Hello Jawwad,

In my lab, I also do RNA extraction from rodent tissues already stored in TRIzol from the field, and I also use the Qiagen RNeasy kit. You do not need to use liquid nitrogen. With the protocol, RNA purity is as high as 1.9 out of a possible 2.

Its basically as follows:

1. Crush the tissues with beads in 700ul of TRIzol and using a tissue lyser such as the one from Qiagen.

2. Spin for 3 mins at max. speed, RT.

3. Take out the clear top (the tissue debris should be at the bottom of the tube) and transfer it into another tube containing 140ul of chloroform. Mix after adding it.

4. Do a cold spinning - 4oC, 10,000 rpm for 10 mins.

5. You should have three phases at this stage - the bottom with TRIzol, the middle brown phase and the top clear part that should contain your RNA. Transfer the clear phase into another tube containing 600ul of AVL (The lysis buffer in the Qiagen kit. You may add BME to the AVL. It is optional though).

6. Add 500ul of absolute EtOH and then transfer into the spin column, transferring max of 650ul each time.

7. Wash the spin column twice using 500ul of the AW1 and AW2 buffer - 10,000rpm for 1 minute, RT.

8. Add 500ul absolute ethanol and spin 1 minute at 10,000rpm, then 3min at max speed to dry the spin column.

9. Elute RNA in 60ul of the AVE buffer (I add 16ul of Linear acrylamide to the AVE buffer though).

10. Confirm the concentration and purity using NanoDrop.

I hope that is helpful.

Dear Testimony J. Olumade. First I thank you very much for such a detailed protocol. However, I would like to add that I have tried, as a pilot trial, storing samples in TriZol, then pipetting out TriZol and adding RLT Lysis Buffer added with B-mercaptoethanol (Qiagen Kit). Crushed samples in the same tube with a plastic pestle and carried on with the kit protocol. The purity I got was between 1.8 - 2.0.

As you have done phase separation in TriZol, I would like to try that now.

Big Thanks-

Yes you can isolate the RNA from Trizol stored sample by using this kit. We have isolated the RNA and its yield is good. Follow the protocol of manufacturer. The only thing is thaw Trizol sample fastly.

Hey Jawwad Mirza ,

Thank you for informing me of the RLT buffer. Does it come with the Qiagen kit? because I am not familiar with that buffer.

I think I should also mention that I was facing some challenges with extraction using Commercially purchased Phenol. I tried to prepare the water saturated and adopt a protocol using the water saturated phenol and chloroform before adopting the Qiagen protocol. After addition of chloroform and cold centrifugation, there are no phase separations. I wonder is the problem is from my preparation of the phenol or the protocol I am using.

P:S. I prepared the phenol by heating phenol crystals to 65oC in a water bath and adding nuclease-free water then mixing. After allowing to cool, i further added an extra 100ml of water.

Any suggestions will be appreciated.

Sanpreet Singh I thank you for your valuable suggestion.

Dear Testimony J. Olumade , yes the RLT lysis buffer was provided by the Qiagen in the kit and we were advised to add B-mercapto- according to protocol.

The phase separation through phenol-chloroform extraction is a problem for me too. Sometimes, we do not see a clear phase-separated but I took these samples till qRT-PCR step and good satisfactory results.

Based on human psychology, these minor things in a protocol make us feel comfortable with our procedure and possible results. We miss 1 sign (phase separation, unseen pellet, too much pellet, low nanodrop conc. etc) and all our samples go at stake unless tested through different ways (gel electrophoresis, cDNA lib, conventional PCR through house keeping genes etc).

Here I would suggest you, based on my experience, to standardize your initial volume of phenol according to your sample size. Do not be greedy when taking supernatant during phase separation. Lastly, test your final elute with a cDNA reaction after DNase treatment.

I hope all things go best for both of us.

Cheers-

Thank you so much Jawwad Mirza . You have been a blessing.

I will check for the RLT buffer.

I also totally agree wit you. I also got some good qRT-PCR results with the protocol.

Thanks once again.

Best regards.

RLT buffer comes in Qiagen RNeasy Extraction Kit and AVL in the Qiagen QiAmp viral RNA mini kit.