Comparison of amplification methods for transcriptomic analyses of low abundance prokaryotic RNA sources.
Microarrays have established as instrumental for bacterial detection, identification, and genotyping as well as for transcriptomic studies. For gene expression analyses using limited numbers of bacteria (derived from in vivo or ex vivo origin, for example), RNA amplification is often required prior to labeling and hybridization onto microarrays. Evaluation of the fidelity of the amplification methods is crucial for the robustness and reproducibility of microarray results. We report here the first utilization of random primers and the highly processive Phi29 phage polymerase to amplify material for transcription profiling analyses. We compared two commercial amplification methods (GenomiPhi and MessageAmp kits) with direct reverse-transcription as the reference method, focusing on the robustness of mRNA quantification using either microarrays or quantitative RT-PCR. Both amplification methods using either poly-A tailing followed by in vitro transcription, or direct strand displacement polymerase, showed appreciable linearity. Strand displacement technique was particularly affordable compared to in vitro transcription-based (IVT) amplification methods and consisted in a single tube reaction leading to high amplification yields. Real-time measurements using low-, medium-, and highly expressed genes revealed that this simple method provided linear amplification with equivalent results in terms of relative messenger abundance as those obtained by conventional direct reverse-transcription.
Francois
Patrice
P
Genomic Research Laboratory, Service of Infectious Diseases, University Hospitals of Geneva, CH-1211 Geneva-14, Switzerland. patrice.francois@genomic.ch
Garzoni
Christian
C
Bento
Manuela
M
Schrenzel
Jacques
J
D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000592:standards; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D013211:Staphylococcus aureus / Q000235:genetics*
D012329:RNA, Bacterial