)計(jì)及條件優(yōu)化

1、Roche Applied Science1qPCRqPCR實(shí)驗(yàn)設(shè)計(jì)及實(shí)驗(yàn)設(shè)計(jì)及條件優(yōu)化條件優(yōu)化Roche Applied Science2n檢測模式及應(yīng)用檢測模式及應(yīng)用n對照的設(shè)置對照的設(shè)置n相對定量相對定量 n如何選取看家基因如何選取看家基因n相對定量實(shí)驗(yàn)設(shè)置相對定量實(shí)驗(yàn)設(shè)置n實(shí)驗(yàn)評判標(biāo)準(zhǔn)實(shí)驗(yàn)評判標(biāo)準(zhǔn)主要內(nèi)容主要內(nèi)容Roche Applied Science3Real-TimeReal-Time PCRPCR ExperimentsExperimentsData Analysis depending on ApplicationAmplification Analysis for Quan

2、tification and Qualitative DetectionMelting Curve Analysis for Product Identification, Mutation Detection, andGene ScanningDye 1Dye 1Dye 2Dye 2Endpoint Detection for Genotyping (Dual Color)Real-time PCR is the combination of PCR and detection within one reaction vessel. Roche Applied Science4Amplifi

3、cation AnalysisAmplification Analysisfor Quantification and Qualitative DetectionnTypical ApplicationsTypical ApplicationsnDetection and quantification of Detection and quantification of bacteria and virusesbacteria and virusesnQuantification of gene Quantification of gene expressionexpressionnSuita

4、ble Detection FormatsSuitable Detection FormatsnSYBR Green ISYBR Green InProbe-based formatsProbe-based formatsHydrolysis Probes, e.g. Universal ProbeLibrary Probes (UPL)Hybridization ProbesRoche Applied Science5Melting Curve AnalysisMelting Curve AnalysisEstablished and New ApplicationsLabeled Prob

5、es for GenotypingLabeled Probes for GenotypingHigh Resolution Melting Dye for Gene High Resolution Melting Dye for Gene ScanningScanningSYBR Green I for product identificationSYBR Green I for product identificationRoche Applied Science6Amplification Endpoint AnalysisAmplification Endpoint Analysisfo

6、r SNP Genotyping with Hydrolysis ProbesnDual color approachnTypical dye combinations: FAM VIC/HexnColor compensation not requiredRoche Applied Science7LightCyclerLightCycler 480480 ReagentsReagentsOptimized for each ApplicationnLightCyclerLightCycler 480 SYBR Green I Master 480 SYBR Green I MasternL

7、ightCyclerLightCycler 480 Probes Master 480 Probes Masternfor qPCR with hydrolysis probes, incl. endpoint genotypingfor qPCR with hydrolysis probes, incl. endpoint genotypingnLightCyclerLightCycler 480 Genotyping Master 480 Genotyping Masternfor melting curve-based genotypingfor melting curve-based

8、genotypingusing HybProbe or SimpleProbe probesusing HybProbe or SimpleProbe probesnLightCycler 480 High Resolution Melting MasterLightCycler 480 High Resolution Melting MasternLightCycler 480 RNA Master Hydrolysis ProbesLightCycler 480 RNA Master Hydrolysis ProbesnRT step done within 3 minRT step do

9、ne within 3 min Roche Applied Science8Universal Probe Library (UPL)Universal Probe Library (UPL)What is it? Universal ProbeLibrary (UPL) a unique combination Universal ProbeLibrary (UPL) a unique combination of of prevalidated hydrolysis probesprevalidated hydrolysis probes and free online and free

10、online assay design that allows you to design assay design that allows you to design custom gene custom gene expression assaysexpression assays to quantify virtually any transcript to quantify virtually any transcript in in any genomeany genome. . Prevalidated Prevalidated Hydrolysis ProbesHydrolysi

