2012-荧光定量PCR-Workflow

©2010,PromegaCorporation.ConfidentialandProprietary.NotforFurtherDisclosure.2012BillSUNTechnicalSalesSpecialistConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.PromegaWorld-WideConnection•Founded1978•Over50distributorsinEurope,Asia,AfricaandtheAmericas•~1,000employeesworldwide•ISO13485certified•ManufacturinginUS,KoreaandShanghai•Over2,000productsforlifescienceresearchandappliedscienceincludingdrugdiscoveryPromegaBioSystemsSunnyvale,Inc.,CAPromegaBioSciences,SanLuisObispo,CAPromegaHeadquartersandManufacturingMadison,WisconsinPromegaBioSystems,Seoul,KoreaShanghaiPromega,ChinaConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.PromegaProvidesOutstandingLifeScienceResearchToolsSystemsBiologyProteomicsGenomicsCellomicsDNARNAProteinCellTissue•CellularAnalysis•ProteinProteinInteraction•DrugScreeningResearchTools•LittleAnimalImaging•Cell-FreeProteinExpression•DNA,RNAPurificationandAnalysis•EpigeneticsResearchToolConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.qPCR实时荧光定量PCR：Workflow反转录定量纯化RNAgDNA定量纯化ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.RNA质量判断1.RNA浓度：1OD260=40μg/ml2.RNA纯度：OD260/OD280≥1.73.RNA完整性：取约1μgRNA，在1%琼脂糖胶中电泳典型的结果如右图所示：ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.RNA纯化方法柱膜法：SVTotalRNAIsolationSystem,Z3100裂解细胞加入异丙醇，震荡5秒准备吸附柱1.裂解物上柱离心；2.DNA酶消化；3.清洗RNA洗脱至DEPC水特点Trizol法柱膜法RNA产量++++++RNA纯度++++++DNA污染+++-RNA完整性++++++操作时间长短RQ1RNase-FreeDNase(Cat.#M6101)ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.RNA反转录——一般注意事项1.扩增前与扩增后的处理分开在不同的区域进行，并使用专用的移液器；2.戴手套操作并经常更换；3.使用带滤芯枪头；4.使用无菌、无核酸酶、低挂壁的管子；5.GoScript™ReverseTranscriptase,GoScript™5XReactionBuffer和PCRNucleotideMix置于冰上融化，勿加热融化；ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.Protocol——GoScript反转录变性退火延伸失活RNA+引物加入RTMixPCR反应或-20/-80℃保存ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.ComponentsAmountExperimentalRNA(upto5µg/reaction)XμlOligo(dT)(0.5µg/reaction)and/orRandomPrimer(0.5µg/reaction)orgene-specificprimer(20pmol/reaction)]XμlNuclease-FreeWaterXμlFinalvolume5μl条件优化：模板RNA与引物引物选择：1.随机引物可以反转录总RNA；OligodT引物只能反转录带polyA尾的mRNA，不能反转录真核生物的部分无polyA尾的RNA（18s等）、原核生物RNA（细菌）；2.加入随机引物+OligodT组合可以提高反转录效率；3.基因特异的引物：与目的基因3’端配对的一条引物；4.引物浓度：OligodT0.01–0.125µg/µl，RandomPrimer0.01–0.1µg/µl，Gene-specificprimer0.5–1µM。模板量：根据待测基因的丰度调整：1pg–5μg。ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.ComponentsAmountNuclease-FreeWater(toafinalvolumeof15µl)XμlGoScript™5XReactionBuffer4μlMgCl2(finalconcentration2.5mM)2μlPCRNucleotideMix(finalconcentration0.5mMeachdNTP)1μlRNasin®RibonucleaseInhibitor(finalconcentration1U/μl)(optional)0.5μlGoScript™ReverseTranscriptase1μlFinalvolume15μl条件优化：RTMix镁离子浓度：1.GoScript™5XReactionBuffer不含镁离子，需要加入试剂盒中提供的25mM镁离子。2.绝大多数的反转录反应，推荐镁离子终浓度2.5mM，即2μl/20μl体系。3.优化镁离子浓度时，建议每0.25mM为一个梯度，在1.5mM-5mM范围内优化。ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.条件优化：RT程序变性：1.推荐70℃，在65℃-75℃范围内优化；2.复杂二级结构RNA升高温度以打破二级结构；长片段RNA降低温度保证RNA质量。3.“warm-start”cDNA合成：复杂二级结构RNA的延伸，可在70℃变性后直接加入RTMix进行42℃延伸。4.低灵敏度可以接受的话，取消变性阶段，直接将RNA模板、引物、与RTMix混合后延伸。退火：延长退火时间至5min可以提高检测灵敏度。延伸：1.延伸时间可以缩短至5min；2.绝大部分cDNA合成在15min内可以完成；3.长于12kb的RNA，可降低延伸温度至42℃-37℃；4.复杂二级结构RNA，延伸温度升高至42℃-55℃；5.延长延伸时间1hr，可提高cDNA的产量；6.将延伸条件（42℃，60min）改为两步可提高反转录效率（如改成37℃1min、50℃1sec、40cycle）。ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.GoScript™ReverseTranscriptionSystem最适温度GoScript™ReverseTranscriptase最适延伸温度37℃-42℃7.5KbtranscriptRNA:oligod(T)cDNA延伸温度37℃42℃ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.GoScript™ReverseTranscriptionSystem复杂二级结构RNA：50℃孵育NocturninLoadingOrder:1-NTC,2-noRTcontrol,3-GoScript,4-CompanyAControls:B2MKanamycinSampleNocturninB2MGoScript21.8±0.416.7±0.3CompanyA24.1±0.516.6±0.4RealTimePCR定量普通PCR，产物跑胶分析复杂结构RNA，可调整孵育温度至50℃-55℃ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.GoScript™ReverseTranscriptionSystem抵抗抑制剂RT0Ethanol(%)0.10.51.051020GoScript+++++++++++++++++++CompanyA+++++++++++++RT0Hematin(mM)1510204060GoScript++++++++++--CompanyA+++++++--低浓度抑制剂存在时，不影响反转录效率！ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.GoScript™ReverseTranscriptionSystem8.9Kb模板.500ng100ng20ng4ng800pg160pg32pgntc长达8.9Kb的mRNA也能高效反转录ConfidentialandProprietary.NotforFurtherDisclosure.ConfidentialandProprietary.NotforFurtherDisclosure.GoScript™ReverseTranscriptionSystem灵敏度：扩增中等丰度基因(LaminA)Real-timeqRT-PCRdetectionofLaminAfromcDNAsynthesizedbydifferentRTs20304010,0001000100101InputCellNumberCy