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膀胱癌患者算清microRNA实时荧光定量PCR法检测中内参基因的筛选及验证.docx

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膀胱癌患者算清microRNA实时荧光定量PCR法检测中内参基因的筛选及验证 Title:ScreeningandValidationofReferenceGenesfortheDetectionofmicroRNAbyReal-TimeFluorescentQuantitativePCRinBladderCancerPatients Introduction: Bladdercancerisoneofthemostcommonmalignanttumorsoftheurinarysystem.Theearlydiagnosisandtreatmentofbladdercancerarecrucialforpatientprognosis.microRNAs(miRNAs)havebeenshowntoplayimportantrolesintheoccurrenceanddevelopmentofvariouscancers,includingbladdercancer.Real-timefluorescentquantitativePCRisawidelyusedmethodforthedetectionofmiRNAsduetoitshighsensitivityandspecificity.However,accuratequantificationofmiRNAsrequirestheuseofsuitableinternalreferencegenestonormalizetheexpressionlevels.TheaimofthisstudyistoscreenandvalidateappropriatereferencegenesforthedetectionofmiRNAsinbladdercancerpatientsusingreal-timefluorescentquantitativePCR. Methods: 1.Samplecollection:Bladdercancertissuesamplesandadjacentnon-canceroustissuesampleswillbecollectedfrombladdercancerpatientswhoundergosurgery.ThesampleswillbeimmediatelystoredinliquidnitrogenuntilRNAextraction. 2.RNAextractionandcDNAsynthesis:TotalRNAwillbeextractedfromthetissuesamplesusingacommercialRNAextractionkit,andthencDNAwillbesynthesizedusingareversetranscriptionkit. 3.Referencegeneselectionandprimerdesign:Severalcommonlyusedreferencegenes,includingU6,SNORD48,RNU1-1,andRNU6B,willbeselectedforevaluation.SpecificprimersforthereferencegenesandmiRNAsofinterestwillbedesignedusingbioinformaticstools. 4.Real-timefluorescentquantitativePCR:Real-timePCRexperimentswillbeconductedusingafluorescence-basedPCRsystem.TheexpressionlevelsofbothmiRNAsandreferencegeneswillbedetectedsimultaneously. 5.Dataanalysis:Thestabilityofreferencegeneswillbeassessedusingstatisticalalgorithms,suchasgeNorm,NormFinder,andBestKeeper.Themoststablereferencegene(s)willbeselectedfornormalizationofmiRNAexpressionlevels. Results: 1.Evaluationofreferencegenes:Theexpressionlevelsoftheselectedreferencegeneswillbeanalyzedusingreal-timePCRinbladdercancertissuesandadjacentnon-canceroustiss