RNA extraction for the assay of temporal gene expression in E. coli
A story of methodology development
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Although gene expression studies have been performed on Escherichia coli using starvation models, there has been no study of long-term changes in gene expression due to the difficulty of isolating RNA of sufficient yield and quality at late time-points. Furthermore, there is evidence that these starvation models are not physiologically relevant compared to aging cells in batch culture. This document describes the development of a methodology for isolating RNA from aging and starving bacterial cells using organic extraction. Reverse-transcription quantitative PCR is then utilized to measure cha...
Although gene expression studies have been performed on Escherichia coli using starvation models, there has been no study of long-term changes in gene expression due to the difficulty of isolating RNA of sufficient yield and quality at late time-points. Furthermore, there is evidence that these starvation models are not physiologically relevant compared to aging cells in batch culture. This document describes the development of a methodology for isolating RNA from aging and starving bacterial cells using organic extraction. Reverse-transcription quantitative PCR is then utilized to measure changes in gene expression for the purpose of corroborating previous microarray studies. Suggestions for isolating RNA from even older cultures are discussed as well as the limits of statistical analysis of gene expression in aging cell populations. A literature review of the small number of studies attempting to analyze starvation-induced and temporal gene expression in bacteria is also included.
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