相对定量实验设计及条件优化

1、Roche Applied Science1qPCRqPCR实验设计及实验设计及条件优化条件优化Roche Applied Science2n检测模式及应用检测模式及应用n对照的设置对照的设置n相对定量相对定量 n如何选取看家基因如何选取看家基因n相对定量实验设置相对定量实验设置n实验评判标准实验评判标准主要内容主要内容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通用探针库通用探针库技术要点技术要点n把传统的水解探针由25-35nt减至8-9nt，报告基团用Fluorescein标记，并使用了低背景的BHQ（Black hole quencher）淬灭基团n熔解温度取决于所用的LNA（locked nucleic acid technology，锁定核苷酸）技术

12、核苷的2、4位形成环氧亚甲基桥结构，提高短探针Tm值，使其Tm值高于引物达到qPCR的要求。n其检测特异性由引物和探针共同来决定。n通过网络设计中心选取合适探针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 d

16、esign Probe Finder Software - SpecificityProbe Finder Software - SpecificityRoche Applied Science13Universal ProbeLibraryUniversal ProbeLibraryOnly 165 probes for over 5 billion assays种 属实验数量覆盖率HumanHomo sapiens 639 50099%MouseMus musculus 509 50099%RatRattus norvegicus 364 00098%PrimatesPan troglod

17、ytes 519 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

18、%总总 数数 5 000 000 Roche Applied Science14n检测模式及应用检测模式及应用n对照的设置对照的设置n相对定量相对定量 n如何选取看家基因如何选取看家基因n相对定量实验设置相对定量实验设置n实验评判标准实验评判标准主要内容主要内容Roche Applied Science15ControlsControlsEnable you to understand unexpected results and verify the experimentnNo template control (NTC)contains all components of the PCR

19、reaction 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

20、 controlis 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 reve

21、rse transcriptase 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

22、 in bacteriology/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检测模式及应用检测模式及应用n对照的设置对照的设置n相对定量相对定量 n如何选取看家基因如何选取看家基因n相对定量实验设置相对定量实验设置n实验评判标准实验评判标准主要内容

23、主要内容Roche 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

24、hot stove 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 Science19参比基因的选择n理想的参比基因:n表达水平在所有样品中是持续稳定的：正常组织 vs. 肿瘤组织n表达水平不受实验条件变化的影响：处理样本 vs. 未处理的样本n看家基因(House

25、keeping genes)：编码有基础细胞功能的蛋白质n同一样品中目标基因表达的浓度与看家基因表达的浓度相关；n校正了样品间质量和数量的差异。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-actinmul

26、tigene family; 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 pseudo

27、genes : 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.Roch

28、e Applied 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细胞处理或非处理巨噬细胞处理或非处理Perform an absolute Perform an absolute quantification quantification Selecti

29、on of a 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-gam

30、ma/PBGDMonitor 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

31、a HK Genewith 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 t

32、o identify 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

33、.Roche Applied 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, Dat

34、a obtained 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 Re

35、search samples obtained from subjects undergoing from subjects undergoing chemotherapychemotherapyAMLAML：急性髓细胞白血病：急性髓细胞白血病Roche Applied Science质量控制质量控制 qPCR qPCR 数据的标准化数据的标准化geNorm & NormFinder Roche Applied ScienceSoftwareSoftwarengeNORM:geNORM:nhttp:/ NormFinder: nhttp:/www.mdl.dk/publications

36、normfinder.htm http:/www.multid.se/genex/hs410.htmnbestkeeperbestkeepernhttp:/www.gene-quantification.de/bestkeeper.html27Roche Applied Science没有任何基因的表达是永久恒定的没有任何基因的表达是永久恒定的在每种生物学环境下，总是存在一组基因，其表达量的差异较在每种生物学环境下，总是存在一组基因，其表达量的差异较常见或通用的内参基因更小。常见或通用的内参基因更小。建议选择建议选择 生物学环境特异的内参基因生物学环境特异的内参基因RefGenes RefGe

37、nes 推荐的基因往往比常见的内参基因表现更稳定推荐的基因往往比常见的内参基因表现更稳定RefGenes RefGenes 访问路径：访问路径： http:/http:/. .质量控制质量控制 qPCR qPCR 数据的标准化数据的标准化towards validating reference gene empirically Roche Applied Science29Reference Gene SelectionReference Gene SelectionnNormalization of gene expression measurements in tumor tissues:

