||Improved Method for Recovery of mRNA from Aquatic Samples and Its Application to Detection of mer Expression.
Jeffrey, W. H. ;
Nazaret, S. ;
Von Haven, R. ;
||University of West Florida, Pensacola. Center for Environmental Diagnostics and Bioremediation. ;Lyon-1 Univ., Villeurbanne (France). Lab. d'Ecologie Microbienne.;Environmental Research Lab., Gulf Breeze, FL.
Messenger RNA ;
Aquatic microbiology ;
Bacterial genes ;
Drug metabolic detoxification ;
Genetic transcription ;
Artificial membranes ;
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Previously described methods for exraction of mRNA from environmental samples may preclude detecting transcripts from genes that were present in low abundance in aquatic bacterial communities. By combining a boiling sodium dodecyl sulfate-diethylpyrocarbonate lysis step with acid-guanidinium extraction, the authors improved recovery of target mRNA from both pure cultures and environmental samples. The most significant advantage of the new protocol is that it is easily adapted to yield high recovery of mRNA from 142-mm-diameter flat filters and high-capacity cartridge filters. The lysis and extraction procedures are more rapid than previously described methods, and many samples can be handled at once. RNA extracts have been shown to be free of contaminating DNA. The lysis procedure does not damage target mRNA sequences, and mRNA can be detected from fewer than 10 to the sixth power bacterial cells. The authors used the new method to examine transcripts of genes responsible for detoxification of mercurial compounds. Induction of merA (specifying mercuric reductase) transcripts in stationary-phase Pseudomonas aeruginosa containing Tn501 occurred within 60 s of HgCl2 addition and was proportional to the amount of Hg(II) added. The new technique also allowed the detection of merA transcripts from the microbial community of a mercury-contaminated pond. (Copyright (c) 1994, American Society for Microbiology.)