If you have spin columns with you, try this protocol. After the collecting the aqueous phase of the Trizol add equal volume of 96% Ethanol and mix by gentle pipetting. Transfer the mixture to spin column (Any DNA/RNA Extraction kit spin column will work). Incubate for a minute. Centrifuge and wash the column first with 320 microliters of 3M Sodium acetate and then with 400 microliters of 70% Ethanol. Centrifuge at full speed for 1 min to dry the column and then add 50 microliters of pre-warmed Rnase-free water to the center of the column and incubate for 3 mins. Centrifuge to elute.

Unfortunately this RNA looks very degraded. The heavy blob at the bottom is low molecular weight RNA fragments and these exceed the intensity of the 18S and 28S bands several fold, so most of your RNA is fragmented. If you tell us more about your sample type and processing we might be able to help you work out why this happened.

I agree with laura but it depends on what you want to do with it

Like Laura said your dominant species is degraded but you do have a small amount of undegraded RNA as indicated by intact ribosomal subunits

Thus for qualitative RT PCR and say sequencing in my experience that would work

However for qualitative work where you are measuring gene expression - say real time PCR - I would not use that RNA prep

Try and figute out the source of your degradation

I might be able to help with details of extraction etc

Dear Laura Leighton , Thanks a lot for your important suggestions. My sample is fish intestine and I am using trizol reagent and following their kit protocol for RNA extraction. I am not sure why this is happening recently with my sample. Any kind of suggestions is much appreciated. Thanks a lot again.

Dear Syed

Attach your protocol and i will take a look and comment some more

Dear Laurence Stuart Dawkins-Hall , thanks a lot for your valuable suggestions. I am using trizol and following their kit protocol for RNA extraction. I have one question for you. During RNA wash with alcohol should I vortex my RNA pellets?

The purpose of the alcohol wash is to remove contaminants (mostly phenol and some salt) and most of that is actually on the sides of the tube. You might get more pure RNA if you are really thorough with the wash steps (eg vortexing the pellets) but that probably won't make a difference to RNA integrity.

You said happening recently - have you previously been extracting RNA from the same sample type and using the same protocol that was not degraded? How do you store tissue before extracting RNA from it?

Dear Syed

A brief vortex will more effectively desalt

One you have vortexed in 70% ethanol, respin your tube for 5 min at full speed before removing alcohol

Dear Laurence Stuart Dawkins-Hall , Thank you for the information. I have attached my RNA extraction protocol. Let me know if you have any comments or suggestions. Thanks in advance.

Dear Laura Leighton , Thank you again for the information. Previously I also used the same sample and same protocol but did not have degradation. I have attached an image for previous RNA extraction example.

I put 800-1000 ul trizol in 2ml tube and put the sample immediately in trizol after sampling and then to liquid nitrogen immediately. Then I put the sample in -80C freezer.

I want your suggestion on how to air-dry the RNA pellet. How do you do it normally? Thank you in advance.

Dear Syed. I will. I have doing this since 1988

Dear Laurence Stuart Dawkins-Hall, so kind of you :)

Dear Syed

Just looked at your protocol

In principle standard and ok but could be finessed to reduce contaminants which might contribute to your RNA degradation. For example:

1. Make sure your lysis solution is pre cooled and perform initial lysis on ice

2. Having added trizol to your lysate, vortex for 30 sec to 1 minute. Shaking for 15 second is not enough mixing

3. Having removed the upper aqueous phase perform a second chloroform extraction

4. Remove upper aqueous phase and add that to isopropanol

5. When you spin down DNA from iso ppt remove supernatant and add back 1 ml of room temp 70% ethanol.

6. Vortex for 20 seconds

7. Spin for 5 minutes to recover pellet

Etc

Hope that helps!

If you have spin columns with you, try this protocol. After the collecting the aqueous phase of the Trizol add equal volume of 96% Ethanol and mix by gentle pipetting. Transfer the mixture to spin column (Any DNA/RNA Extraction kit spin column will work). Incubate for a minute. Centrifuge and wash the column first with 320 microliters of 3M Sodium acetate and then with 400 microliters of 70% Ethanol. Centrifuge at full speed for 1 min to dry the column and then add 50 microliters of pre-warmed Rnase-free water to the center of the column and incubate for 3 mins. Centrifuge to elute.

Your protocol looks fine to me. I would guess that the degradation is happening early; before you actually do the RNA extraction - there is still quite a bit of degraded RNA visible in your good gel even though the 18S and 28S bands look much clearer. What type of sample is this? For storage of samples frozen in Trizol, they need to be fully homogenised before storage. If you are dropping a piece of tissue or similar into Trizol and then freezing that slowly (ie by putting it into a freezer, rather than into a dry ice bath or liquid nitrogen) then the middle of the tissue chunk is getting chewed by RNases.

For the actual protocol I agree with Laurence's suggestions. I always use chilled trizol when I can, and vortex samples to mix. For drying pellets, I find it really helps to spin the tubes a second time. Eg: Centrifuge final alcohol wash, remove supernatant (~500uL). Spin tubes again for 10-30 seconds in benchtop quick-spin centrifuge to pull down the droplets of alcohol from the sides of the tube. Use smaller pipette to remove supernatant (~20uL). This lets them dry much faster and more evenly. Watch for the pellet to begin to change colour as it dries (from white to clear) - when it is clear around the edges, add water. If you over-dry the pellets they won't redissolve as easily and you might have to use heat to dissolve the RNA (which can damage it.)

You can use RNAlater for storage of your specimen. You can place your specimen directly into RNAlater and no homogenization is required. Just use smaller amounts of your specimen. I do agree with Laura Leighton but the problem I faced during homogenization in trizol is that it creates a mess and will eventually lead to Rnase contamination, as Rnases are abundantly found in our environment. You can also use Rnase zap to clean your work area before proceeding with your extraction. After collecting the aqueous phase of Trizol, just use a spin column based approach for washing and drying, as it will be hassle free and you wont be loosing your RNA during removal of your supernatant. However, the protocol suggested by Laura Leighton and Laurence Stuart Dawkins-Hall is the appropriate way for RNA extraction, I am just providing an easy alternative.

Dear Syed

A couple months ago I had the same problem as you, so I tried to change the protocol stage by stage, I suggest adjust the below stages and see how is the results:

6. add 800u isopropranol and incubate for 20 minutes at -20 (overnight incubation is the best)

7. centrifuge 12000 for 10min -4

try to wash for two times or more

I believe air-dry is a critical stage, note that extracted RNA shouldn't be dried completely. Moreover, the extra amount of liquid could increase the contamination. For more information, I extracted RNA from rat ovarian and uterine tissues and the results were perfect.

good luck.

Dear Mohammad Samare-Najaf , thanks a lot for your suggestion. Another person also suggested me to do keep the sample in -20C for overnight after adding isopropanol. I will try to do this and see if it actually helps my situation. For overnight, how many hours do I need to keep it in -20C?

Thank you.

Dear Navonil Gogoi , Thanks a lot for your suggestions.

Dear Laurence Stuart Dawkins-Hall , Thanks a lot for your suggestions.