38、 comparison 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,

39、 Day PJ.Anal 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 referenc

40、e genes in real-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,

41、 Peelman LJ.BMC Dev Biol. 2005 5: 27. Roche Applied Science30n检测模式及应用检测模式及应用n对照的设置对照的设置n相对定量相对定量 n如何选取看家基因如何选取看家基因n相对定量实验设置相对定量实验设置n实验评判标准实验评判标准主要内容主要内容Roche Applied Science31相对定量实验的样本命名和类型设定相对定量实验的样本命名和类型设定样本名样本名看家基因看家基因 （引物对（引物对1 1）靶基因靶基因A A （引物对（引物对2 2）靶基因靶基因B B （引物对（引物对3 3）Calibrator(Calibrator(

42、对照组样本对照组样本) )0 0Reference CalibratorReference Calibrator Target Target CalibratorCalibratorTarget Target CalibratorCalibratorNegative(Negative(阴性对照阴性对照) )NTCNTCReference NegativeReference NegativeTarget NegativeTarget NegativeTarget NegativeTarget NegativeStandard(Standard(标准品标准品1)1)STD1STD1Referenc

43、e StandardReference StandardTarget StandardTarget StandardTarget StandardTarget StandardStandard(Standard(标准品标准品2)2)STD2STD2Reference StandardReference StandardTarget StandardTarget StandardTarget StandardTarget StandardStandard(Standard(标准品标准品3)3)STD3STD3Reference StandardReference StandardTarget S

44、tandardTarget StandardTarget StandardTarget StandardStandard(Standard(标准品标准品4)4)STD4STD4Reference StandardReference StandardTarget StandardTarget StandardTarget StandardTarget StandardUnknown(Unknown(实验组实验组1 1样本样本) )1 1Reference UnknownReference UnknownTarget UnknownTarget UnknownTarget UnknownTarge

45、t UnknownUnknown(Unknown(实验组实验组2 2样本样本) )2 2Reference UnknownReference UnknownTarget UnknownTarget UnknownTarget UnknownTarget UnknownUnknown(Unknown(实验组实验组3 3样本样本) )3 3Reference UnknownReference UnknownTarget UnknownTarget UnknownTarget UnknownTarget UnknownUnknown(Unknown(实验组实验组4 4样本样本) )4 4Refere

46、nce UnknownReference UnknownTarget UnknownTarget UnknownTarget UnknownTarget UnknownUnknown(Unknown(实验组实验组5 5样本样本) )5 5Reference UnknownReference UnknownTarget UnknownTarget UnknownTarget UnknownTarget UnknownUnknown(Unknown(实验组实验组6 6样本样本) )6 6Reference UnknownReference UnknownTarget UnknownTarget U

47、nknownTarget UnknownTarget UnknownUnknown(Unknown(实验组实验组7 7样本样本) )7 7Reference UnknownReference UnknownTarget UnknownTarget UnknownTarget UnknownTarget UnknownRoche Applied Science32“10+10”“10+10”的加样方案的加样方案Primers+Taq Mix(5uL)=10uLPrimers+Taq Mix(5uL)=10uLDNA template+Taq Mix(5uL)=10uLDNA template+T

48、aq Mix(5uL)=10uLSample1Sample1Sample2Sample2Sample3Sample3Sample4Sample4Sample5Sample5引物对引物对A A (Reference)(Reference)引物对引物对B B (Target1)(Target1)引物对引物对C C (Target2)(Target2)引物对引物对D D (Target3)(Target3)引物对引物对E E (Target4)(Target4)Roche Applied Science33nOne to One: One target is paired with one refe

49、rence according 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

50、(all) = (T2/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 配对法则Roche Applied Science34n检测模式及应用检测模式及应用n对照的设置对照的设置n相对定量相对定量 n如何选取看家基因

51、如何选取看家基因n相对定量实验设置相对定量实验设置n实验评判标准实验评判标准主要内容主要内容Roche Applied Science35实验评判标准实验评判标准n特异性nMelting curve analysis for SYBR Green I assaysnNegative controlsn灵敏度和动力学范围nDilution series n效率nStandard curven重复性nReplicates, statisticsRoche Applied Science36Detection Format SYBR Green IDetection Format SYBR Gree

52、n IConfirm Specificity 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 elevate

53、d temperature 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 synthesisnPC