Isolation of Escherichia coli mRNA and comparison of expression using mRNAand total RNA on DNA microarrays

Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mR NA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3 ' -termini, We have developed a method to isolate Escherichia coil mRNA by polyadenylating it in crude cell extracts with E. coil poly(A) polymeras e I and purifying it by oligo(dT) chromatography. Differences in lacZRNA le vels were similar with purified mRNA and total RNA in dot blot hydridizatio ns for cultures grown with or without gratuitous induction of the lactose o peron, More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E. coil genome. Comparable signal int ensities were obtained with only 1% as much oligo(dT)-purified mRNA as tota l RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA. These and additional studies of genome-wide expression with DNA microarray s provide evidence that in vitro poly(A) tailing works universally for E. c oil mRNAs, (C) 2001 Academic Press.