DIG system - Northern blot (Jan/23/2008 )
Please, I have a question. Im going to use the DIG system from Roche to do my northern blot experiments.
I will label my oligos using one of the kits. There are two kits there that I would like to use.
One add a 3' ddUTP and the other add a 3' DIG-dUTP tail.
The one that adds the tail looks to be more sensitive.
I would like to use this one but Im afraid that the tail add to the small oligonucleotide could mess up the hybridization.
Please, someone knows anything about that??????
Thanks in advance.
The tailing kit would probably give higher sensitivity but there might be doubts on the specificity of the probes. Considering that the use of such probes has not been reported thus far for miRNA northerns, I think it's possible that the reviewers of your work will rise the doubt. If you will use the tailing kit, I would suggest to have ready the possibility of some sort of validation, for instance by real time pcr or by using a tailed probe on some miRNA with a known expression profile.
It's just my opinion, as I didn't try. I wish that someone with a direct experience will share it because detecting small RNAs by non-radioactive northern is probably a bottleneck for many labs now.
My concern is probe with polyU tail might easily anneal to polyA tail of mRNA...
For better result, using synthezed RNA oligo or LNA oligon with DIG would be better, even though they are still less sensitive compare to radio isotop probe.
Thank you very much for the answer.
Maybe It is a better deal to use the dUTP since it looks to work.
i there...just wondering in out instructions the final step on our steps is to "record the concentration of each visible dot" after looking at how many spots have occured...we were given a membrane with various spotted concs. on them (and we no wat that conc is to)...so wat im wondering is how can i record a conc. from wat i see--am i just recording the concentration from the ones that were given to us...and just seeing at what concentration the dot appears at?....also anyone now of anygood websites/books that have info on this kinda stuff..
thanks in advance,