use of lysozyme in RNA extraction - (Aug/21/2008 )
I have a very low yield of RNA after extracting from S. aureus cells using Ambion bacteria kit. I think I need to use an additional step to break down the cell wall using lysozyme. I have researched this and I need to use 3ug/ml lysozyme (lyophilised powder) made up in TE. This should be added to the cell pellet after centrifugation and digested for 20-40 mins. I got this from Agilant RNA isolation protocol and I am assuming it would be OK to use prior to the method I am using, but I am wondering if addition of the lysozyme will cause the cells to overexpress some genes and produce different levels of RNA (I am using this RNA for RT-PCR). Also, the Ambion kit uses RNAwiz which, apart from breaking down the cell wall, also protects the RNA from RNases. Since the RNAwiz is not added until after the lysozyme step I am worried that during the lysozyme treatment my RNA will be degraded.
My questions are:
-Should I use RNA later for the lysozyme step or will freezing in liquid nitrogen prevent the lysozyme changing the expression of genes? If so, when should I add this/freeze the cells
-Should I use an RNase inhibitor for the lysozyme step? If so, which one and when should I just add it to the TE/lysozyme
I would be grateful for any help anyone can give me. Thanks in advance.
late reply, but anyway.
I would first check this paper.
and personnaly, I would use an extraction procedure that makes use of chaotropic reagents, so you are sure you RNA won't be degraded.