High yield extraction of E. coli RNA from human whole blood

Studies of bacterial transcriptomics during bloodstream infections are limited to-date because unbiased extraction of bacterial mRNA from whole blood in sufficient quantity and quality has proved challenging. We addressed the key aspect of this issue and investigated approaches for the lysis of the human blood cells and the stabilisation of bacterial RNA in human whole blood samples containing E. coli cells. We compared four lysis agents: Triton X-100, saponin, ammonium chloride and the commercial MolYsis lysis buffer CM for their ability to yield high amounts of undegraded RNA and investigated their impact on the bacterial gene expression. For low cell numbers the best mRNA yields were obtained if the commercial RNAprotect reagent was added directly to the blood sample without prior lysis of the blood cells. Using this protocol, significant amounts of human RNA were co-purified but did not have a negative impact on qPCR detection of the bacterial mRNA. Importantly, at low concentrations of bacterial RNA, moderate amounts of co-purified human RNA had a positive effect on the yield of bacterial mRNA. Among the tested lysis agents Triton X-100 was the fastest in lysis and reduced the human mRNA background by three to four orders of magnitude compared to stabilisation without prior lysis. Treatment with lysis agents was found to be associated with changes in bacterial gene expression. However, those changes were below 1.5 fold for the genes investigated. This study provides a framework for the choice of the most suitable sample preparation method to study bacterial bloodstream infections.