11、s Probes+ +Free Online Assay Free Online Assay Design CenterDesign Center= =UPLUPLRoche Applied Science9Universal ProbeLibraryUniversal ProbeLibrary通用探針庫通用探針庫技術(shù)要點(diǎn)技術(shù)要點(diǎn)n把傳統(tǒng)的水解探針由25-35nt減至8-9nt，報告基團(tuán)用Fluorescein標(biāo)記，并使用了低背景的BHQ（Black hole quencher）淬滅基團(tuán)n熔解溫度取決于所用的LNA（locked nucleic acid technology，鎖定核苷酸）技術(shù)

12、核苷的2、4位形成環(huán)氧亞甲基橋結(jié)構(gòu)，提高短探針Tm值，使其Tm值高于引物達(dá)到qPCR的要求。n其檢測特異性由引物和探針共同來決定。n通過網(wǎng)絡(luò)設(shè)計(jì)中心選取合適探針Roche Applied Science10UniversalUniversal ProbeLibrary ProbeLibrary How does it work? UniversalWithin the human transcriptome Within the human transcriptome Each probe can hybridize to over 7000 expressed gene, each tran

13、script could be detected by at least 16 probes. Randomly picked 8-9 mer oligonucleotide in transcriptomeRoche Applied Science11Universal Probe Library Universal Probe Library LNA technique enable UPL Higher TMLNA technique enable UPL Higher TM ProbeProbe TargetTarget Perfect match 3-acgaccac-5 Singl

14、e mismatch Single mismatch 3-acg 3-acgg gccac-5ccac-5 T Tm m DNA 8-mer 5-tgctggtg-3 Tm= 35C T Tm m= 25= 25C C 10 10C C LNA 8-mer LNA 8-mer 5-TGC 5-TGCT TGGTG-3GGTG-3 T Tm m= 71= 71C C T Tm m= 45= 45C C 2626C CUPL probes have Tm compatible with real-time PCRUPL probes have Tm compatible with real-tim

15、e PCRUPL probes show excellent single-mismatch discriminationUPL probes show excellent single-mismatch discriminationRoche Applied Science12primer primer UPL probeUPL probe primer primerSepcificity is abtained by the comnination of primers and the probe.Primer & Probe design Primer & Probe design Pr

16、obe Finder Software - SpecificityProbe Finder Software - SpecificityRoche Applied Science13Universal ProbeLibraryUniversal ProbeLibraryOnly 165 probes for over 5 billion assays種 屬實(shí)驗(yàn)數(shù)量覆蓋率HumanHomo sapiens 639 50099%MouseMus musculus 509 50099%RatRattus norvegicus 364 00098%PrimatesPan troglodytes 519

17、 50096%DrosophilaDrosophila melanogaster 253 50099%ArabidopsisArabidopsis thaliana 199 00098%C. elegansCaenorhabditis elegans 134 00095%MaizeZea mays 61 50094%RiceOryza sativa 898 50098%ZebrafishDanio rerio 630 00098%AnophelesAnopheles gambiae 193 00098%YeastSaccharomyces cerevisiae 42 00095%總總 數(shù)數(shù) 5

18、 000 000 Roche Applied Science14n檢測模式及應(yīng)用檢測模式及應(yīng)用n對照的設(shè)置對照的設(shè)置n相對定量相對定量 n如何選取看家基因如何選取看家基因n相對定量實(shí)驗(yàn)設(shè)置相對定量實(shí)驗(yàn)設(shè)置n實(shí)驗(yàn)評判標(biāo)準(zhǔn)實(shí)驗(yàn)評判標(biāo)準(zhǔn)主要內(nèi)容主要內(nèi)容Roche Applied Science15ControlsControlsEnable you to understand unexpected results and verify the experimentnNo template control (NTC)contains all components of the PCR reaction

19、 except the templatenDetection of contaminating nucleic acidnDetection of primer dimer formation in SYBR Green I assaysnPositive controlis a sample that is positive for the target sequence of the assaynFor initial assay validation (specificity, sensitivity, efficiency determination)nNegative control

