Northern blot hybridization is a useful tool for analyzing transcript patterns. To get a picture of what really occurs in vivo, it is necessary to use a protocol allowing full protection of the RNA integrity and recovery and unbiased transfer of the entire transcripts population. Many protocols 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, alkaline transfer, and hybridization to riboprobes. Use of this protocol resulted in sharp and intense hybridization signals relative to long mRNAs previously difficult to detect.

Tagliavia, M., Taravella, A., Marineo, S., Puglia, A.M., & La Farina, M. (2010). Optimized RNA Extraction and Northern Hybridization in Streptomycetes. BIOLOGICAL PROCEDURES ONLINE, 2010 [10.1007/s12575-010-9027-7].

Optimized RNA Extraction and Northern Hybridization in Streptomycetes

TAGLIAVIA, Marcello;MARINEO, Sandra;PUGLIA, Anna Maria;LA FARINA, Mario
2010

Abstract

Northern blot hybridization is a useful tool for analyzing transcript patterns. To get a picture of what really occurs in vivo, it is necessary to use a protocol allowing full protection of the RNA integrity and recovery and unbiased transfer of the entire transcripts population. Many protocols 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, alkaline transfer, and hybridization to riboprobes. Use of this protocol resulted in sharp and intense hybridization signals relative to long mRNAs previously difficult to detect.
Settore BIO/19 - Microbiologia Generale
Settore BIO/18 - Genetica
Tagliavia, M., Taravella, A., Marineo, S., Puglia, A.M., & La Farina, M. (2010). Optimized RNA Extraction and Northern Hybridization in Streptomycetes. BIOLOGICAL PROCEDURES ONLINE, 2010 [10.1007/s12575-010-9027-7].
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10447/46855
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