微阵列技术和转录组测序技术在肺腺癌病人基因表达改变中的应用

TheComparativeMicroarrayandRNA-seqAnalysisofGeneExpressionChangesin10PatientsofLungAdenocarcinomaandItsMatchedAdjacentNoncancerTissuesAbstractDNAmicroarrays,alsoknownasoligonucleotidearray,isaproductofthegradualimplementationoftheHumanGenomeProject(HGP)andtherapiddevelopmentandapplicationofmolecularbiology,whichisinspiredbythewidelyusedofcomputerchip.It——whichconnectedfinancialmicroelectronics,lifesciences,computerscienceandphotoelectrochemicaltogether,developedonthebasisoftheoriginalnucleicacidhybridizationasanewtechnology——isoneofabio-chip.Theprincipleofthistechniqueisintegratedknowngeneprobesequenceonasolidsurface,alargenumberofthenucleicacidsequenceswhicharetestedinbiologicalcellsortissueshybridizedwiththeprobearrayhybridized,bydetectingthepositionofahybridizationprobetoachievegeneticinformationrapidly.TheobjectofRNA-seqisstudyingthesumofallRNAtranscriptomesequencingcomefromaspecificcellinafunctionalstatethatcanbetranscribedout,includingmRNAandnon-codingRNA.RNA-seqisthefoundationandstartingpointofthefunctionandstructureofgenes,throughanewgenerationofhigh-throughputsequencing,toobtainthespecies-specifictissuesororgansinastateofalmostalltranscriptssequenceinformationfullyandquickly,whichhasbeenwidelyusedinbasicresearch,clinicaldiagnosisanddrugdevelopmentandotherfields.ThepurposeofsuchexperimentaldesignistosupposethatweaimtoperformacomparativemicroarrayandRNA-seqanalysisofgeneexpressionchangesin10patientsoflungadenocarcinomaanditsmatchedadjacentnoncancertissues.Pleasedesignastudyonthiscomparativeanalysis.Keywords：Microarray、RNA-seq、lungadenocarcinoma、geneexpressionchanges.MICROARRAYBackgroundAstheadventofmicroarraytechnologysince1995,itisimmediatelyappliedtocancerresearch,andhasmademanyimportantadvances,alsohasbroughthopeandpossibilitiesforrevealingtheoccurrenceanddevelopmentofthetumorultimately.Microarrayhasaverywiderangeofapplications,suchasthedetectionofgeneexpression,DNAsequencing,findinganewgene,thedetectionofmutantsandpolymorphism,drugscreening,diseasediagnosis,andgenelibraries,andthusithasagreatpotentialandvalueabouttheapplicationofcancerresearch,especiallyitshigh-speedproductivityandwealthofinformation,itwillplayanimportantroleindeterminingthetumorriskfactors,earlydiagnosisandtimelytreatmentandprognosis.TheapplicationofcDNAmicroarrayscanfindspecificgeneindifferenttumoranddifferentperiodoftinequickly,andprovideusefulcluesforcancerprevention,diagnosisandtreatment.Material

1.ChipUsingchipswhichhave18mm(64lines),18mm(64columns)spottingareaandcontain5000targetgenesprovidedbyacompany.
2.TissuesamplesTenpatientswithlungadenocarcinomafromtheFirstAffiliatedHospitalofChongqingMedicalUniversity,cancertissueandnormaltissuefarfromthecancerweretakenfromsurgicalspecimens,andremovedinliquidnitrogen,thenmovedto-80℃refrigeratorforRNAextraction.Cancertissueandnormaltissuesamplesweremixedwithrelatedreagentsrespectivelyassamplecuvettes,theaboveRNAsampleswerepreparedforthecancergroupandthecontrolgrouprespectively.Method

1.Pre-hybridizationWeputthewell-preparedpre-hybridizationsolutionandthechipsinawaterbathafewminutesfordegeneration,dryoutthechips,thenaddthedenaturedpre-hybridizationsolutionintotothesamplingareasofthechips,thenputthemintothehybridization-boxfor5to6hforhybridizing.2.LabeledtheprobeWeusethedirectlabelingmethodoftotalRNA.ReversetranscriptasewasaddedatRTreactionsystem,thenaddingthereversetranscriptasebuffer,DTT,dNTPs,then,labelingtheexperimentalgroupwithCy5dCTPinadarkroomafterreversetranscriptasewasadded,labelingthecontrolgroupwithCy3dCTPgroupinthesameway,purifyingDNAbyDNApurificationcolumn,collectingthepurifiedsample,finallyalabeledreagentisadded3.HybridWeaddthehybridizationreagentIinprobetube,mixthemthoroughly,dissolvetheprobe.ThenaddhybridizationreagentII,mixthem,takeoutthechipusedaspre-hybridizationforreserve,degeneratetheprobeandchip.Wetakeoutthechipanddipthemintoethanol,puttheprobeontheiceafterremovingitquickly.Thenweputtheprobeonthechip,placetheminhybridizationcompartment,sealtheminthehybridized-boxovernight.4.WashingWerinsechip.Preparingtwodyeingtankswhichareequippedwithtwokindsofwashingagents,immersethechipintheabovetwodyeingtankwashinginturn,dryandscanthechip.5.ImageProcessingUsingScanArray4000scannertoscantheimage,theimageprocessingsoftwareisGenePixPro3.0.6.DataProcessingScreeningdatawhichtheRatio(cy5/cy3)greaterthan2orlessthan0.5asdifferentiallyexpressedgenesFlowchartPutthepre-hybridizationsolutionandthechipsinawaterbath(degeneration)↓dryoutthechips,addthepre-hybridizationsolutionPutthemintothehybridization-box，5to6h↓addthereversetranscriptaseAddthereversetranscriptasebuffer,DTT,dNTPs↓LabeltheexperimentalandcontrolgroupwithCy5dCTPandCy3dCTPseparately(darkroom)↓purifyingDNA(DNApurificationcolumn)AddthehybridizationreagentIinprobetubeI、II,degeneratetheprobeandchip↓takeoutthechipanddipthemintoethanolPuttheprobeonthechip,placetheminhybridizationcompartment↓sealtheminthehybridized-box,overnightTwodyeingtanks(equippedwithtwokindsofwashingagents)Immersethechipintheabovetwodyeingtankwashinginturn↓dryt