分段书写3mm d pcr颅底软骨细胞分离和培养

WesternBlot分析COL2A1RNATotalRNAwasextractedfromCBSCsusingTrizol(TaKaRa,Japan)accordingtothemanufacturer’sspecifications.TheyieldofRNAwasdeterminedusingaNanoDrop2000spectrophotometer(ThermoScientific,USA),andtheintegritywasevaluatedusingagarosegelelectrophoresisstainedwithethidiumbromide.ficationwasperformedwithatwo-stepreactionprocess:reversetranscription(RT)andPCR.EachRTreactionconsistedof0.5μgRNA,2μlofPrimerScriptBuffer,0.5μlofoligodT,0.5μlofrandom6mersand0.5μlofPrimerScriptRTEnzymeMixI(TaKaRa,Japan),inatotalvolumeof10μl.ReactionswereperformedinaGeneAmp®PCRSystem9700(AppliedBiosystems,USA)for15minat37℃,followedbyheatinactivationofRTfor5sat85℃.The10μlRTreactionmixwasthendiluted×10innuclease-waterandheldat-20℃.Real-timePCRwasperformedusingLightCycler®480ⅡReal-timePCRInstrument(Roche,Swiss)with10μlPCRreactionmixturethatincluded1μlofcDNA,5μlof2×LightCycler®480SYBRGreenIMaster(Roche,Swiss),0.2μlofforwardprimer,0.2μlofreverseprimerand3.6μlofnuclease-water.Reactionswereincubatedina384-wellopticalte(Roche,Swiss)at95℃for10min,followedby40cyclesof95℃for10s,60℃for30s.Eachsamplewasrunintriplicateforysis.AttheendofthePCRcycles,meltingcurveysiswasperformedtovalidatethespecificgenerationoftheexpectedPCRproduct.TheprimersequencesweredesignedinthelaboratoryandsynthesizedbyGenerayBiotech(Generay,PRC)basedonthemRNAsequencesobtainedfromtheNCBIdatabase(Table1)TheexpressionlevelsofmRNAswerenormalizedtoGAPDHandwerecalculatedusingthe2-ΔΔCtmethod(LivakandSittgen,2001).表3各扩增使用的引物序列、循环数和产物大Table3NucleotidesequencesoftheprimersandprobesusedforRT-产物大小代及传代过程中软骨相关Col1a1，Col2a1，Col10a1及Sox9的表达量变化。液，吹打至清亮，转移至EP1mlTrizol200ul15s，常温静15min；412000g15min500ul，颠倒混匀，10mi（RNA提取效率℃12000g10min，RNA1ml75%乙醇（DEPC水配制混匀。4℃7500g离心5min，去上清，样品干燥5-10min，用RNA-水（DEPC水1：1000稀释）20-50ul30minNanoDrop2000分光光度计测定浓OD260/OD280RNA完整性。转录反应完全后，85℃5s终止反应。逆转录完毕后加入90µlNuclease-H2O稀释至100µl在-20℃冰箱，用于后续实验。引物设计：引物采用RocheLCPDS2软件设计并由捷瑞生物工程合成，各扩增使用的引物序列、循环数和产物大小见表3。PCRLightCycler®480SYBRGreenIMasterLightCycler®4802×LightCycler®H2O，3.6μl。PCR程序：95℃10min；95℃10s，60℃30s，40个循环。循环结束后利用熔解曲线检测产物特异性，从60℃缓慢升温至97℃，每℃5次荧光信号。相对表达量，ΔCt=Ct目的-Ct内参，ΔΔCt=ΔCtP1-P4样品-ΔCtP0样品，相对表达量=2-ΔΔCt。应用test单因素方差分析（One-wayysisofvariance，ANOVARNATotalRNAwasextractedfrom(inputthetypeofyoursamples)using(inputthekitusedforRNAextraction)accordingtothemanufacturer’sspecifications.TheyieldofRNAwasdeterminedusingaNanoDrop2000spectrophotometer(ThermoScientific,USA),andtheintegritywasevaluatedusingagarosegelelectrophoresisstainedwithethidiumbromide.ficationwasperformedwithatwo-stepreactionprocess:reversetranscription(RT)andPCR.EachRTreactionconsistedof0.5μgRNA,2μlofPrimerScriptBuffer,0.5μlofoligodT,0.5μlofrandom6mersand0.5μlofPrimerScriptRTEnzymeMixI(TaKaRa,Japan),inatotalvolumeof10μl.ReactionswereperformedinaGeneAmp®PCRSystem9700(AppliedBiosystems,USA)for15minat37℃,followedbyheatinactivationofRTfor5sat85℃.The10μlRTreactionmixwasthendiluted×10innuclease-waterandheldat-20℃.Real-timePCRwasperformedusingLightCycler®480ⅡReal-timePCRInstrument(Roche,Swiss)with10μlPCRreactionmixturethatincluded1μlofcDNA,5μlof2×LightCycler®480SYBRGreenIMaster(Roche,Swiss),0.2μlofforwardprimer,0.2μlofreverseprimerand3.6μlofnuclease-water.Reactionswereincubatedina384-wellopticalte(Roche,Swiss)at95℃for10min,followedby40cyclesof95℃for10s,60℃for30s.Eachsamplewasrunintriplicatefor ysis.AttheendofthePCRcycles,meltingcurveysiswasperformedtovalidatethespecificgenerationoftheexpectedPCRproduct.TheprimersequencesweredesignedinthelaboratoryandsynthesizedbyGenerayBiotech(Generay,PRC)basedonthemRNAsequencesobtainedfromtheNCBIdatabaseasfollows:(inputyourprimerTheexpressionlevelsofmRNAswerenormalizedto(inputthereferencegene,e.g:GAPDH,ACTB)andwerecalculatedusingthe2-ΔΔCtmethod(LivakandSittgen,TotalRNAwasisolatedusingTRIzolreagent(Invitrogen,Carlsbad,CA)accordingtothemanufacturer’sinstructions.Afterreversetranscriptionreaction,realtimereversetranscription-polymerasechainreaction(PCR)wasperformedbyaRoc