Dear Stephen,

I would suggest Phenol/Chloroform (pH 5.2) extraction with a previous lysozym treatment. If you want to stick with a kit I can recommend RiboPure from Ambion. After that: DNA digestion and then try to get rid of rRNA with beat based kits (both Ambion). Work always on ice and use special RNA storage solutions (Trisodium citrate, RNase free), freeze on -80 °C. You can maybe try to harvest cells in the log phase due to higher expression activity than in the stationary phase.

Best regards

Ingo

Dear Stephen,

we routinely prep RNA from mycobacteria using bead beating (0.1 mm Zirconium/silica beads, FastPrep machine) in Trizol (Life Tech). We usually get good quality RNA with not too much DNA contamination suitable for qRT-PCR or RNA sequencing. We have prepped RNA form a lot of different growth and stress condition with similar quality. We finally treat the RNA with the TurboDNAfree kit (Ambiot/LifeTech) to remove DNA.

Good luck,

Fredrik

Dear Stephen,

we work in S. epidermidis with the Fast prep machine in combination with the Qiagen RNeasy kit. We do not perform DNase digestion on the column as this step usually does not remove all DNA. We use a separate classical DNase step afterwards. As lysostaphin digests glycin-glycin bridges in S. epidermidis we add glycin to the growth media. In S. aureus glycin should be predominant in the cell wall precursor side chain. However, individual strains might behave differently.

For RNA extraction we use lysostaphin for a short time before Fast prep cell disruption or go directly into the Fast prep machine to avoid influence of the incubation on the RNA.

Best regards

Johannes