my samples are disrupted cells in RNAprotect + PBS. I have tryed to perform RNA extraction using Trizol but i get litle RNA concentration and pourly clean. Any suggestions about removing the RNA protect? Thank you
Add an excess of TRIzol. For every 0.1 mL of cell pellet lysate, add 2 mL of TRIzol instead of 1 mL. Then proceed with usual TRIzol procedure from there. Double the chloroform addition to 400 uL, then lift 1100 uL of the resulting water layer at that step, add 1 mL of propanol (instead of 500 uL) at that step - and proceed, it should be fine.
Hopefully you have at least 5 million disrupted cells per 100 uL of lysate? If not - lower the final amount of nuclease-free water you add back to the final RNA pellet to 30 uL on the low end. Ideally, you'd add back about 70-100 uL nuclease-free water back to the final RNA pellet...
I recently had problems with this as well. I was using TRI instead of Trizole but it's a very similar protocol. What I ended up doing was just trying it again and again and again until I finally got some good RNA. A few things I did change was using a bio safety hood instead of a chemical hood, increase the culture by 50%, wash everything with ethanol before RNase away, and replacing my reagents. There are so many things that can go wrong that it is hard to say exactly what is happening.
we do a simple sqeezing of cell mass whether animal or plant in sterile kimwipes, remove as many as liquid within the cells and then proceed with kit protocol;
Thank you all for your answers. I have to remark that i do not have a cell pellet: all my cells (ca. 5000 cells) are broken and in ca 1000ul RNAprotect+PBS. I have tried to divide my samples in two (500ul and 500ul) and use Trizol in 1:10, glycogen and dissolving the final RNA in 20ul DEPCH2O, obtaining random values on RNA for my triplicates (50-80ng/ul) which is not enough.
The expected RNA yield from 5000 cells (if mammalian/human) is actually about 7.5 ng/uL (when diluting the final RNA pellet in the 20 uL that you add back at the end and assuming 100% recovery at the rate of 30 pg total RNA/cell). So you are actually getting readings (50-80 ng/uL) that exceed (by about 7 to 11X) what would be expected from this small number of cells. You should try to increase the number of cells you are isolating to ~5 million.
NanoDrop readings in your current range could be a little shaky - so perhaps that is where your 'random' concentration values are coming from with these relatively low RNA concentrations. Where you are getting the 10-fold higher RNA than expected is curious...
If you actually are talking about RNA from 2500 cells, then you are getting anywhere from 13 to 21X more RNA than would usually be expected here...
Thank you Jack. I unfortunately can’t increase the amount of cells because they are sorted cells from a FACS machine. I also assume that the nanodrop is no very accurate at this level of concentration; maybe I should quantify my RNA by pcr. But what intrigues me is that when performing qpcr for these samples (100ng RNA) and GAPDH I obtain to high Ct levels (double than usually for GAPDH from other human cell samples).
100 ng RNA in a (20?)uL [apparently] one-step qPCR is too much RNA: e.g., 5 ng/uL. Adding so much RNA to such a one-step qPCR would inhibit the RT enzyme and/or the DNA-dependent DNA polymerase used. Normally, 1 ng RNA/uL and lower would suffice for the qPCR aspect; even lower RNA amounts for GA3PDH should work well. If you didn't dilute your samples out far enough prior to qPCR, carry-over inhibitors in the RNA samples could have caused this problem as well....
If the Ct value is later than you expect then possibly you have an inhibitor present in that mixture. I'm a fan of mixing experiments to test whether a nucleic acid prep has inhibitors present.
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