The biggest problem with s aureus is breaking open the cell wall. A lot of methods recommend some kind of enzymatic digestion (lysostaphin/lysozyme) but I am not personally fond of adding a 30 minute incubation at 37C to my protocol (especially for gene expression analysis). I recommend using some kind of glass bead method. I have personally had excellent result using the FastPrep instrument in combination with Trizol. The instrument is relatively expensive but a good investment if you plan on doing many RNA preps in the future.

You may want to try the Machery-Nagel Nucleospin RNA kit (http://www.mn-net.com/tabid/1324/default.aspx) if you can get it. I haven't tried it personally but have often had better results with Machery-Nagel kits than with Qiagen kits. This paper (http://www.sciencedirect.com/science/article/pii/S0378111911007591) seems to like the kit and proposes a few alternate methods.

The biggest problem with s aureus is breaking open the cell wall. A lot of methods recommend some kind of enzymatic digestion (lysostaphin/lysozyme) but I am not personally fond of adding a 30 minute incubation at 37C to my protocol (especially for gene expression analysis). I recommend using some kind of glass bead method. I have personally had excellent result using the FastPrep instrument in combination with Trizol. The instrument is relatively expensive but a good investment if you plan on doing many RNA preps in the future.

The answer is simple and that is use higher quantity of biomass. Nothing is wrong with those kits you mentioned, unless these have become outdated.

I agree with Jesper, breaking open the cells could be a limiting factor there, but as Jai said, what about increasing the amount of cells? A cheap method also would be just boiling, centrifuge, phenol/chloroform, add DNAases to get rid of the DNA? At the end you can concentrate using a speed-vac.. So you can run multiples tubes of the same sample and concentrate them into one.

I also agree with Jesper, using glass beads (

Hi, I was happy with the method I used during my postdoc. I will link to the paper.

http://www.ncbi.nlm.nih.gov/pubmed/23963175

Thank you all for your suggestions. I have tried using trizol and phenol: chloroform, in the second method, incubated the cell suspension at 95C for 10 min and got RNA with high amount of DNA. I can not increase the biomass, since i am infecting the epithelial cells with 20 MOI of organism.

Hi Nithya

Following phenol/chloroform extraction try precipitating with LiCl (http://www.lifetechnologies.com/dk/en/home/references/ambion-tech-support/rna-isolation/general-articles/the-use-of-licl-precipitation-for-rna-purification.html). This specifically precipitates only RNA and not the DNA. Alternatively, treat your samples with a DNAse prior to phenol extraction.

Jesper

As Jesper said in his comment it all comes down to how you break open the bacteria and the only method that will work for S. aureus beadbeating/fastprep. As for the genomic DNA contamination, all RNA preps, no matter which isolation protocol you use, will have some degree of genomic DNA contamination. To remove it try using a DNase like the Ambion Turbo DNA free kit. This works very well with S. aureus RNA preps for qPCR and RNA-seq.

if using fast prep 24 instrument for breaking cell wall of S. epidermidis, what is the cycle time, pulse and time interval? Is it essential to cover the holder with dry ice? I covered the samples with dry ice but it was frozen.  can anyone please help me with this. Also i m using Qiagen RNeasy Mini kit. 

Hi Murugesan! Protocols on fast prep can vary accord to your organism, amount of cells, etc. I suggest you make some tests... 4 or 6 cycles (4 - 5 m/s, 30s - 1 min), intercalating with ice bath ( 1 min). After that, continue the protocol of the kit (RNA Cleanup with DNA digestion).

Thank you Isabela bonfim