20、is a sample that is negative for the target sequence of the assaynTo verify the specificity of the assayRoche Applied Science16Controls, Controls, continuedcontinuednRT (reverse transcriptase) minus controlfor gene expression assays.Prepare a RT minus control by omitting the addition of reverse tran

21、scriptase to the cDNA synthesis reaction, or use your untranscribed sample RNAnto indicate false positive results due to DNA in the RNA samplenInternal controlcontrol sequence is amplified in the same reaction as the target and detected with a different dye (duplex approach)e.g.; for samples in bact

22、eriology/virology to verify the PCR reactionnas a control for the sample preparation if added during NA isolation, and/ornas a control for the amplification reaction (PCR inhibition)Roche Applied Science17n檢測模式及應(yīng)用檢測模式及應(yīng)用n對照的設(shè)置對照的設(shè)置n相對定量相對定量 n如何選取看家基因如何選取看家基因n相對定量實(shí)驗(yàn)設(shè)置相對定量實(shí)驗(yàn)設(shè)置n實(shí)驗(yàn)評判標(biāo)準(zhǔn)實(shí)驗(yàn)評判標(biāo)準(zhǔn)主要內(nèi)容主要內(nèi)容Roch

23、e Applied Science18www.roche-applied-Everything in Life is Relative Everything in Life is Relative it just depends it just dependsWhen you sit with a nice girl for two hours, When you sit with a nice girl for two hours, it seems like two minutes. it seems like two minutes. When you sit on a hot stov

24、e for two minutes, When you sit on a hot stove for two minutes, it seems like two hours thats relativity. it seems like two hours thats relativity. Albert Einstein Albert EinsteinRoche Applied Science19參比基因的選擇n理想的參比基因:n表達(dá)水平在所有樣品中是持續(xù)穩(wěn)定的：正常組織 vs. 腫瘤組織n表達(dá)水平不受實(shí)驗(yàn)條件變化的影響：處理樣本 vs. 未處理的樣本n看家基因(Housekeeping

25、genes)：編碼有基礎(chǔ)細(xì)胞功能的蛋白質(zhì)n同一樣品中目標(biāo)基因表達(dá)的濃度與看家基因表達(dá)的濃度相關(guān)；n校正了樣品間質(zhì)量和數(shù)量的差異。Roche Applied Science20www.roche-applied-Gene Expression Level - ExampleGene Expression Level - Exampleh-PBGD in Tissue Samples and Cell LinesRoche Applied Science21www.roche-applied-Housekeeping GenesHousekeeping Genes-actinmultigene f

26、amily; 20 genes; 1 active locus : hormones of tyroid gland20 pseudogenes : stomach tumorg g-actinmultigene family; pseudogenesGAPDHmultigene family; 10-30 genes; 200 in mouse : lung, pancreatic, colon cancermostly pseudogenes : insulin, EGF5.8S,18S, 28S RNApseudogenes2-microglobulinno pseudogenes :

27、Non-Hodgkin lymhoma abnormal expression in tumorsG6PDHno pseudogenes : kidney, stomach tumor : hormones, oxidant stress, growth factorsPBGDno pseudogenesaldolasepseudogenesHPRTpseudogenesU3, U8, .Pseudogenesornithin : tumorsdecarboxylase.GeneGenomic structure / pseudogenesRegulation e.g.Roche Applie

28、d Science22www.roche-applied-Level of gene Level of gene expressionexpressionIFN-gamma: IFN-gamma: targettargetHPRT: housekeeperHPRT: housekeeper2M: housekeeper2M: housekeeper+ 脂多糖脂多糖+ + 地塞米松地塞米松巨噬細(xì)胞處理或非處理巨噬細(xì)胞處理或非處理Perform an absolute Perform an absolute quantification quantification Selection of a

