Isolation of Escherichia coli mRNA and comparison of expression using mRNA and total RNA on DNA microarrays.
Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3'-termini. We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E. coli poly(A) polymerase I and purifying it by oligo(dT) chromatography. Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon. 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. coli genome. Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA. These and additional studies of genome-wide expression with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E. coli mRNAs.
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720, USA.
V F
VF
Wendisch
D P
DP
Zimmer
A
A
Khodursky
B
B
Peter
N
N
Cozzarelli
S
S
Kustu
D002457:Cell Extracts / Q000032:analysis; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D016680:Genome, Bacterial; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012329:RNA, Bacterial / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification
D002457:Cell Extracts; D012329:RNA, Bacterial; D012333:RNA, Messenger; D007544:Isopropyl Thiogalactoside