Prokaryotic RNA preparation methods useful for high density array analysis: comparison of two approaches.
High density oligonucleotide arrays have been used extensively for expression studies of eukaryotic organisms. We have designed a prokaryotic high density oligonucleotide array using the complete Escherichia coli genome sequence to monitor expression levels of all genes and intergenic regions in the genome. Because previously described methods for preparing labeled target nucleic acids are not useful for prokaryotic cell analysis using such arrays, a mRNA enrichment and direct labeling protocol was developed together with a cDNA synthesis protocol. The reproducibility of each labeling method was determined using high density oligonucleotide probe arrays as a read-out methodology and the expression results from direct labeling were compared to the expression results from the cDNA synthesis. About 50% of all annotated E.coli open reading frames are observed to be transcribed, as measured by both protocols, when the cells were grown in rich LB medium. Each labeling method individually showed a high degree of concordance in replica experiments (95 and 99%, respectively), but when each sample preparation method was compared to the other, approximately 32% of the genes observed to be expressed were discordant. However, both labeling methods can detect the same relative gene expression changes when RNA from IPTG-induced cells was labeled and compared to RNA from uninduced E.coli cells.
Rosenow
C
C
Affymetrix Inc., 3380 Central Expressway, Santa Clara, CA 95051, USA. carsten_rosenow@affymetrix.com
Saxena
R M
RM
Durst
M
M
Gingeras
T R
TR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D016680:Genome, Bacterial; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007763:Lac Operon / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*
D018076:DNA, Complementary; D012329:RNA, Bacterial; C022571:adenosine 5'-O-(3-thiotriphosphate); D007544:Isopropyl Thiogalactoside; D001710:Biotin; D000255:Adenosine Triphosphate