Northern Advice - What DNA vs cRNA probes (Aug/09/2005 )
I have ~20 probes to screen for setting up a diagnostic test. I need to do northerns (both PAGE and AGE) and am debating whether to use a DNA probe or an RNA probe.
Pros of DNA probe: Easy, random priming of plasmid, can piggy-back some-one elses 32P supply
Cons of DNA probe: Not as sensitive
Pros of RNA probe: High sensitivity
Cons of RNA probe: Have to do twice as many labellings since need probes in both directions, need to get specific reagents.
I've *maybe* decided on using a DNA probe first to sort the probes a bit (hopefully chuck out some that are as useful as a bicycle is to a fish), then go to cRNA probes, but I'd like to hear anyones opinion before I start.
Also: I've read some literature about Ambion's ULTRAHyb and how it can "make your DNA probe as good as an RNA probe" - anyone tried this? Are the claims justified?
Cheers for any input.
Is there any reason why you are doing your screening via Northerns? Couldn't you just use a slot blot? This would be much simpler.
Yeah, a slot blot would be much simpler, but I do need to use northerns to check the hybridisation patterns. If any of the probes are specific for our application I will be (hopefully) switching to slot-blot to do the large scale analysis. The reason I'm going to do northerns first is because I need to make sure that the probes are hybridising to the correct RNA. (A previous worker on the project found that some (previously analysed) probes don't behave in a logical, or even analysable (is that a word?) manner, so the more information I can get on them, the better).
It's going to take me a while to plow through them all, but hopefully I'll keep sane while screening .
If specificity is going to be a problem then it might be better to use oligonucleotide probes- you can screen quickly through them to find specific regions. If you need to get highly label density with oligo probes then you might want to use 3' tailing with alpha P32.
Improved automated DNA sequencing