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ID:18154860
大小:1.05 MB
页数:58页
时间:2018-09-14
《全反式维甲酸对脐血粒单系、红系和淋巴系祖细胞hoxb基因表达的影响》由会员上传分享,免费在线阅读,更多相关内容在教育资源-天天文库。
1、thawedina37°Cwaterbath,dilutedinwarmIMDMmediacontaining10%FBS,washed(×2)withIMDM,andthenpelletedforRNAisolation.TotalRNAwasisolatedbytheguanidiniumthiocyanate/acidphenolmethodusingTrizolreagentinaccordancewiththemanufacturer'sstandardmethod.RNApelletswerewashedwith70%ethanolcontainingdiethylpolyca
2、rbonate(DEPC),airdried,andredissolvedin0.01%DEPCbyheatingat55°C.TheintegrityandpurityoftheRNAwasassessedbyagarosegelelectrophoresis.FollowingthequalityassessmentoftheRNA,aliquotswerequantitatedbyabsorbanceat260and280nm.6.ThemRNAwastranscribedintocDNAbyusingoligoprimer.ThevariableHOXB6genewaschosen
3、toamplifyinvitrorealtimefluorogenicquantitativeinstrumenywithgenespecificpeimerbyFluorogenicQuantitativeReverseTranscriptionPolymeraseChainReaction(FQ-RT-PCR).FQ-RT-PCRwascarriedoutusingTaqManprobe-basedchemistry.Thischemistryprovidesforahighlevelofspecificitythroughthedesignofcomplementaryoligonu
4、cleotideprimersand5'-reporter/3'quencherfluorogenicprobes.DuringthenormalPCRprocessthefluorescentprobeiscleavedbythenative5'-exonucleaseactivityofTAQpolymerasethatreleasesthereporterfromthequencher,resultinginthegenerationofasequence-specificsignal.Thegradingdilutedpositivereservetranscribedproduc
5、tionofHOXB6geneandgluceraldehydephosphatedehydrogenase(GAPDH)genewasusedtofacturetheirstandstandardcurves.ThethresholdofeachsamplewererecordedandusedtocalculatetheinceptcDNAtemplate.Eachadditionalcycleresultsinthereleaseofreportermoleculesfromtherespectiveprobes.Thefluorescenceintensityisrelatedto
6、theinitialnumberofRNAcopies,whichcanbeassessedbydeterminingthethresholdcycle(CT).AllHOX-specificprimersandprobesweredesignedagainstGenBank-publishedsequencesinassociationwithPrimerExpress(AppliedBiosystems).EndogenouscontrolswerepurchasedasRNA-specificPreDevelopedAssayReagents.7.Amplificationswere
7、performedfollowinganinitial2-minuteincubationat55°Ctoallowuracil-N-glycosylase(UNG)todestroyanycontaminatingRNA,followedbytreatmentat94°Cfor10minutestoinactivatetheUNGenzymeandactivatetheDNApolymerase.Thiswasfoll
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