Quenching of microbial samples for increased reliability of microarray data.
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 degrees 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.
Pieterse
Bart
B
Wageningen Centre for Food Sciences, Diedenweg 20, 6700 AN Wageningen, The Netherlands.
Jellema
Renger H
RH
van der Werf
Mariët J
MJ
D007778:Lactobacillus / Q000235:genetics; D000432:Methanol; D040081:Protein Array Analysis / Q000379:methods*; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D015203:Reproducibility of Results; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D012333:RNA, Messenger; D000432:Methanol