29、HK GeneSelection of a HK Genewith a Known Starting Template AmountRoche Applied Science23www.roche-applied-Relative Relative RatioRatioMacrophage cells with or without treatmentMacrophage cells with or without treatmentIFN-gamma/HPRTIFN-gamma/HPRTIFN-gamma/2MIFN-gamma/2MIFN-gamma/PBGDIFN-gamma/PBGDM

30、onitor target/reference ratio.Monitor target/reference ratio.Must find at least 2 housekeeping Must find at least 2 housekeeping genes that give the same ratio genes that give the same ratio variation among different samplesvariation among different samplesSelection of a HK GeneSelection of a HK Gen

31、ewith an Unknown Starting Template Amount+ LPS.+ + DexamethasoneRoche Applied Science24N Normalization of Quantitative RT-PCRormalization of Quantitative RT-PCRReference Gene(s) Carefully select reference gene(s) for accurate normalizationuse genes from a wide variety of cellular processes to identi

32、fy reference genes unaffected by experimental conditionsone or more reference genes can be usedaverage of multiple pre-selected housekeeping genes is a solution in case no single reference gene is appropriate This quantification method corrects for quantity and quality differences in samples.Roche A

33、pplied Science25www.roche-applied-Correlation of Results Using G6PDH and cABL Correlation of Results Using G6PDH and cABL as Reference Gene in Cancer Research Studiesas Reference Gene in Cancer Research StudiesData obtained from Dr. Martin Weisser, Med. Klinik III., Klinikum Grosshadern, Data obtain

34、ed from Dr. Martin Weisser, Med. Klinik III., Klinikum Grosshadern, GermanyGermanyMonitoring of t(8;21) Monitoring of t(8;21) translocation (AML1-ETO) translocation (AML1-ETO) relative to two different relative to two different housekeeping geneshousekeeping genesResearch samples obtained Research s

35、amples obtained from subjects undergoing from subjects undergoing chemotherapychemotherapyAMLAML：急性髓細(xì)胞白血病：急性髓細(xì)胞白血病Roche Applied Science質(zhì)量控制質(zhì)量控制 qPCR qPCR 數(shù)據(jù)的標(biāo)準(zhǔn)化數(shù)據(jù)的標(biāo)準(zhǔn)化geNorm & NormFinder Roche Applied ScienceSoftwareSoftwarengeNORM:geNORM:nhttp:/ NormFinder: nhttp:/www.mdl.dk/publicationsnormfinder.h

36、tm http:/www.multid.se/genex/hs410.htmnbestkeeperbestkeepernhttp:/www.gene-quantification.de/bestkeeper.html27Roche Applied Science沒有任何基因的表達(dá)是永久恒定的沒有任何基因的表達(dá)是永久恒定的在每種生物學(xué)環(huán)境下，總是存在一組基因，其表達(dá)量的差異較在每種生物學(xué)環(huán)境下，總是存在一組基因，其表達(dá)量的差異較常見或通用的內(nèi)參基因更小。常見或通用的內(nèi)參基因更小。建議選擇建議選擇 生物學(xué)環(huán)境特異的內(nèi)參基因生物學(xué)環(huán)境特異的內(nèi)參基因RefGenes RefGenes 推薦的基因往往比

37、常見的內(nèi)參基因表現(xiàn)更穩(wěn)定推薦的基因往往比常見的內(nèi)參基因表現(xiàn)更穩(wěn)定RefGenes RefGenes 訪問路徑：訪問路徑： http:/http:/. .質(zhì)量控制質(zhì)量控制 qPCR qPCR 數(shù)據(jù)的標(biāo)準(zhǔn)化數(shù)據(jù)的標(biāo)準(zhǔn)化towards validating reference gene empirically Roche Applied Science29Reference Gene SelectionReference Gene SelectionnNormalization of gene expression measurements in tumor tissues: comparison

38、of 13 endogenous control genesde Kok JB, Roelofs RW, Giesendorf BA, Pennings JL, Waas ET, Feuth T, Swinkels DW, Span PN.Lab Invest. 2005 85(1): 154-159nReference genes identified in SH-SY5Y cells using custom-made gene arrays with validation by qPCRHoerndli FJ, Toigo M, Schild A, Gotz J, Day PJ.Anal

