Hi all,
I had a bad experience on extracting total RNA from frozen human brain tissues. The PMI for all tissues is from 3 to 22 hours. I used the Qiagen RNeasy Lipid kit and tissueruptor to homogenize the tissues. But after running the RNA samples on the agarose gel, the 18s and 28s bands are quite blurry and huge degradation is observed. I am afraid the RIN might be below 5.0. I think if the tissues are preserved well, there is higher chance to get good quality RNA from. But is there any way to improve the results? thank you all!
Not an expert in brain tissue deterioration after excision but the first thing I did not like in your protocol is the tissueruptor. This can be cool and efficient or a major contributor to RNA degradation. It depends how quickly you get the tissue with the metal beads into the disruptor.
If you aske around you will find a lot of "old-fashioned" folks who will strongly recommend the use of liquid nitrogen and manual disruption and lysis. Grind the tissue up in a mortar. Should be easy with brain tissue. This raw powder should be kept "super-cold" when transferring and handling to avoid any further degradation of the RNA (from previous improper handling, freezing to late, too slowly etc etc).
Add you lysis buffer and do manual disruption and lysis with a syringe or similar device depending on volume. I say that since the time between the melting of the tissue in for example Qiazol and complete lysis of the cells should be minimized. Otherwise, what was the point of grinding up the tissue so well? Biceps training? Basically: immediately mix and mix and run through the syringe after you initiated tissue melting/defrosting.
If you have the feeling, that you already took care of this issue and my advice is not of relevance, you may have to consider whether your starting material is already messed up pretty well. There is yet no miracle treatment to make degrade RNA whole again during the extraction process. If you have access to mouse brains, use one and immediately freeze or extract RNA even without freezing as your positive control for RNA quality.
As always: RNA work is about speed and temperature. The warmer and slower the conditions the less quality you get. Since you are not in control of the first step of the procedure (the harvesting of the tissue), you only can improve the subsequent steps. But you can check whether the harvesting was improper by using a freshly frozen mouse brain. Does that make sense?
Bad RNA is one of the very frustrating issues in the lab. Controls for your procedure may help you find the culprit of the degradation. That is why scientists invented controls. I mention this from time to time: controls are 50% of the experiments, 40% is labeling, the rest is the actual experiment. Sounds silly, but how often have you encountered hard to interpret results or found racks of samples labeled 1,2,3,4,5...
Good luck.
I agree with all of above. I used to work on brain tissue a long time ago (albeit on HIV DNA not RNA) and the issue of time from PM was always an issue which cannot be resolved. Most degradation probably has started before you have even got to the tissue. I do not think you need a macerator unless you are dealing with large amts of tissue. We had blocks of tissue
Excellent point by Ian. The RNA quality can become almost irrelevant depending what your read-out/assay is. I do a lot of microRNA work and can use highly degraded RNA from FFPE tissues. Does not really matter as long as you control correctly. For mRNA things are different. If you do qRT-PCR you need a lot of normalizers to account for the differences in RNA degradation. Or you only compare similarly degraded material. All not good but as Ian mentioned, sometimes it is very very hard to get good staring material. Ever tried to get whale skin!?
When you run your gel, do you have a positive control, some RNA from cultured cells, where you know it should be pristine? Could be degradation also occurs during the run. Unlikely.
So, I totally agree with Ian. I think he points in the right direction.
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