荧光定量PCR实验数据分析.ppt

标准曲线-v2.0原始多通道荧光信号-v2.0原始多通道荧光信号-v2.0质控报告-V2.0理想的实验结果可疑的扩增曲线01案例1:是否存在扩增？02一些形状和正常的扩增曲线有区别的曲线，该如何判断呢？03正常的扩增曲线04平台期很低如何判断它们是真正的扩增？这些曲线都有典型的指数增长期，而且和其他的曲线平行（虽然比较短）。如何判断它们是真正的扩增？同时也具有特征性的三个增长阶段。12通常如果模板的起始浓度太低,反应体系会形成大量的引物二聚体。大量引物二聚体的形成使得引物很快消耗完，从而造成扩增曲线的平台期很低。12可能是目标模板的浓度太低。是什么原因造成平台期很低?****Howaboutinthiscase?Atoughercall.Let’sseewhy.Iftheseareallamplifiedusingthesameassay,it’sprettysafetosaythatthecurvesontherightarenotrealamplification.Unlessthesesampleswereextractedinatotallydifferentmanner,suchthattheonesontherightcontainedMUCHmoreinhibitorthanthoseontheright,thenthisisnotrealamplification.Iftheseareallamplifiedusingthesameassay,it’sprettysafetosaythatthecurvesontherightarenotrealamplification.Unlessthesesampleswereextractedinatotallydifferentmanner,suchthattheonesontherightcontainedMUCHmoreinhibitorthanthoseontheright,thenthisisnotrealamplification.Thesuspectcurves(ifindeedtheyareamplificationcurvesatall)areveryclosetothenoise.Assuch,theyarehighlysuspect.Also,remindeveryonethattheYaxisisaLOGscale.Soeverystepupisactuallyanorderofmagnitude.Thismakesthenoiselookalotclosertorealamplificationthanitreallyis.Togetabetterperspectiveonthings,youcanswitchtoaLINEARview.Thereisstillaveryshallowriseinthesuspectsamples,butitdoesn’tappeartobeamplification,asthereisnogeometric(curving)risetothesuspectsamples.Wemaybeseeingsomegradualbreakdownoftheprobe,which,ifithappens,doessoinalinearfashion.Butthereisnoevidenceofamplification.*Jaggedcurvesshouldneverbequantified.Ifabsolutenumbersareneeded,moretemplatemustbeused(iflowtemplateisindeedtheproblem).Also,keepinmindthatquantificationofsampleswhosereplicatesarenottightduetolowcopynumberisalwayssuspect.Rememb