瘤胃细菌RNA提取,核糖体RNA去除剂反转录方法

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1、Protocol:BacterialRNAextractionandtranscription1.Rumenfluidsamplecollection（1）Rumencontentsweretakenfromeachcowthroughrumenfistulaandwerefilteredwithfourlayersofcheeseclothtoacquiretherumenliquid.（2）Thealiquotsoftheliquidsweredispensedinmicrotubesandfrozeninliquidnitrogenimmediately.

2、2.RNAextraction（RNeasy®PlusUniversalHandbook，Appendix1）（1）ThefrozenrumenfluidsamplesweregrindedusingRetschCryoMill。Theprogramareasfollows：ProgramTimePrecool10minCycles5Runtime2minCooltime2minFrequency28/s（2）Thegrindedrumenfluidsampleswerethendisruptionin1mlQIAzolLysisReagenttogetatot

3、alvolumeof1.5ml。（3）Thehomogenizedsampleswerevortexedfor2mintomixthesamplesandQIAzolwell。（4）Placethetubecontainingthehomogenateonthebenchtopatroomtemperature(15–25°C)for5min.Thissteppromotesdissociationofnucleoproteincomplexes.（5）Add100μlgDNAEliminatorSolution.Securelycapthetubecontai

4、ningthehomogenate,andshakeitvigorouslyfor15s.AdditionofgDNAEliminatorSolutionwilleffectivelyreducegenomicDNAcontaminationoftheaqueousphase,makingfurthertreatmentwithDNaseunnecessary.（6）Add180μlchloroform.Securelycapthetubecontainingthehomogenate,andshakeitvigorouslyfor15s.Thoroughmix

5、ingisimportantforsubsequentphaseseparation.（1）Placethetubecontainingthehomogenateonthebenchtopatroomtemperaturefor2–3min.（2）Centrifugeat12,000xgfor15minat4°C.Aftercentrifugation,heatthecentrifugetoroomtemperature(15–25°C)ifthesamecentrifugewillbeusedinthelaterstepsofthisprocedure.Aft

6、ercentrifugation,thesampleseparatesinto3phases:anupper,colorless,aqueousphasecontainingRNA;awhiteinterphase;andalower,red,organicphase.Fortissueswithanespeciallyhighfatcontent,anadditional,clearphasemaybevisiblebelowthered,organicphase.Thevolumeoftheaqueousphaseshouldbeapproximately6

7、00μl.（3）Transfertheupper,aqueousphase(usually600μl)toanewmicrocentrifugetube(notsupplied).（4）Add1volume(usually600μl)of70%ethanol,andmixthoroughlybypipettingupanddown.Donotcentrifuge.Proceedimmediatelytostep11.Note:Thevolumeoflysatemaybelessthan600μlduetolossduringhomogenizationandce

8、ntrifugation