This is the first time I'm doing Northerns, and my task is simple: I need to describe the dynamics of Sindbis virus replication. That means detecting the amount of positive and negative strand RNAs, and, if possible, the amount of the subgenomic RNA...
But I do have questions...
First, the oligo design looks relatively simple: just a 40 or so nucleotide long complementer sequence to the positive or negative strand -right?
How do I validate them? (To show that they indeed are specific.)
How do I show the different types of RNAs, and their ratios? I was thinking of a probe that spans both the subgenomic RNA and the complete RNA, hence it would detect all three forms: complete, and the subgenomic. Is that a valid approach?
Some people use RNA probes instead of DNA ones - how should I choose one or the other? The problem is that I need to do it quick and dirty - I have to characterize my system, so I can actually start "THE" experiment, which will take months or more to perform and evaluate, so I don't have many shots at it. My funding runs out in 18 months. (No pressure, of course.)
It was suggested that I dig up oligos by people who used them before, but after a day reading paper after paper I still have not been able to find probe sequences -if someone happen to know them, I'd be greatful.
Thank you for your time.
Edited by Andras, 05 April 2011 - 09:09 AM.