I just did a total RNA extraction using TRIZOL and the 260/230 values of all samples is between 0.50 and 0.90! I also noticed that for some of them the pellet did not dissolve completely. Also, I can smell phenol in some, although it is a faint odour. How can I purify this? What do I do to dissolve the pellets fully? In the meantime, can I store these RNA tubes at -80C?
Thank you!
Hi Kunal,
More information my friend so that I can try and help. Can you describe your protocol for RNA purification? Let us know exactly what you did, including how many cells you started with, and what the yield was (you told us the purity but not how much).
Without knowing any of that information, your 260/230 information indicates that you have a high amount of contaminants. You must be very careful when using TRIZOL (or during any phenol:chloroform extraction) not to take up any of the interphase/organic phase when you are pipetting off your harvested RNAs. If I have 500ul of aqueous phase, I will pipette off only 400ul to leave behind a safe amount to avoid interphase contamination (watch very carefully by eye as you pipette!).
You should practice with only one sample. Be very careful not to take any interphase, and don't forget to wash your pellet with 75% ethanol. Until you get high yield, high quality (260/280 of ~1.9-2.0) RNAs, you shouldn't do any more multi-sample processing.
See the attached TRIZOL reagent handbook for protocols and troubleshooting tips as well.
Best of luck! Ask any more questions and keep us updated!
Hi, Nathaniel explain you the probably causes of your problem. In general phenol contamination reduce the yield of RNA extraction, and overdried your pellet after ethanol washing can cause problem in the dissolving of the sample.
For answer to your question, if you need to save your samples, this discussion can help you:
https://www.researchgate.net/post/What_s_the_best_way_to_remove_salts_phenol_and_other_contaminants_from_my_RNA_samples
Then, in general you can dissolve the pellet if you put it at 55-60°C, but it is better if you remove first the contaminants. You have to store your RNA at -80°C, and it is better to make aliquotes of your RNA to avoid cycles of freeze and thaw.
Thank you everyone. Yes indeed, Nathaniel. I always take only 400ul of the aqueous phase. The amusing part is that I've done this so many times but this was the first time ever that I got such poor values. I do the following:
Macerate tissue in 1ml Trizol with an electronic homogenizer.
Add 200ul Chloroform and mix.
Centrifuge at 14,500 rpm for 15mins
Transfer aqueous phase to new tube and add equal volume of isopropanol and mix.
Let it stand for 5minutes.
Centrifuge for 20mins at 14,500 rpm.
Discard supernatant and add 60ul 70% ethanol.
Centrifuge for 10mins at 14,500 rpm.
Discard ethanol and air dry pellet.
Resuspend pellet in nuclease free water.
I think you over dried your pellet. I have always heated up my samples to 37 or 42 degrees for a couple minutes after adding the nuclease free water to help get the pellet back into solution. I have a tendency to overdry pellets as well. Good luck
Hi Kunal
As per the value you told for A260/A230, indicating there is polysaccharides contamination. As you also noted there is still smell of phenol means that pellet didn't washed completely and overdried thus not dissolved.
First you should go for the right protocol of homogenization of the tissue you using.
Aspirate only upper aqueous phase without distrubing the interphase so not to try for complete
Aprirate to minimise contamination.
Repeat one more washing of Ethanol to remove phenol contamination.
Not to overdry the pellet and incubtae at 55-60°C for 5 to 10 min to completely dissolve pellet.
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