PNNL Scientists Improve Quality of Global Transcriptome Studies
Contact: Weiwen Zhang
Contact: Dave Culley
Contact: Fred Brockman
Transcriptome data are only as good as the quality of the input RNA. The completion of a draft genome sequence for the microorganism Methanosarcina barkeri has enabled use of high-throughput technologies such as microarrays and mass spectrometry of proteins to detect global changes that occur in response to experimental treatments. However, because M. barkeri has a complex cell wall, produces a thick outer layer of polysaccharide, and grows in large aggregates of cells rather than as individual cells, standard RNA extraction protocols give incomplete lysis of cells and result in low yields of poor-quality RNA. Under these conditions, transcriptome data could reflect how a minority of cells is responding, rather than how the entire population is responding.
Methanogenic organisms such as M. barkeri are of interest because they are involved in the anaerobic breakdown of organic matter under sulfate-limiting conditions and are essential for recycling carbon compounds and maintaining Earth's global carbon flux. These organisms are the major source of atmospheric methane, an important greenhouse gas that directly contributes to climate changes and global warming. Methanosarcina species have been isolated from such diverse methane-generating environments as mud samples taken from freshwater lakes and the rumen of cattle.
To quantitatively isolate RNA from the entire population of M. barkeri, Pacific Northwest National Laboratory (PNNL) researchers tested modifications of standard RNA extraction methods to optimize efficiency and minimize the number of unlysed cells remaining after extraction. To further test the quality of the resulting RNAs, the researchers performed replicate microarray analyses. The results showed that three modifications were required to quantitatively isolate RNA and yield the most reproducible microarray results for M. barkeri: a liquid nitrogen-grinding pre-treatment, addition of a chaotropic chemical present in the reagent Trizol, and a 6-minute bead-beating treatment.
The results, which will appear in the Journal of Microbiological Methods, make it clear that the methods used to isolate RNA can have a significant impact on the variability, trend, and accuracy of microarray data. In addition, analysis of the microarray results obtained with RNA from the optimized protocol showed that, as expected, the genes involved in methane production (methanogenesis) were among the most highly expressed genes in M. barkeri.
The work was done under PNNL's Biomolecular Systems Initiative. The project team includes Weiwen Zhang, Dave Culley, and Fred Brockman, PNNL; and Bill Kovacik, University of Cincinnati.
Culley DE, WP Kovacik, FJ Brockman, and W Zhang. 2006. "Optimization of RNA isolation from the archaebacterium Methanosarcina barkeri and validation for oligonucleotide microarray analysis." Journal of Microbiological Methods (in press) doi:10.1016/j.mimet.2006.02.014.