Comparison of amplification methods for transcriptomic analyses of low abundance prokaryotic RNA sources

Francois, Patrice; Garzoni, Christian; Bento, Manuela; Schrenzel, Jacques (2007). Comparison of amplification methods for transcriptomic analyses of low abundance prokaryotic RNA sources. Journal of microbiological methods, 68(2), pp. 385-91. Amsterdam: Elsevier 10.1016/j.mimet.2006.09.022

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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.

Item Type:

Journal Article (Original Article)

Division/Institute:

04 Faculty of Medicine > Department of Haematology, Oncology, Infectious Diseases, Laboratory Medicine and Hospital Pharmacy (DOLS) > Clinic of Infectiology

UniBE Contributor:

Garzoni, Christian

ISSN:

0167-7012

ISBN:

17112614

Publisher:

Elsevier

Language:

English

Submitter:

Factscience Import

Date Deposited:

04 Oct 2013 14:59

Last Modified:

04 May 2014 23:17

Publisher DOI:

10.1016/j.mimet.2006.09.022

PubMed ID:

17112614

Web of Science ID:

000244382400026

URI:

https://boris.unibe.ch/id/eprint/25689 (FactScience: 60476)

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