Ambion microbexpress bacterial mrna enrichment kit

RNAs from WT/EV, sleC mutant/EV, spo0A mutant/EV, spoVAC*/EV, spoVAC*/spoVAC, dpaAB mutant/EV, and dpaAB mutant/dpaAB strains grown for 18 h on 70:30 sporulation medium containing thiamphenicol (5 μg/ml) were extracted for quantitative real-time PCR (qRT-PCR) analyses of the spoVAC, spoVAD, spoVAE, dpaA, and dpaB transcripts. The RNA was extracted using a FastRNA Pro Blue kit (MP Biomedical) and a FastPrep-24 automated homogenizer (MP Biomedical). Contaminating genomic DNA was depleted using three successive DNase treatments, and mRNA enrichment was done using an Ambion MicrobExpress bacterial mRNA enrichment kit (Invitrogen), and samples were reverse transcribed as previously described (40 (link)).

RNA from WT, spo0A^–, sigF^–, spoIIQ^–, sigE^–, spoIIIA^–, spoIIIAH^–, and sigG⁻strains grown for 17 hrs on 70:30 sporulation media was extracted for qRT-PCR analyses of spoIIIAA transcript. RNA from WT, spo0A^–, sigF^–, spoIIQ^–, sigE^–, spoIIIA^–, and sigG⁻strains grown for 25 hr on 70:30 sporulation media was extracted for qRT-PCR analyses of spoVT, CD1430, and spoVAD transcripts. RNA from WT/EV, spo0A^–/EV, spoIIIAA^–/EV, spoIIIA^–/spoIIIA operon, and spoIIIA^–/spoIIIAK167A operon complementation strains grown for 17 hr on 70:30 sporulation media was extracted for qRT-PCR analyses of spoIIIAA transcript. RNA was extracted using a FastRNA Pro Blue Kit (MP Biomedical) and a FastPrep-24 automated homogenizer (MP Biomedical). Contaminating genomic DNA was depleted using three successive DNase treatments and mRNA enrichment was done using an Ambion MICROBExpress Bacterial mRNA Enrichment Kit (Invitrogen). Samples were tested for genomic DNA contamination using quantitative PCR for rpoB. Enriched RNA was reverse transcribed using Super Script First Strand cDNA Synthesis Kit (Invitrogen) with random hexamer primers.