RNA and poly(a) RNA extraction problems - (May/12/2006 )

Hi all, i've been experimented a lot o problems doing the extraction of my RNA, which is from Cervical Ganglia neurons of the rat, the dissection is really terrible an also i need to sacrifice a lot of rats in order to get some tissue to work with, after the dissection i put my tissue in RNAlater, anf then i do an enzymatic digestion, i am really afraid to use liquid nitrogen instead to homogenize the tissue because is really small amount, so i perform a papain and cystein digestion and after that i centrifuge and take out the enzyme solution and then i follow the RNA extraction protocol by using trizol....,after that i use a poly(A)RNA extraction by using the MicroPoly(A)Purist of ambion, because i need mRNA to do a northern blot!!!!. But.............. when i want to know the yield and quality of my RNA, following the denaturing agarose gel electrophoresis (protocol from ambio) i can not be able o see anything!!!!!!!!!!, just a bright line in the middle of the gel!!!!!!, somebody can help me, i don't really want to waste my "rna" sample doing this control experiments...


I am really new in this kind of experiment ( i used to do celular biology), this is my first northern blot!!!!, soo i really need help and in my lab nobody do molecular biology so imaging my big problem!!!!, please help

hi
well what is the OD of your sample ? did you checked that point?
How do you stain your gel ? do you add EtBr or Sybrgreen on sample or in the gel?

*Can't you do a lysis directly by trizol?

hi fred, i didn't measure it because i realized that was not so much of material ( that is what i think....), so a friend of mine that used to work with dna told me that is better to check the concetration of the nucleic acids by gel when the concetration is so low. That's why i run a gel with the protocol of ambion but i couldn't be able to see anything and y used EtBr.
I can not be able to lysis directly by trizol, the tissue is so hard to lysate

i've never tried thatl, i am going to do it on monday, i have some extra rats so after that i'll tell you what happen... thanks!!!!

preheat trizol at 50°. But no more than 55° cause it's a denaturing condition for phenol.

Hi fred, i am going to run a rna gel again, with the sample that i already have, but with a different protocol. After that i am going to try the rna extraction again, with trizo at 50 C. and i'll let you know what happen.

Ok, i'll wait for your reply
claws crossed
fred

i was wondering, what is the size of the RNA you're looking for?
May you switch for acrylamide gels which are more convenient for tranfert... (just thinking of the next part of the exp...)

so sweett!!!, my rna is degraded, but at least i don't have DNA contamination, so i need to sacrifice more rats, but the important thing is that before that i really need to have a nice protocol for the extraction, i think that the main porblems is the quick disrruption of the tissue, i am thinking that maybe would be better if if i culture the neurons and after that do the RNA extraction from the cells and not from the tissue, but...... well i need to think about it
any suggestion????

Thanks!!!

Minerva

Ok, i'll wait for your reply
claws crossed
fred

i was wondering, what is the size of the RNA you're looking for?
May you switch for acrylamide gels which are more convenient for tranfert... (just thinking of the next part of the exp...)