I'm running a PCR to look for gene expression in cDNA and the gene may be expressed at a low level.
The RNA is trizol extracted, cDNA made with 1ug in the ABI high-capacity RT kit. Housekeeping gene for the cDNA shows good amplification.
My basic problem is that I found some samples that were expressing the gene, but then I can't reproduce the result. I've tried replacing all of the reagents, changing [Mg], and increasing Tm. The control sample expresses at a high level, but a 1:100 and 1:1000 dilution of the control cDNA in water only amplify sometimes at 40 or 45 cycles.
The PCR spans an intron and the correct band size is 244bp. We have sequenced multiple cases and everything at that size is our gene of interest. There is consistent nonspecific bands with increased cycles at ~400bp that matches a pseudogene and at ~200 that matches from the first exon into the following intron.
Sample 5 and sample 11 have both been positive in the past, but you can see that at two different amounts of Mg, they aren't both amplifying. I've only attached this one picture to demonstrate that sometimes the case is clearly positive with a strong band and no non-specific amplification, and sometimes there's just no amplification at all.
We have tried increasing the amount of template, but still have the same problem. We aren't getting a faint band sometimes, it's just all or nothing.
Thanks in advance.