3 bands on RT-PCR - (Mar/21/2005 )
I had all the nuclear accids of a tissue and did RT-PCR using primers for a specific mRNA gene. I developed l electrophoresis on agaroze gel and while I was expecting one band I finally got three. Any ideas?
I am not sure what you mean by 'zone', but if you are talking about three bands on the gel, I would think some of them might be non-specific products.
Either that, or your protein has some splice forms that you pick up in your RT-PCR
Did you make your primers to span intron/exon boundaries, to ensure that you would not get genomic products if there was any DNA carryover? What do you mean by 'all the nuclear acids'?
There are two posibilities that can make you RT-PCT generate more than 1 band.
First, your primer not specific enough. Not so much during PCR but more important during your RT step. As we know, RT step has to be carry out at lower temperature (~50oC). This might be how the unspecific band come about.
Secondly, some of your RNA had degraded into smaller fragment. Thus the small fragment actually serve a "primer" during PCR. So now you got "extra primer" in your solution. That why you can get multiple band.
1. You can do a BLAST to check if your primers are really specific to that gene. Pay more attention to 3' region of your primers.
2. Then try to increase the annealing temperature in your PCR step.