I solved the issue. I usually did not wash cells, before adding trizol, but I observed that if I wash cells with PBS (or HBSS) and I put Trizol, the RNA was no more degraded.

Thank everybody for supporting me.

I have had problems with RNA degradation when extracting from tissues like the liver if it was not immediately snap frozen in LN2 but cell culture is generally unproblematic. Are you using cells in standard cell culture? I usually wash the cells once in PBS to reduce media/serum carryover. Are you using Trizol from Life Tech, another company or home made? I had some issues with another company's Trizol "equivalent" that was resolved by going back to Trizol. Also, how do you know your RNA is degraded? Are you running a gel or do you just assume it is from the lack of RT-PCR product. In the latter case, it could be something in your RT/PCR. Finally, how are the cells treated? I had an entire experiment where I had to use kit purification because the treatment I was using (poly-anionic compound) carried over from the Trizol prep and affected downstream RT/qPCR.

I am using normal cell medium (like DMEM with 10% FBS and PSG) and according to TRIZOL protocol, I remove growth medium and directly add trizol into the plate. But yesterday I tried to wash once with HBSS and then added Trizol and RNA was still degraded but a little bit less. I do not know if it makes any sense! So today I am trying to do more washing steps. I know that RNA is degraded because I am running it on an agarose gel and in parallel I run always a positive control which gives me the canonical 2 bands of RNA ribosomal, while my RNA produces a smear.

I agree that Trizol does not mention washing but I do it anyway and ensure that I get all the PBS off by leaving the plate tilted for 10-15 seconds and then aspirating again. I assume you are avoiding the interphase after the first spin so you just get the aqueous phase don't get genomic contamination as this may affect the appearance on the gel. I error on the side of getting less aqueous phase rather than risking a little interphase contamination. Also, when adding the isopropanol, be sure to invert the tube a few times since it may not mix well following pipetting.

If I think of anything else, I'll let you know.

Good luck!

Thank you very much!

How do you take the samples with Trizol? do you wash the cells with PBS? How long are the cells without medium?. Do you work on ice?

How do you test the RNA integrity?

Please try to add beta-mercaptoethanol to the lysis buffer in 1:100 ratio. This step will help to stabilize the tissue during extraction by killing RNAase. Give a try, I hope it will help. Thanks

RT PCR

Firstly, what are the 260/280 and 260/230 ratios of your RNA suspensions? This could help to know where is the problem from... Secondly, I had exactly the same problem some months ago. I tested all the reagents one by one and nothing seemed contaminated. However, I recovered my previous results by changing the Trizol solution (but you already tried it) AND overall the tubes! Make sure that tubes are certified for RNA extraction... Moreover, I now always store RNA precipitated in ethanol at -80°C and resuspend it just before using it fors tests and retro-transcription. Finally, resuspension is really very important and not so easy to achieve (for example, RNA must be dried but not too much because it decreases solubility). Hope you'll solve the problem and wait for news...

Good luck !

Do you see that happened in one cell line? Have you tried other samples? I used Trizol before and now I switch to use RNAzol RT. I am pretty happy with it. You should try RNAlater as mentioned in earlier post. Hope this will help. Good luck.

I had degradation problems with in vitro synthesized RNA. Sometimes, if you dry the RNA pallet for too long, the degradation may occur. Also, I am guessing you work on ice!!!

Best,

Sagar

To maintain the integrity of your rna samples:

1) mince your desirable rna extracted tissues into small pieces and store at -80

2) If -80 not available , use rna later or store in liquid nirogen tank till tissue processing.

3) work in complete aseptic condition (laminar flow cabinet) which is most important step you should consider

4) store the extracted RNA in -80 also till cDNA biosynthesis process or store it lyopholized at -20

I hope this will satisfy your question

thanks

I used Trizol for RNA extraction of tissue samples. It usually work well. However, quality of RNA depends a lot on handling, environment, samples, etc. Therefore, care must be taken at each steps, from sample handling to storage of the RNA products. Your working bench must be 'cleaned', your apparatus must be RNase free by DEPC treated or 4-6 hrs baked to eliminate RNase. When you disrupt the cell, you should do it as quick as possible. I think pelleted RNA in 70%EtOH stored at -80C is a good condition.

I solved the issue. I usually did not wash cells, before adding trizol, but I observed that if I wash cells with PBS (or HBSS) and I put Trizol, the RNA was no more degraded.

Thank everybody for supporting me.

magnifecent idea I also used this technique ; hogenize using PBS then use trizol than is the most appropriate ay for estraction

If you continue to have these issue after you have tried all the advice above, you may want to switch to column or bead-based extraction protocol. I have abandonned the trizol technique for years now because the 260/230 ratio almost always suggested that there is a solvent carryover which usually need to be addressed depending on your downstream application by doing another cleanup step before using the RNA. Hope this helps.

Now it is working perfectly, thanks

Thank you Gareth, I solved it. The problem was trivial and basically the degradation of RNA seems to happen when the cells are not washed prior trizol.

In my opinion TISSUE DISPERSION must be carried out at the earliest so that the cells do not die slowly on their own.

I am working on a similar issue, as I work on RNA extraction but on whole blood, l can tell you that after finish your extraction you must store it at -80 C ASAP, also not store it for a long time as it is also degraded, it is better stored as cDNA for long time preservation.

I agree with Gareth and Sandip. Be sure to use 10 volumes Trizol to one volume cells. Resuspend pellet in water and perform spectrophotometry with one volume 10mM Tris pH 8. If the Abs 260/280 is less than 2.0, add 1 volume 5M ammonium acetate and spin 15K x g. Recover supernatant, avoiding pellet, precipitate with 2 volumes ethanol to remove carry over RNAses.

will RNA degrade if I put the tissue straight to -80oC without throwing them to Liq-N2?

Thanks

RNAzap is good but did u really wash it with ddh2o after application of  of zap reagent, and secondly if your freeze though cycles are irratic, then thee would be obvious rna degradation hence aliquiting samples are of great importance

Did you use PBS made in DEPC water? Every time I do RNA extraction I get degradation. But I haven't washed the cells with PBS. I am going to try washing with PBS.

I was reading all your interesting comments and I wonder whether you wash the cells with PBS previously deattached of the flasks or during culture. Moreover, if you have the cells in suspension, would a centrifugation in between the washings modify the gene expression?