Ask: Dr. Metz
Thanks for the questions. Based on my experience at the bench, as well as what I have seen with the thousands of samples we receive here, the following method of isolating total RNA from tissue is the best.
If the tissue is tough (like muscle, mammary gland, etc), it should be pulverized in liquid nitrogen using a mortar and pestle. The resulting powder should be added to Trizol and subjected to homogenization with a Tissue Homogenizer. For soft tissues (like liver or brain), the sample can just be added to Trizol and homogenized.
Then just follow the Trizol protocol with the following points:
- Do not try to get ALL of the aqueous phase – settle for something like 80% of it (300 µl if it is 400µl).
- Do not dry the final pellet after the 80% EtOH wash. Do a quick spin and remove as much liquid as you can with a P200.
- Immediately add 100µl water and resuspend the pellet well. The pellet will be virtually invisible – it will look like a piece of plastic wrap and you might be able to tell it’s there by distortions in the light passing through the tube.
- Do not store the RNA in this stage – Immediately go to next step.
Qiagen Rneasy cleanup:
- I’ve consistently seen nothing but good results with this kit. Samples I’ve seen that have been cleaned up or isolated from other kits (Zymo, Thermo, etc) almost always still have DNA in them.
- The first step of the RNA cleanup protocol is to increase the volume to 100µl. That’s why you resuspend in 100µl above.
- Include the RNase-free DNase set step and follow the instructions exactly as the protocol says (note that the spinning speeds change!).
- If you expect low yields, elute in 30µl water. You can also take the 30µl elutant, place it back in the column and perform a second elution without increasing the volume if you want to try to eke out as much RNA as possible.
- If you expect a normal yield, just elute in 50µl water.
- If you expect a high yield, elute in 30µl water, then add another 30µl water and repeat for a total of 60µl.
For cells, just use the Qiagen Rneasy kit. Scrape the cells off the plate in lysis buffer using a scraper like this:
Just follow the protocol and do the Dnase step as described. Yes, you do need to Dnase treat.
Hope this helps.