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Ask for help about the RNA purification protocol by phenol/chlorform methods


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#1 veer2

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Posted 11 May 2009 - 09:14 AM

Ask for help about the RNA purification protocol by phenol/chlorform methods

I just start to work on the miRNA qRT-PCR fields and try to establish some assay, the method is firstly to add poly (A) at the 3-end of miRNA, then synthesis the first cDNA strand by reverse transcription

After the isolation of total RNA (Trizol methods from invitrogen), add poly (A) at the 3-end of miRNA, the phenol/chloroform procedure is needed. What is the detailed protocol of phenol/chloroform purification? and after purification, the small RNA should still be included in the total RNA.

Thanks a lot in advance

Veer2

#2 mastermi

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Posted 11 May 2009 - 11:57 AM

Usually it's just the same like a DNA phenol-chloroform extraction, except you use acid phenol instead of alkaline (we use phenol from QBiogene).
I use make two phenol-steps followed by one chloroform-step.
The precipitation is done with ethanol and Na-acetate. You can also add glycogen which should increase precipitation.

#3 veer2

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Posted 12 May 2009 - 11:54 AM

Thanks a lot




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