I work with Rosa hybrida tissues, and total RNA extraction from it is so hard. There are other plant species which gives good quantity and intact total RNA. So, what is the reason behind the Roses tissues that influence RNA extraction? And how can i customize the conventional protocols to ensure good quantity of RNA from Roses?
Thank you so much Sir David Farringdon Spencer
. Infact now I realize why I was getting good results in species other than Rosa. So the phenolic compounds are my enemies.I always keep the sample frozen and never leave to thaw. I extract RNA under RNAse free environment; I change gloves after each step, and never touch any other substance. Their is a very good (but lengthy) protocol for Rosa, called 'Hot borate method'. But problem with that method is small RNAs are not isolated, and thus it is useless for me. In our lab, there is not enough financial support for using kits that are very easy and fast. For extracting tRNA for miRNA downstream applications, TRIzol method (Phenol + Guanidine Thiocyanate) is mostly used. It gives very good results with Arabidopsis and Tobacco, but fails in Rosa. So, i was advised by my lab seniors to use the extraction reagent called 'pBiozol' containing β-Mercapto-Ethanol + Sodium Azide. Its protocol is just like that of TRIzol, having one additional step of NaCl mixing. Our lab has customized the it a bit, adding Phenol: chloroform step. It gives some tRNA, but not good enough for downstream uses (actually the 28s is dimmer than the 18s). I have been trying it now for more than 1.5 years, no success.
I am confused, whether to use phenol:chloroform or only chloroform? as Rose tissues have phenols, so does it make any sense to include phenol during cleaning step???
i extracted total RNA and somehow i got good bands. Then I used DNAse1 to remove gDNA, it failed completely, erasing the RNA bands while gDNA was still there. Then I used reverse transcription to make cDNA. I checked it with UBI, there was no cDNA at all. Don't know whats the issue.
One thing that irritates me is the color of RNA pellet, that is still prominent in the diluted RNA, and cDNA as well. May be the problem is that RNA has some impurities, or it isn't washed completely. What do you experts say?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA