Dear colleagues, so the question is in the title... Background: we've spent a few days trying to isolate RNA from an adherent cell culture grown in a 6-well plate. Isolating RNA from various sources has never been a problem for us until now. We just get fully or almost fully degraded RNA, although we've already cleaned everything with RNAseZap, changed plastic, all reagents except Qiazol (the color seems to be normal, and it was properly stored, though it's not new) and even the operator. Please note, that we do trypsinize the cells prior to isolation, as Qiazol (and the alike reagents) contains phenol which should dissolve the plate plastic. Thorough googling has given a hint that this degradation might be due to trypsinization and that some labs seem to add the lysis reagent directly to plastic flasks. There are no direct evidences for that, however. Obviously, this is due to the fact that all manufacturer's protocols state that adding the lysis reagent to the plastic flasks is misbehavior. So could you please tell me if you've ever tested this forbidden procedure? Or maybe you see another source of the problem (although please consider that we've already taken all the necesseary standard decontamiation and anti-RNAse steps). P.S.: To be sure it's not about our DEPC-treated water we dissolved the pellet in pure formamide. Degradation is still on. P.P.S.: isolating RNA from the same source with the Promega SV 96 Total RNA Isolation System works just fine. This procedure does not require cell detachment. However, it worked when done after trypsinization. So maybe our Qiazol is just dead? How can it be? What are the actual symptoms of dead Qiazol/Tri Reagent/Trizol?
I always used TRizol directly to my 6w plate, without trypsinize the cells... never had an issue... I just take my plate, wash PBS, add 1ml Trizol (under the hood for the smell), wait 5mn, collect the sample and transfer to an 1.5ml eppendorf tube and continue the protocol or put my sample at -80*C...
I always used TRizol directly to my 6w plate, without trypsinize the cells... never had an issue... I just take my plate, wash PBS, add 1ml Trizol (under the hood for the smell), wait 5mn, collect the sample and transfer to an 1.5ml eppendorf tube and continue the protocol or put my sample at -80*C...
Hi Peter the wash step with PBS or sodium citrate is critical to obtain good quality RNA. The exopolysaccharides secreted by cells used to attach to surfaces block the trizol action.
Hi Peter, I am using the Triazol/ Qiazol in a similar was as Valentine mentioned:
1) Remove medium
2) Wash cells with PBS
3) Add Triazol/ Qiazol directly to the cells
4) Pipette up and down
5) Transfer into eppendorf tube
and it works!
Direct lysis monolayer of cells for RNA with Trizol reagent, should not cause have any degradation of RNA without tripsin, however, trypin does degrade RNA. Please see reference Anal Biochem. 2014 Oct 15;463:38-44. doi: 10.1016/j.ab.2014.06.017. Epub 2014 Jun 28.
To avoid trypsin and your worry of the dissolving plastic ware, you can use cell scraper, after rinse cells with PBS, scrape all cells off the surface, and then do trizol from there.
For adherent cell RNA extraction, we performed this in the following stepwise protocol:
1.Remove loose cells (planktonic/not attached cells) by 3x shake-washing on a shaker and subsequent discarding of loose cells.
2. Detach cells (attach to glass beads) in Trizol by gentle shaking in the hand.
Note: It is normal to get much lesser RNA in attached cells compared to the planktonic cells.
3. Purify RNA.
4. Check RNA quality.
Trizol works well directly on the dish. You should NOT use Trypsin as this could cause degradation. Also washing with PBS is not recommended. Simply remove the medium and put the Trizol on the cells, follow exactly the recommended protocol. Dependent on the brand of plastic and coating it is possible that you will get some extra peak at 230nm at the Nanodrop, but normally no degradation. Anyway it is possible that somebody managed to contaminate your stock bottle, try a fresh one.
Dear colleagues, thank you all very much! You helped a lot, because first of all we now understand that trypsinizing the cells is a bad, senseless and obsolete idea. Secondly, I see that some of you wash the cells and consider it critical, while the others and some protocols (including the one by Chomczynski and Sacchi) never recommend it.
Now, discussing Christopher's mention, here are examples of protocols stating that plastic be avoided:
Sigma TriReagent: https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/t9424bul.pdf
I've worked with this one previously, not with cultured cells.
Qiagen Qiazol: https://www.qiagen.com/us/resources/download.aspx?id=61c3ddbd-69c1-4b68-ab89-a428f14a9245&lang=en
As you can see in the handbook and the quick-start protocol, the manufacturer avoids any discussion of the cultured cells.
As TriReagent and Qiazol are principally similar, I thought that the limitations of the former apply to the latter.
Considering Trizol (I've never tried this one), I felt like it's all about glass petri dishes of varying diameter, because no classical plastic flasks were specified at all, yet dishes' diameters were listed thoroughly.
Once again - thanks to all of you!
Hi Peter
You can use TRIzol directly. TRIzol Reagent is a ready-to-use reagent.TRIzol works by maintaining RNA integrity during tissue homogenization, while at the same time disrupting and breaking down cells and cell components.
Best of luck
-
Nilesh
Yes, use Trizol directly into the plates and transfer the homogenate into a tube, as the chloroform will dissolve the plastic
Ya,, We also do the same just remove the media, wash 2 times with cold PBS, add trizol, scratch the cells, transfer into 1.5 ml epp.tube and make mechanical destruction of the cells by pippetting seveal times and vortex just for 0 .5 min an dkeep the tube for 5 min in the RT and then you can keep at -80
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