TY  - JOUR
TI  - Purification of Bacterial RNA from Infected Macrophages
AU  - Lobel, Lior 
AU  - Sigal, Nadejda 
AU  - Pasechnek, Anna 
AU  - Herskovits, Anat A.
VL  - 5
IS  - 22
PY  - 2015
DA  - 2015/11/20
SP  - e1660
C1  - Bio-protocol 2015;5:e1660
DO  - 10.21769/BioProtoc.1660
UR  - https://doi.org/10.21769/BioProtoc.1660
AB  - Studying the transcriptome of bacterial pathogens during infection is a very informative and effective tool for discovering genes that contribute to successful infection. However, isolating bacterial RNA from infected cells or tissues is a challenging process due to the much higher amounts of host RNA in the lysates of infected cells. We have optimized a method for isolating RNA of Listeria monocytogenes (L. monocytogenes) bacteria infecting bone marrow derived macrophage cells (BMDM). After infection, we lyse the cells and filter the lysates through 0.45 µm filters to discard most of the host proteins and RNA. Next, we resuspend the bacteria and extract RNA following DNase treatment. The extracted RNA is suitable for gene expression analysis by real-time PCR or microarray. We have successfully employed this protocol in our studies of Listeria monocytogenes gene regulation during infection in vitro (Lobel et al., 2015; Lobel et al., 2012; Kaplan Zeevi et al., 2013; Rabinovich et al., 2012).
KW  - Listeria monocytogenes
KW  - Bacterial RNA extraction
KW  - Intracellular bacterial RNA
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False