We have tried to isolate RNA from mouse skin samples. We usually use a tissue lyser to homogenize our tissue samples and Trizol to isolate RNA. But for skin samples this doesn't work. If the homogenisation works well than the RNA is not intact after the RNA isolation process. We also tried to homogenize the tissue using mortar and pestle and liquid nitrogen but this doesn't work as the tissue samples are very small. Does anyone have a suitable protocol for RNA isolation from small skin samples?
For small samples, I use a Fastprep. The two main issues in my opinion, especially for RNAse-rich tissues such as skin and pancreas, are 1) to quickly disrupt the tissue in TRIZOL before it has time to thaw (thus don't do too many samples at a time) and
2) to use a sufficient amount of TRIZOL to denature all RNAses.
Thank you for your answer. I think that our problem is the tissue disruption process. We tried to homogenize the tissue at different intensities (Hz). More intense homogisation resulted in higher RNA isolation efficiencies but also in higher RNA degradation (RNA agarose gel). But there was no effect of incubation time on the degradation process. Therefore I don't think that RNase activity is the biggest problem.
I forgot to mention that you should avoid heating while crushing the tissue. Some devices allow you to add dry ice (or precool the rotor) to maintain the tubes at a low temperature while shaking. Alternatively, you can make pulses, separated by breaks, to prevent overheating caused by continuous shaking.
Also, when the amount of material is a problem, like in your mortar/pestle test, you can add a carrier such as acrylamide, glycogen or yeast tRNA to prevent RNA loss during extraction and precipitation.
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