Hi everyone,
I am isolating RNA from pine cambium and am having problems getting clean pure RNA. If I use a miRvana kit I get around 10ng/ul RNA and 0,5 ng/ul DNA and by LiCl precipitation I get clean RNA samples but also higher contaminating DNA.
If I use DNase treatment (thermofisher AM1906) it only works on LiCl extracted RNA but does not efficiently remove DNA (from 5 to 1 ng/ul, after 2 hours doubling enzyme and buffer). With this conditions RNA starts to degrade.
Reading extensively I have seen that using acidic phenol can remove DNA from RNA samples because at pH 4,5 DNA moves to the organic phase and RNA stays in the aqueous one.
Does anybody have any experience on that and knows how risky it is? I assume removing phenol afterwards will be tough.
I attach a gel image of my RNA extractions using LiCl to precipitate.
Thanks in advance
Diego
You can use acidic phenol for RNA extraction. After addition of acid phenol and mixing it properly, perform centrifugation. This will bring your RNA to the upper aqueous phase, please be careful while extracting that. Try not to be close towards the interphase.
Attached a good source about (Acid) Phenol-Chlorophorm extraction.
What do you mean with risky? Handling phenol is hazardous and should be done carefully. There is no "risk" for you samples in terms of downstream enzymatic reactions and usage. Traces of phenol can be removed with subsequent chloroform extractions.
This method is very effective in purifying RNA with minimum DNA contamination. Acid Phenol-Chloroform extraction can be repeated multiple times till the desired purity is reached. Contamination of phenol in your sample are easily removed with 1-2 extractions in Chloroform.
Our usual routine, starting from cell pellet:
3x extractions in Phenol (pH4,5-5): Chloroform : Isoamilic Alcohol (50:10:1). (1-2x are likely enough if you start from kit purified RNA)
2x extractions in Chloroform
Isopropanol/Etanol precipitation.
(I can provide you a detailed protocol if required)
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