Good day.
I would like to extract total RNA using a kit from MRSA and candida for gene expression. i tried sub-culturing standardized inoculum (50ul) with 500ul of my plant extract sub-MIC and 500ul of LB broth. I haven't gotten anything when i checked on nanodrop. is it maybe that my inoculum volume was less or is there any method that is best in preparation of samples for total RNA extraction where you have to have samples treated with extract and the one not treated with extract. and the RNA kit came with DNase I, is it necessary for me to buy another one or that's only for manual method?
Does the kit have separate protocols for gram positive bacteria/fungi?
They're much harder to lyse than gram negative bacteria and mammalian cells and you get very minimal nucleic acid release from detergents/denaturants alone. Usually you need to pretreat them by bead beating them to physically break them or briefly treat them with an enzyme that destroys their cell walls before moving onto chemical lysis steps.
Alexandra Johnson No the kit doesn’t have extra protocol for gram positive. So meaning i can try bead beating before i add my lysis buffer?
Mariah Muhongo Seems like you are mixing two different things. For the nucleic acids (NA) extraction, it does not matter what treatment/experimental design you are using. If your plant extract have suppression property on expression or the whole bacterial growth, obviously there will not be enough biomass to get sufficient NA.
There must be a minimum amount of biomass required for any kit, and if not, you should not expect good results. Also, since you did not mention the name of the kit you are using, any trouble-shooting for the protocol is not possible here. But, follow the protocol and modify as and when needed.
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