39、 Biochem. 2004 335(1): 30-41nDetermination of an internal control to apply reverse transcription quantitative PCR to study stress response in the lactic acid bacterium Oenococcus oeni.Desroche N, Beltramo C, Guzzo J.J Microbiol Methods. 2005 60(3):325-33.nEvaluation of potential reference genes in r

40、eal-time RT-PCR studies of Atlantic salmon.Olsvik PA, Lie KK, Jordal AE, Nilsen TO, Hordvik I.BMC Mol Biol. 2005 Nov 17;6:21.nSelection of reference genes for quantitative real-time PCR in bovine preimplantation embryos.Goossens K, Van Poucke M, Van Soom A, Vandesompele J, Van Zeveren A, Peelman LJ.

41、BMC Dev Biol. 2005 5: 27. Roche Applied Science30n檢測模式及應(yīng)用檢測模式及應(yīng)用n對照的設(shè)置對照的設(shè)置n相對定量相對定量 n如何選取看家基因如何選取看家基因n相對定量實(shí)驗(yàn)設(shè)置相對定量實(shí)驗(yàn)設(shè)置n實(shí)驗(yàn)評判標(biāo)準(zhǔn)實(shí)驗(yàn)評判標(biāo)準(zhǔn)主要內(nèi)容主要內(nèi)容Roche Applied Science31相對定量實(shí)驗(yàn)的樣本命名和類型設(shè)定相對定量實(shí)驗(yàn)的樣本命名和類型設(shè)定樣本名樣本名看家基因看家基因 （引物對（引物對1 1）靶基因靶基因A A （引物對（引物對2 2）靶基因靶基因B B （引物對（引物對3 3）Calibrator(Calibrator(對照組樣本對照組樣本)

42、)0 0Reference CalibratorReference Calibrator Target Target CalibratorCalibratorTarget Target CalibratorCalibratorNegative(Negative(陰性對照陰性對照) )NTCNTCReference NegativeReference NegativeTarget NegativeTarget NegativeTarget NegativeTarget NegativeStandard(Standard(標(biāo)準(zhǔn)品標(biāo)準(zhǔn)品1)1)STD1STD1Reference StandardRe

43、ference StandardTarget StandardTarget StandardTarget StandardTarget StandardStandard(Standard(標(biāo)準(zhǔn)品標(biāo)準(zhǔn)品2)2)STD2STD2Reference StandardReference StandardTarget StandardTarget StandardTarget StandardTarget StandardStandard(Standard(標(biāo)準(zhǔn)品標(biāo)準(zhǔn)品3)3)STD3STD3Reference StandardReference StandardTarget StandardTarge

44、t StandardTarget StandardTarget StandardStandard(Standard(標(biāo)準(zhǔn)品標(biāo)準(zhǔn)品4)4)STD4STD4Reference StandardReference StandardTarget StandardTarget StandardTarget StandardTarget StandardUnknown(Unknown(實(shí)驗(yàn)組實(shí)驗(yàn)組1 1樣本樣本) )1 1Reference UnknownReference UnknownTarget UnknownTarget UnknownTarget UnknownTarget UnknownUnk

45、nown(Unknown(實(shí)驗(yàn)組實(shí)驗(yàn)組2 2樣本樣本) )2 2Reference UnknownReference UnknownTarget UnknownTarget UnknownTarget UnknownTarget UnknownUnknown(Unknown(實(shí)驗(yàn)組實(shí)驗(yàn)組3 3樣本樣本) )3 3Reference UnknownReference UnknownTarget UnknownTarget UnknownTarget UnknownTarget UnknownUnknown(Unknown(實(shí)驗(yàn)組實(shí)驗(yàn)組4 4樣本樣本) )4 4Reference UnknownR

