First of all, are you sure you got 5ug/uL based on "RNA concentration in a sample was 5-16µg/µL"? I've worked with cell lines and tissue cells, that is bucket load of RNA that would make me very happy. Did you mean 5 ng instead of ug? That is a factor of 1000 difference.

Regarding the A260 ratios, they just indicate your RNA is (relatively) free of protein/salt/extraction chemical contaminants which some downstream assays could be sensitive to. You can have degraded RNA and still get a clean extraction free of protein and extraction chemicals because the extraction was good.

Lastly, are we looking at human RNA? We don't see sharp 28S + 18S bands which indicate with your RIN values you stated that the RNA seems to be degraded. How was the sample extracted and stored? RNA, unlike DNA is very sensitive to storage. You can leave out DNA on benchtop for a day without a problem, not so much with RNA unless it is in a preservative solution like RNAlater or FFPE etc.

https://worldwide.promega.com/resources/pubhub/methods-of-rna-quality-assessment/

@Can

The concentration was 5 microgram/microliter

Hi, I totally agree with Can Kiessling. You can achieve a relatively clean extraction of degraded RNA. In our lab, we encountered a similar issue with some samples from stranded marine tetrapods. However, without further information regarding your samples and methods, our ability to assist you might be limited. For instance, if you are working with protostome animals or other non-metazoan eukaryotes, you may observe a "hidden break" in the 28S rRNA, requiring careful interpretation of your RIN values.

Natsidis, P., Schiffer, P.H., Salvador-Martínez, I. et al. Computational discovery of hidden breaks in 28S ribosomal RNAs across eukaryotes and consequences for RNA Integrity Numbers. Sci Rep 9, 19477 (2019). https://doi.org/10.1038/s41598-019-55573-1

@Clei

The tissue was rat ovary.

And extraction was done by trizol reagent method. And washing by 75% ethanol...

I've used the Trizol method, that method will yield high quality RNA so i don't think it's a method problem. If your Trizol extraction was a problem, you would probably have low A260/230 and possibly A260/280 too. My first suspect would be the sample tissue, was it fresh/preserved using something like RNAlater? I'm also assuming to rule out RNAse-related problems.

@Can

Tissue was fresh rat ovary which was kept at -80 for 24 hrs and then utilized for RNA extraction. When pellet was obtained I did 2 washes with 75% ethanol and then incubate for 30 min at -20 with 1 ml ice cold solution of absolute ethanol+ ammonium acetate (10:1 ratio). Then again I did 3 washing with ice cold 75% ethanol. After this finally air dry the pellet @ room temperature because I did not getting dry in ice so I dry on room temperature for some time. Then suspend the pellet in 50 microliter RNAse free water.

Hi Mehjabeen Javed, I think your protocol is okay. In our lab, we only use Ammonium Acetate when we have a limited amount of tissue. Typically, we wash the pellet twice with 75% ethanol and then dry it at 40°C. However, we have encountered issues with RNA integrity when the initial incubation (lysis homogenate) exceeds 10 minutes. I would recommend avoiding more than 5 minutes, especially if you are planning downstream analyses such as RNA sequencing (RNASeq).

Best regards,

you likely have RNase contamination somewhere in your process. I recently had to deal with this issue too. autoclaving does not get rid of RNase. you need to wash with soap and water, triple rinse with 18megaohm water then soak 30minutes in 1N NaOH for glassware. We also ordered Obliterase, a spray reagent to clean metal labware and surfaces. if you think that your beakers/tubes/etc are clean - one use items - then ensure that you are not leaving the RNA in trizol too long. tissue needs to be manually homongenized with mortar/pestle, dounce homogenizer, or tissuelyzer first. you may also want to perform a lysis like for protein but add in RNase inhibitor like RNase OUT from Thermo then perform Trizol extraction

@ Julie

It was difficult to homogenise the rat ovary in pestle mortar and also by homogeniser. So I did sonicate that for around 5 min with trizol at 37 degree temperature.

My sonicator did not work temperature lesser than this so fix this temperature.

@Clei

Drying the pellet @40degree will not degrade the RNA?

Mehjabeen Javed I've never had any issues, but you need to be careful not to overdry the pellet. If you're concerned, it's okay to let it dry at room temperature. Based on its results, your RNA has been coming out quite clean from the extraction. I would be more worried about degradation, as mentioned earlier by colleagues Julie and Can.

Best regards

Mehjabeen Javed I would NOT sonicate your sample, this likely sheared your RNA. try grinding with mortar and pestle with liquid nitrogen to cool the mortart and pestle, add Trizol, slowly allow to thaw, then immediately transfer to dounce homogenizer for 33 strokes. could also pass through a 31G needle if you don't have a dounce homogenizer.

I agree with Julie on many of her points. Would also recommend passing through gauge needles, in my PhD student times I used 18 & 21 gauge needles for heart samples that were stored in RNAlater which made them very hard to break down using mortal and pestle. With the needle method, I got good RNA yield (but I can't say you will get good protein for your sample). I used the 18 gauge first which was relatively easy, then moved to 21 which is requires considerable hand-finger coordination and strength. Also, can we be sure that sonication isn't causing your RNA to fragment? Overall, you usually have problems with either sample storage, or sample isolation. If you think your sample was properly stored, then it's likely you are having issues at the sample isolation part. That could be many things, Julie has given good leads to rule out some of them.

@Can

I simply stored the sample in 50 microliter of RNAse free water àt -80.