I am trying to extract RNA from urine pellet for RNA seq. However, when I used TRIzol protocol, the RNA had low A260/280 (~1.3), while with QIAgen kit, they had low A260/A230 (~0.5)
I am wondering if we could conduct further RNA clean up (reprecipitation or wash) to improve the ratios
Thank you!
Do the ethanol 75% washing step twice. My samples are generally around 2 doing that when using Trizol. But I do not find a big difference in RTqPCR improving this ratio, honestly. 1.4 samples works fine. 260/280 is more important imo
Yes washing twice may also help but have you tried to extract DNA via phenol-chloroform extraction? and also have you used your extracted DNA for a PCR reaction using any desired gene specific primers?
You can try LiCl precipitation whcih removes removes carbohydrates and gross DNA contamination. However, please bear in mind that the concentration of RNA should be at least 0.2 μg/μL to assure efficient precipitation. Also, LiCl precipitation will not quantitatively precipitate small RNAs such as tRNA and 5S rRNA.
Alternatively, use your Trizol isolated RNA solution with the filter cartridge for improving its quality further.
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