%0 Journal Article
%T Optimized RNA Extraction and Northern Hybridization in Streptomycetes
%A Marcello Tagliavia
%A Anna Taravella
%A Sandra Marineo
%A Anna Puglia
%A Mario La Farina
%J Biological Procedures Online
%D 2010
%I BioMed Central
%R 10.1007/s12575-010-9027-7
%X Northern hybridization, although less sensitive than other methods for RNA analysis, is the only technique providing information about the concentration of specific transcripts among a complex, overlapping RNA population. This information is required in the study of important events of 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, and its efficient and even transfer to the filter.Streptomycetes are largely studied antibiotic-producing soil bacteria, which undergo a complex life cycle characterized by the differentiation of a vegetative and an aerial, spore-producing mycelium. Lysis of these and of other gram-positive organisms is difficult to achieve. Most Streptomyces RNA purification protocols include an initial incubation step during which some RNA degradation pathways active in vivo may keep on going in vitro. This often results in RNA degradation. To overcome these artifacts, an efficient lysis must be promptly achieved and quickly followed by addition of a fully denaturing agent which protects RNA integrity.A second point that must be considered is the loss of low M.W. transcripts occurring when the RNA is purified by column chromatography.Finally, blotting in the presence of a neutral buffer results in a non-efficient transfer of high M.W. transcripts to the filter.Thus, we set up an optimized protocol for studying mRNA processing and decay in streptomycetes which could overcome these limits. This procedure allowed to gain information on the expression of Streptomyces coelicolor dnaK operon, indicating that early published data (see Figure 2 in [1], probably reflected a selective loss of specific transcripts leading to a misinterpretation of what really occurs in vivo.R5 Agar: 103 g/l sucrose, 0.25 g/l K2SO4, 10.1 g/l MgCl2.6H2O, 10 g/l glucose, 0.1
%K streptomycetes
%K total RNA purification
%K RNA processing
%K RNA degradation
%K RNA glyoxylation
%K alkaline blotting
%K northern hybridization
%U http://www.biologicalproceduresonline.com/content/12/1/27