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Quenching of microbial samples for increased reliability of microarray data

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Author: Pieterse, B. · Jellema, R.H. · Werf, M.J. van der
Type:article
Date:2006
Institution: TNO Kwaliteit van Leven
Source:Journal of Microbiological Methods, 2, 64, 207-216
Identifier: 239127
doi: doi:10.1016/j.mimet.2005.04.035
Keywords: Biology · Biotechnology · Microarray data · Microbial samples · Quenching · messenger RNA · transcriptome · article · cytolysis · gene · hybridization · image analysis · Lactobacillus plantarum · microarray analysis · nonhuman · operon · priority journal · RNA isolation · steady state · technique · Lactobacillus · Methanol · Protein Array Analysis · Reproducibility of Results · RNA, Bacterial · RNA, Messenger · Transcription, Genetic · Lactobacillus plantarum

Abstract

Messenger RNA levels change on a minutes scale due to both degradation and de novo transcription. Consequently, alterations in the transcript profiles that are not representative for the condition of interest are easily introduced during sample harvesting and work-up. In order to avoid these unwanted changes we have validated a - 45°C methanol-based quenching method for obtaining reliable and reproducible 'snapshot' samples of Lactobacillus plantarum cells for transcriptome analyses. Transcript profiles of cells harvested with the quenching method were compared with transcript profiles of cells that were harvested according to two different commonly applied protocols. Significant differences between the transcript profiles of cells harvested by the different methods from the same steady-state culture were observed. In total, 42 genes or operons were identified from which the transcript levels were altered when the cells were not immediately quenched upon harvesting. Among these, several have previously been associated with cold-shock response. Furthermore, the reproducibility of transcript profiles improved, as indicated by the fact that the variation in the data sets obtained from the quenched cells was smaller than in the data sets obtained from the cells that were harvested under non-quenched conditions. © 2005 Elsevier B.V. All rights reserved.