Optimized RNA Extraction and Northern Hybridization in Streptomycetes 英文参考文献.docVIP

Optimized RNA Extraction and Northern Hybridization in Streptomycetes 英文参考文献.doc

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Optimized RNA Extraction and Northern Hybridization in Streptomycetes 英文参考文献

BiolProcedOnline(2010)12:27–30 DOI10.1007/s12575-010-9027-7 OptimizedRNAExtractionandNorthernHybridization inStreptomycetes MarcelloTagliaviaAnnaTaravellaSandraMarineo AnnaMariaPugliaMarioLaFarina Received:16December2009/Accepted:6February2010/Publishedonline:17March2010 #TheAuthor(s)2010.ThisarticleispublishedwithopenaccessatS Abstract Northern blot hybridization is a useful tool for analyzingtranscriptpatterns.Togetapictureofwhatreally occursinvivo,itisnecessarytouseaprotocolallowingfull protectionoftheRNAintegrityandrecoveryandunbiased transferoftheentiretranscriptspopulation.Manyprotocols suffer from severe limitations including only partial protection of the RNA integrity and/or loss of small sized molecules. Moreover, some of them do not allow an efficient and even transfer in the entire sizes range. These difficulties become more prominent in streptomycetes, where an initial quick lysis step is difficult to obtain. We present here an optimized northern hybridization protocol to purify, fractionate, blot, and hybridize Streptomyces RNA. It is based on grinding by a high-performance laboratory ball mill, followed by prompt lysis with acid phenol-guanidinium,alkalinetransfer,andhybridizationto riboprobes. Use of this protocol resulted in sharp and intense hybridization signals relative to long mRNAs previouslydifficulttodetect. 1Introduction Northern hybridization, although less sensitive than other methodsforRNAanalysis,istheonlytechniqueproviding information about the concentration of specific transcripts among a complex, overlapping RNA population. This informationisrequiredinthestudyofimportanteventsof gene expression regulation, such as RNA transcription termination, processing, and degradation. The quality of a northern hybridization protocol depends on three main points: protection of RNA integrity, unbiased recovery of the entire transcripts population,andits efficientandeven transfertothefilter. Streptomycetes are largely studied antibiotic-produc

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