Hello everyone. I have some problems with a RNA extraction. I used the trizol reagent to extract RNA from tissues of leaves and roots under drought stress. When I dissolve the RNA in water, RNA from leaves is colorless, but the RNA from roots is brown and dense (similar to oil). I washed the roots to clean soil residues but I believe that this coloration is caused by the oxidation of sugars present in the roots by the drought stress. I need to recover and clean this RNA because is very important to my research. I need to make cDNA for real time PCR but if I use this "dirty" RNA I have inhibiton problems with the reference gene (housekeeping) . I would like to precipitate the RNA with Isopropyl alcohol or with ethanol but I fear I could lose the RNA. Can someone help me? Any tips please? Thanks for your time. Have a nice day.
You might want to try Ambion 'plant RNA isolation aid' solution (cat # 9690).
You can add the solution to the lysis buffer in the Ambion RNAqueous kit or to
the lysis buffer in the Qiagen RNeasy kit. It works great!.
Why are you not precipitating the RNA with isopropanol - that is the standard procedure.
Have a look at the following webpage
https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf
It says:
After homogenizing the sample with TRIzol™ Reagent, chloroform is added,and the homogenate is allowed to separate into a clear upper aqueous layer (containing RNA), an interphase, and a red lower organic layer (containing the DNA and proteins). RNA is precipitated from the aqueous layer with isopropanol. DNA is precipitated from the interphase/organic layer with ethanol. Protein is precipitated from the phenol-ethanol supernatant by isopropanol precipitation. The precipitated RNA, DNA, or protein is washed to remove impurities, and then resuspended for use in downstream applications.
• Isolated RNA can be used in RT-PCR, Northern Blot analysis, Dot
Blot hybridization, poly(A)+ selection, in vitro translation, RNase
protection assay, and molecular cloning.
• Isolated DNA can be used in PCR, Restriction Enzyme digestion,
and Southern Blots.
• Isolated protein can be used for Western Blots, recovery of some
enzymatic activity, and some immunoprecipitation.
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