I am trying to extract total RNA from bacteria (Salmonella) using the mirVana kit. I am interested in studying the total trasncriptome, inlcuding small RNAs. But after extraction (I elute in ultrapure H2O preheated to 95°C) I obtain only small RNAs, and I don´t see the typical bands for 23s and 16s ribosomal RNAs in the 1% agarose gel. Anyone knows why does this happen? If I do the extraction using RNeasy (Qiagen) I see perfectly the rRNAs bands in the gel (see photo attached. Lanes 1, 2 and 4- RNA obtained with RNeasy, lane 3 RNA obtained with mirVana. 1kb DNA ladder Fermentas, only for approx reference, because I have not RNA ladder). The problem with RNeasy is that it purifies only RNA molecules longer than 200 nucleotides.
But the results are the same (only RNAs of small size, less than 200nt) also when the samples btained with mirVana kit are analyzed with the bioanalyzer. In addition, why the RNAs extracted with RNeasy kit do run well in the agarose gel? (in these cases I see perfectly both bands corresponding to 16 and 23S rRNAs)
Hi Lucia
are you using the first or the second protocol?
if you are using the second you'll get only small size RNA
if you're using the first, are you using the dilution solution with the kit?
I can see traces of the two bands in the third well, therefore I doubted that you used small size RNA protocol.
do you use the same water with RNeasy?, why don't you try using DEPC treated water for dilution?
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