RNA extraction protocol development for the assay of temporal gene expression in batch-cultured Escherichia coli K-12

Abstract

<p>The sigma S subunit (RpoS) of RNA polymerase acts as the master regulator of stress in <em>Escherichia coli</em>, allowing adaptation and survival under unfavourable conditions such as nutrient deprivation. RpoS regulates and integrates the signals of hundreds of genes (about 10% of the genome) organized into complex networks and modules that govern the response to stress and entry of the cell into stationary phase. Although microarray studies have been performed on starvation models in <em>E. coli</em>, the expression of the RpoS regulon has not been studied in long-term cultures due to the difficulty of isolating RNA from starving cells. In this study, the development of a protocol for isolating RNA from stationary phase cells, employing hot acid phenol without the use of DNase, is described. In addition, the expression of several genes during different phases of growth is analyzed by RT-qPCR in order to validate preliminary microarray data obtained from 24 and 48 hour-old cultures. Although the results obtained by RT-qPCR agree well with the literature, they do not corroborate preliminary microarray data at the 24 and 48 hour timepoint.</p>