Hello, I would like to isolate RNA from RIPA lysed cells treated in vitro. Has anyone done this? Can I just use a kit (Qiagen RNeasy, or other) to perform this or are there additional steps required?
Thanks,
I just successfully did this and wanted to add some info to this useful comment string. I had cells frozen in 100 ul of RIPA buffer, and wanted to extract RNA because my RNAProtect-frozen samples were accidentally discarded. I took 50 ul of the cell/buffer mix, added 300 ul of the lysis buffer from the Qiagen RNeasy kit, and then continued on with the rest of the standard Qiagen extraction protocol. The resulting RNA was good quality and decent quantity, and I successfully used it for qPCR. My PCR results look exactly as I would expect them and comparable to previous similar experiments where RNAProtect was used instead.
Now, my cell samples were frozen at -80 degrees C for only a few days before extraction. But this does seem to be possible should it be necessary.
I've also extracted RNA from a cell pellet frozen dry and stored for 6+ months. In that case the extracted RNA wasn't great, but was still ok for PCR. I wouldn't recommend doing this if you can avoid it.
I also once had cells frozen in media instead of RNAprotect, and thawed them, spun them down, removed the sups and did RNA extraction on the cell pellet. That did NOT work. The freeze-thaw must have lysed the cells, and I probably lost most of the RNA in the sups.
Just trying to help people out who find themselves in similar situations. Science does that sometimes!
Over the years I extracted RNA from pretty much any lysis buffer. As long as the lysis is done on ice, then diluted with 10 volumes of Trizol (or similar), you can get decent RNA out using chloroform/precipitation or column-based methods. Obviously, make sure there is no RNase in the lysis buffer conponents :-)
RIPA has many variations, but most of the ones I know are perfectly suitable. To be on the safe side you can supplement with RNase Inhibitor to manufacturer's recommended concentration (unit definitions are all over the place).
Good luck!
Hello Blake,
I have used RIPA buffer to lysate cells and collect the proteins for western blot for several years.
To obtain good quality RNA for qPCR, sequencing or even microarrays you should do something different. You can use the Trizol approach or the Qiagen kits, like it was already suggested.
Because of results consistence I used the Qiagen kits but the brand is not an issue. A very important matter is the samples preservation. Before extracting RNA I used to store my samples at -80ºC in RNA-later while I was genotyping or collecting more samples to reach the adequate number of samples.
Good luck and beware of RNA degradation
Thanks AG, JN, FZ, I am well-aware how to isolate RNA. The situation is that a large in vitro study was performed and instead of splitting the cells, they were all lysed with RIPA. I would like to isolate RNA from RIPA lysates and not have to redo the whole study. It is my understanding that this is not the first time that RNA has been isolated from RIPA buffer. It is one part consistency (because the westerns are done and the story would be more robust with transcriptional data) and one part laziness.
thanks to everyone for the suggestions.
it is always the best to have a kit specialized for extraction of RNA and protein at the same time but i agree sometimes samples are precious and they already have been processed by RIPA. i did that today and got RNA but not that much as usual below 100ug/ul so i compare the results with my friend extraction of RNA directly from the same samples. i will let you know if expression studies on both samples produce the near similar resuts or not:)
Hi Blake,
Were you successful in extracting RNA from RIPA buffer?
Thank you.
Hello Blake
I am in a similar situation. Did you get good quality RNA from RIPA pellet?
Thank you
I just successfully did this and wanted to add some info to this useful comment string. I had cells frozen in 100 ul of RIPA buffer, and wanted to extract RNA because my RNAProtect-frozen samples were accidentally discarded. I took 50 ul of the cell/buffer mix, added 300 ul of the lysis buffer from the Qiagen RNeasy kit, and then continued on with the rest of the standard Qiagen extraction protocol. The resulting RNA was good quality and decent quantity, and I successfully used it for qPCR. My PCR results look exactly as I would expect them and comparable to previous similar experiments where RNAProtect was used instead.
Now, my cell samples were frozen at -80 degrees C for only a few days before extraction. But this does seem to be possible should it be necessary.
I've also extracted RNA from a cell pellet frozen dry and stored for 6+ months. In that case the extracted RNA wasn't great, but was still ok for PCR. I wouldn't recommend doing this if you can avoid it.
I also once had cells frozen in media instead of RNAprotect, and thawed them, spun them down, removed the sups and did RNA extraction on the cell pellet. That did NOT work. The freeze-thaw must have lysed the cells, and I probably lost most of the RNA in the sups.
Just trying to help people out who find themselves in similar situations. Science does that sometimes!
@ Sepideh Parsi
Thank oyu for your comment.
Can you please tell us if the transcriptomic analysis went well when you used RIPA buffer?
Thanks
Cheers
Niharika
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