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

Hi Renu,

Have you considered RNA/DNA Shield? It can preserve RNA in any sample for up to 30 days at ambient temperature (indefinitely if stored at -80). The nice thing is that you do not need to remove the reagent when extracting the RNA so you eliminate the potential for sample loss (unlike RNAlater). RNA/DNA shield is readily available in a swab format for you sample collection needs.

https://www.zymoresearch.com/pages/sample-collection

For RNA isolation you could try Zymo's Quick-RNA bacterial/fungal kit. It utilizes bead bashing and the isolated RNA is inhibitor-free and ready for all downstream applications including sequencing, RT-PCR, microarray, etc. Again if you collect your samples in RNA/DNA Shield, you can simply transfer them directly from the collection medium into the purification process without removing the reagent.

https://www.zymoresearch.com/collections/quick-rna-fungal-bacterial-kit/products/quick-rna-fungal-bacterial-miniprep-kit

Hope that helps.