medecinfrancais

Hi all,

I am working on verifying RNAi knockdown of a very large gene (~18,000 nts). The gene is comprised of many repetitive elements, and I have used up almost the entirety of a unique region with my RNAi construct. Despite best efforts to locate a new, entirely unique sequence for my primers, I have been unsuccessful. I have a few questions to try and set up my next attempt:

1) Could I use the primers I originally used from the RNAi construct (to amplify my insert) in the RT-PCR, or will I still see amplification due to my insert?

2) I am able to find partially unique primers for both the 5' and 3' (they are ~50% unique; the 10 interior bases match, 10 exterior bases do not). From the 10th base on the 5' end to the 10th from last base on the 3' end, the transcript which would be amplified matches between this gene and another. Is non-specific amplification likely (so I would see a 220 bp and a 240 bp fragment, if I could even tell a difference between the two)?

3) In the above scenario, would there also be a possibility of hybridization, leading to non-specific amplification (in effect a new primer being created from overhangs on the 220/240 bp frags)?

Thanks,
medecinfrancais