We had some phenol and guanethidine contamination in our RNA samples, providing an inadequate A260/230 ratio. Any tips on how can I overcome that? Actually, how could I reprocess my diluted RNA to eliminate contamination and obtain a better ratio? Thank you in advance!
If your downstream application is reverse transcription, here is a quick protocol for RNA purification:
1. Adjust the volume of your extraction for instance to 100μL with adding RNAse-free water
2. Adjust the concentration of monovalent cations in the sample by adding 1/10th volume of 3 M sodium acetate to the sample (best precipitation: faster and more efficient).
3. Add 1 volume of isopropanol and vortex gently. Leave it on ice for 10 min. Try to shake gently the first two minutes.
4. Recover the RNA by centrifugation (13.2 rpm, 4°C, 5 min) and remove supernatant.
5. Wash the resulting pellet with 70-75% ethanol to remove residual salt (13.2 rpm, 4°C, 5 min). Remove the EtOH with the same procedure (carefully!), wash, spin down and remove the EtOH again. If it is necessary, the washing step can be repeated.
6. Evaporate off residual ethanol (you can leave the tube open). Avoid overdrying!
7. Resuspend the RNA pellet in TE buffer or RNase-free water.
In general, a bad A260/230 ratio is not critical for use in qRT-PCR. Also if you need to precipitate small RNAs and/or yield is very low, you might need to add MgCl2 or use glycogen as a RNA carrier.
This might also help you:
https://www.researchgate.net/post/What_s_the_best_way_to_remove_salts_phenol_and_other_contaminants_from_my_RNA_samples
https://www.researchgate.net/post/How_can_I_improve_RNA_quality_with_low_260_230_ratio
Thank you Julien Delezie
Do you dissolve the sodium acetate in RNAse-free distilled water? Also, I saw in one of these links you send me this description "Add 1/10 vol of NH4OAc, en 2.5 volume 100% cold EtOH (100%) and 1 µl glycogen". Do you add ethanol as well?
We dissolve our sodium acetate in deionized/MilliQ water and filter sterilize it. Hence, no need to use molecular biology grade water unless you are really crazy about RNase contamination and already taking some precautions.
We prefer isopropanol over EtOH mainly to work with smaller volumes.
What is your RNA cc? If you have a good yield, I would suggest to try the quick protocol above. If not, try to perform the precipitation in EtOH or Isopro overnight (Step 3) - this will definitely help.
I personally never used ammonium acetate for RNA prep; the choice of salt for precipitation will depend on your downstream application as the use of glycogen.
Hope this helps.
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