Hi,
We will like to receive advice to improve RNA quality and quantity.
After many trials and calibration, we get a protocol that gives better RNA quality and enough quantity for downstream application (cDNA library preparation for RNA seq).
We work with mouth epithelial cells, and because of the presence of many enzymes in the saliva, it's very difficult to obtain non-degraded RNA (not too much degraded) and enough material.
Today, we have a protocol that gives an RNA output with RIN ~4.5 (tapestation), and ~260 ug/ml (Qubit).
Our Protocol:
Using two swabs (Qiagen) we take mouth cells and throw swabs into a tri-reagent/trizol solution. We load this mixture on a shredder (we try without the shredder step, but the RNA was with lower RIN ~2.5 and 10 times less quantity). Then we load the solution on a column and follow the protocol from the kit direct-zol RNA prep, Zymo.
We try other kits:
-RNeasy (plus) micro from Qiagen, RIN between 1.8 - 2.5 and 1000 - 1500 ug/ml.
-RNeasy micro (and mini) from Qiagen, RIN between 1.8 - 2.5 and 1000 - 1500 ug/ml (similar results).
-Quick RNA prep from Zymo, RIN about 1 and ~500 ug/ml.
We begin with the RNeasy with 1 swab and receive RNA with half concentration.
We also try RNAlatter (Qiagen and Sigma), but the extractions didn't give good results.
Thanks
Laure
I found doing a phase separation step (using chloroform & Maxtract tubes) before loading on to the Zymo column prevented RNA degradation from whole skin biopisies.
First, it is suggested to use suitable swaps to get the largest cell volume. Then we keep the sample at the lowest temperature in the refrigerator to stop the activity of the enzymes (until RNA extraction) and then we use chloroform for the separation steps. Chloroform can also inactivate enzymes. We have a large quantity and quality of pancreatic cells, the pancreas is also rich in RNA-degrading enzymes.
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