Premium
Amplification of low quantity bacterial RNA for microarray studies: time‐course analysis of Leptospirillum ferrooxidans under nitrogen‐fixing conditions †
Author(s) -
MorenoPaz Mercedes,
Parro Víctor
Publication year - 2006
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1111/j.1462-2920.2006.00998.x
Subject(s) - biology , rna , regulon , t7 rna polymerase , transcription (linguistics) , dna , microbiology and biotechnology , dna microarray , gene expression , gene , biochemistry , bacteriophage , escherichia coli , linguistics , philosophy
Summary We have developed a method for the amplification of low quantity total bacterial RNA for DNA microarrays analysis. Current methods are based on the linear amplification by the in vitro transcription from the T7 promoter, similar to that used for eukaryotic mRNA amplification. For the incorporation of T7 promoter, the prokaryotic RNA must be enzymatically modified for the incorporation of a polyA tail at the 3′ end to emulate the eukaryotic mRNA. The method we describe and validate herein avoids this step by the direct and random incorporation of the T7 promoter. From 500 ng of total bacterial RNA, we obtained 130–150 µg of antisense RNA, such products being good substrate for fluorescent labelling and DNA microarray analysis. The method was validated with bacterial samples from which it is very difficult to obtain sufficient amounts and quality of total RNA for global gene expression analysis. This is critical for low cell density growing microorganisms, environmental samples, or many extremophiles where the composition of the cultural media severely affects the RNA yield, like in the case of the acidophile and iron oxidizer Gram‐negative bacterium Leptospirillum ferrooxidans . We further validated our amplification method in parallel experiments with non‐amplified RNA by following the expression of the L. ferrooxidans nif regulon along the time‐course of growth.