Improving the reliability of transcriptomics data; The effect of quenching on RNA transcription profiles

Bart Pieterse1, Renger H. Jellema2, MariŽt J. van der Werf
1Pieterse@voeding.tno.nl, 1. Wageningen Centre for Food Sciences; 2jellema@voeding.tno.nl, 2. TNO Nutrition and Food Research

Expression studies in micro-organisms are concentrating more and more on the expression of multiple genes or even complete genomes. The possibilities of giving a reliable and representative overview of expressed genes and the levels of their corresponding transcripts under a specific condition are still limited by several biological and technical limitations.

We compared the effect of a methanol-based quenching method for cell harvesting and concentrating in order to limit RNA degradation and de novo synthesis during these procedures. This method has previously been described for the isolation of metabolites from yeast (De Koning and Van Dam, 1992).

In a multidisciplinary study, we compared the effect of this harvesting protocol on the transcription profile of Lactobacillus plantarum WCFS1 with that of two other cell harvesting methods. Principal component analyses of our transcriptomics data already enabled us to distinguish between three different harvesting protocols by the first principal component. Hierarchal clustering of the most significant data points indicates that the applied quenching method minimizes alterations in the transcription profile.


Literature:

De Koning, W. and K. van Dam. 1992. Analytical Biochemistry 204:118-123.