Roche公司的RNaseProtectionAssay(RPA)UsingDIG-LabeledRNAProbes下载网址：http://www.roche-applied-science.com/PROD_INF/BIOCHEMI/no1_03/PDF/p22_23.pdf还有一份protocolhttp://www.ualberta.ca/PERINATAL/facilities/pdf/RNase%20Protection%20Assay.pdf可能出现的问题及应对措施1.NoDiscreteBands,OnlySmears－－RNAdegraded,orthetheRNaseDigestionwastooharsh.TryreducingtheconcentrationofRNaseAto1mg/mlordigestforashorterperiodoftime.CheckyourRNAforcondition.2.BandsTooLarge,HighMolecularWeightArtifacts－－TheRNaseDigestionwasinefficientandunabletoeffectivelytrimdowntheRNA:RNAduplexes.TryRNasinglongerorincreasingtheRNaseconcentration./IncompletelylinearizedtemplateDNA.3.BandsintheNegativeControlLane－－InefficentRNaseDigestion(seeabove)/Sensetemplatecontaminatingriboprobe/InsufficentDNasedigestionofriboprobe.4.ManyLowerMolecularWeightBandsUndertheMainBand－－TherecaneitherbeprematurestopsitesintheprobeleADIngtosmallerprobesizes,thereforesmallerproducts.ThiscanalsostemfromoverdigestionbyRNaseAwhichwillbreak-uptheduplexesiftheconcentrationordigestiontimeistoolong.5.NoSignalAtAll:Youdidgenerateanantisenseproberight?
