Amplification of sense-stranded prokaryotic RNA.
Microarray expression analysis has proven to be a valuable methodology. In eukaryotic systems where RNA is limiting, established protocols for amplification of mRNA, which rely on the poly(A) tails, are well established. In contrast, the difficulty in amplifying prokaryotic mRNA has limited the application of microarrays to microbiology. Here we present a method for the Linear Amplification of Prokaryotic Transcripts (LAPT) that is efficient and unbiased. The overhang tailing activity of Moloney murine leukemia virus reverse transcriptase is used to add the T7 promoter to cDNAs during reverse transcription. The promoter addition is uncoupled from the initial priming event allowing the promoter to be attached to the 5' end of the RNA transcript. This enables the amplification of sense-stranded RNA that is representative of the complexity and distribution of the original transcript pool. In microarray assays amplified prokaryotic RNA (10 ng total RNA starting material) showed good Spearman correlations to an unamplified control sample. Using genome-directed primers to bias addition of a T7-promoter to bacterial transcripts allowed amplification of prokaryotic transcripts in the presence of mammalian RNA (at a eukaryotic/prokaryotic RNA ratio of 500 to 1). This technology should facilitate the study of prokaryotic transcriptomes in situations, such as in vivo studies or mixed microbial populations, where the prokaryotic RNA amount is limited and/or the nontarget/target RNA ratios is high.
Lawson
Jonathan N
JN
Program in Molecular Microbiology, University of Texas-Southwestern Medical School, Dallas, Texas, USA.
Johnston
Stephen Albert
SA
D000818:Animals; D017123:Bacteriophage T7; D017931:DNA Primers; D051379:Mice; D008979:Moloney murine leukemia virus / Q000201:enzymology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D011387:Prokaryotic Cells; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D015010:Yersinia pestis / Q000235:genetics; D015011:Yersinia pseudotuberculosis / Q000235:genetics
D017931:DNA Primers; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase