We are having a hard time isolating RNA from Staph Aureus for use with qPCR. We have had poor success with the Life Technologies purelink RNA isolation kit (which has always worked for us), so we switched to thermofisher's TriReagent. We seem to get a good yield with decent purity, we then DNase treat the samples and make cDNA, followed by qPCR with BioRad's iQ Sybr Green. We are having inconsistent results, most of the time the samples do not amplify properly. Is there a better way to isolate RNA from staph? Perhaps the phenol from the TriReagent is interfering? We are careful not to touch the phenol phase, and yet we still have issues.
Hi,
As you are careful about the phenol..it may not be the real culprit
Few suggestions
1) See the integrity of the RNA even after DNAse I treatment....(there should not be any traces of contamination with RNAse)
2) make sure DNAse I is completely inactivated before further experimentation. Even traces of enzyme left will efficiently degrade the primers and the template..
bye
HI Sally, maybe the QuickExtract RNA solution from Epicentre could be a good option for you. It has been tested with S. aureus with good results. You might find interesting the following forum: http://www.epibio.com/docs/default-source/forum-archive/forum-16-2---rapid-rna-extraction-from-cultured-cells-for-rt-pcr.pdf
RNeasy kit works for me https://www.qiagen.com/us/shop/sample-technologies/rna-sample-technologies/total-rna/rneasy-protect-bacteria-mini-kit
You can use also trizol method of RNA extraction. I think it will work well....Best of luck
As suggested above, use a promega kit along with the lysozyme step (this is mentioned in the promega manual however it can be tricky to find, but will increase yield)
As suggested above, use a promega kit along with the lysozyme step (this is mentioned in the promega manual however it can be tricky to find, but will increase yield)
Isolating mRNA from bacteria is notoriously difficult, probably because it has a half-life that is measured in seconds. My colleagues report good results from environmental samples by treating with RNA Later (sorry, I can't remember the manufacturer), which presumably stops all RNase activity.
I would also recommend adding lysostaphin to your lysis buffer, as lysozyme does not work well on staphylococci.
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