RNA extraction of Escherichia coli grown in Lysogeny Broth for use in RT-qPCR

Herein we describe a method used to effectively extract RNA from the Gram-negative bacterium Escherichia coli grown in Lysogeny Broth (LB). We employ a Proteinase K and enzymatic lysis step to facilitate the release of RNA from bacterial cells. A silica-based membrane extraction kit (Qiagen RNeasy) is used followed by an added DNase step (TURBOTM DNase Kit from Ambion). This kit includes guanidine-thiocyanate in the lysis buffer that inhibits RNases in the cell and in the solution. Ethanol is then added as a wash step and promotes binding of the RNA to the silica membrane column. To eliminate DNA contamination, our protocol includes two DNase treatment steps. In the first step, DNase is incubated with a RNA sample bound to a silica membrane during the extraction process. In the second added step, TURBOTM DNase is added to a RNA sample after extraction has been completed. Subsequently, the enzyme and contaminants are removed by high speed centrifugation. This paper outlines a method of RNA extraction of E. coli for later use in RT-qPCR.