RNA extraction - (Nov/30/2007 )
I would like to know if somebody have a protocol that help to get a higher quantity of a RNA which is bond with a lot of protein. By now, I was using trizol reagent, but I get very few of this RNA ... I thought it was because this mRNA was lost in the organic phase because of protein binding... Does someone can help ?
I don't know if this would be the problem, but if it is then I think that the RNA/protein would not go to the organic phase of the tri-reagent but rather to the interface where the DNA goes, so perhaps by just taking the interface and aqueous phase then doing a DNAse digestion you can get more RNA?
You could try a silica column based extraction method and do on-column digestion with DNAse and Proteinase K. I think Qiagen has a RNeasy supplementary protocol for fibrous tissues (e.g. muscle and tendon) that incorporates a protease at some point.
Trizol will release all factors bound to the DNA. That's the point. Try scaling up the cells you are extracting from. Do you notice any degradation of the other RNAs? How are you detecting your mRNA of interest? I work with a small nuclear RNA that is bound in an RNP and I have had no problems extracting my target RNA efficiently with Trizol. You could also pre-treat your sample with proteinase K prior to trizol extraction.
I make up my own AGTC - phenol ingrediets which works fine for me. My colleagues have reported a lot of problems with Invitrogen TriZol; I am not sure which Trizol you are using. Otherwise try Qiagen RNA coulmns.
In fact, that's a new project for me. I never had problem to extract RNA before. But all my members of my new team use proteinase K pretreatment (with no RNA protection ?!?) before going with TRI reagent (sigma). But they obtained RNA degradation... But with Northern, they show that they obtained more of the specific RNA with this pretreatment... I was looking to find another protocol to achieve a maximal recuperation of our RNA without any degradation... Maybe there proteinas K protocol is not right ?
Can you help me with that ?
you want RNA bound to protein... are you working on telomerase?
in any case, trizol works pretty well, and i've used the quiagen spin columns... both are ok. I think i prefer the spin columns.
if you have a method that has worked for you, why not try it and compare with the other memebers of your group. perhaps, there is a comprimise that can be achieved.
Thank you for the tips. I'll try the spin column from Qiagen.... I'm not working with telomerase but with a mutant allele of a gene which is toxic because of abnormal protein binding. I'll try the qiagen column and come back with some news.
Iam new to RNA extraction. I have extracted plant rna by using guanidium thiocyanate method.while quantifing with spectrophotometer I got a ratio of 2.5.but in the denaturing gel I couldn’t observe any bands. Why is that? What should be the minimum concentration of rna should be there to visualize?please could any body guide me
I don't know the minimum concentration, but 0,5-1 micrograms of RNA is clearly visible in a gel stained with ethidium bromide. If the RNA is degraded, it will be less visible as it will not form sharp ribosomal bands.
I would suggest you to post your own question, in this or in the molecular biology forum, instead of adding a question to a thread that was started with a different one. That should bring you more answers.