46、eference UnknownTarget UnknownTarget UnknownTarget UnknownTarget UnknownUnknown(Unknown(實(shí)驗(yàn)組實(shí)驗(yàn)組5 5樣本樣本) )5 5Reference UnknownReference UnknownTarget UnknownTarget UnknownTarget UnknownTarget UnknownUnknown(Unknown(實(shí)驗(yàn)組實(shí)驗(yàn)組6 6樣本樣本) )6 6Reference UnknownReference UnknownTarget UnknownTarget UnknownTarget

47、 UnknownTarget UnknownUnknown(Unknown(實(shí)驗(yàn)組實(shí)驗(yàn)組7 7樣本樣本) )7 7Reference UnknownReference UnknownTarget UnknownTarget UnknownTarget UnknownTarget UnknownRoche Applied Science32“10+10”“10+10”的加樣方案的加樣方案Primers+Taq Mix(5uL)=10uLPrimers+Taq Mix(5uL)=10uLDNA template+Taq Mix(5uL)=10uLDNA template+Taq Mix(5uL)=

48、10uLSample1Sample1Sample2Sample2Sample3Sample3Sample4Sample4Sample5Sample5引物對引物對A A (Reference)(Reference)引物對引物對B B (Target1)(Target1)引物對引物對C C (Target2)(Target2)引物對引物對D D (Target3)(Target3)引物對引物對E E (Target4)(Target4)Roche Applied Science33nOne to One: One target is paired with one reference accord

49、ing to the pipetting scheme,e.g., T1|R1, T2|R2nAll to All: Each target is paired with each reference,e.g., T1|R1, T1|R2, T2|R1, T2|R2nAll to Mean: Pairs each target with all references. The geometric mean of the resulting ratios is calculated,e.g., T1/R(all) = (T1/R1 x T1/R2)1/2 and T2/R(all) = (T2/

50、R1 x T2/R2)1/2nMean to All: Pairs all targets with each reference. The geometric mean of the resulting ratios is calculated,e.g., T(all)/R1 = (T1/R1 x T2/R1)1/2 and T(all)/R2 = (T1/R2 x T2/R2)1/2LightCycler480 SW1.5 配對法則Roche Applied Science34n檢測模式及應(yīng)用檢測模式及應(yīng)用n對照的設(shè)置對照的設(shè)置n相對定量相對定量 n如何選取看家基因如何選取看家基因n相對定

51、量實(shí)驗(yàn)設(shè)置相對定量實(shí)驗(yàn)設(shè)置n實(shí)驗(yàn)評判標(biāo)準(zhǔn)實(shí)驗(yàn)評判標(biāo)準(zhǔn)主要內(nèi)容主要內(nèi)容Roche Applied Science35實(shí)驗(yàn)評判標(biāo)準(zhǔn)實(shí)驗(yàn)評判標(biāo)準(zhǔn)n特異性nMelting curve analysis for SYBR Green I assaysnNegative controlsn靈敏度和動力學(xué)范圍nDilution series n效率nStandard curven重復(fù)性nReplicates, statisticsRoche Applied Science36Detection Format SYBR Green IDetection Format SYBR Green IConfirm S

52、pecificity by Melting Curve AnalysisAmplificationMelting Curve for Product IdentificationPrimer dimer formation in low concentrationsPrimer design ?Roche Applied Science37Detection Format SYBR Green I Detection Format SYBR Green I Improving Specific DetectionSignal acquisition at elevated temperatur

53、e to avoid detection of primer dimersNote:Primer dimer formation has a negative influence on PCR efficiency.A different primer design might improve the assay.Roche Applied Science38Roche Applied Science39PCR QualityPCR QualityAssay Sensitivity, PCR Efficiency, ReproducibilityAssay Sensitivity = Detection LimitPCR Efficiency = PCR QualityInfluencing Factors:nPrimer design, primer annealingnFragment length, amplicon sequence and GC-contentnPurity of sample, inhibitorsnEfficiency of cDNA synthesisnPCR