Dear All,
I am looking for a kit to extract total RNA from MTB which I will later use for RT-qPCR. My initial sample would be very low and cannot afford to loose RNA during the extraction. Also, I would really appreciate if someone could suggest a sample collection buffer with RNA preservative to store MTB cells for a longer time before RNA extraction.
Your help is much appreciated!
Renu
We use Qaigen RNA isolation kit for the same and it works well. But we work with H37Ra strain.
Usually we start with just 5 ml of culture which gives us enough yield of RNA ranging from 500ng/ul to 1 ug/ul. But before using that kit, we treat the cells with lysozyme and break the cells using bead beater during the protocol. Hope it will work for you.
Thanks
I used 1µL/µ1g de RNA Later (Ambion 7020) and my samples were stored at -70º and lasted in good condition about 1 year after colection, and used the extraction kit was EZ-10 spin column animal total RNA mini-preps kit BioBasic Inc. (BS82312)
Dear Ingrid,
Thank you for your inputs. I am planning to collect my samples on swabs. Do you think i could immerse the entire swab in around 500ul of RNA later and freeze them? Also, I am not sure how well RNAlater would permeate mycobacterial cell.
Please suggest.
Dear Manoj,
thank you. We have been using lysozyme for DNA MTB extraction and the yield looks good. I was wondering if we need bead beating. Also, what amount of lysozyme would you recommend for RNA extraction?
Thanks for your help!
Renu
Dear Renu
We first resuspend the pellet (from 5 mL of culture) in 1.5 ml of protoblastic buffer (15 mM tris Cl pH=8, 0.45 M Sucrose, 8 mM EDTA) containing 4 mg/mL of lysozyme for 1 hour at 37 degrees. Afterwards, we take again pellets and resuspend in 1 ml of RLT buffer (supplied by qaigen Kit) and add B-mercaptoethanol. Then we transferred the suspension to a eppendorf tubes which contains 25-50 mg of glass beads. Generally we give 5 cycles in bead beater at 3000 rpm for 5 minutes each with intermittent cooling on ice.
Yes, we need bead beater because release of the bulk of RNA is totally dependent on the shearing of the walls which is solely not occurred by just adding lysozyme in case of H37Ra strains. I don't have experience with H37Rv cells. But i heard that H37Rv cells give good yield even for proteins/RNA like M. smegmatis. The above mentioned protocol we have standardize specially for H37Ra strain.
Thanks
Manoj
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