I am having a problem getting Reverse Transcriptase PCR (for which the plant is genetically engineered) especially in progeny lines, even after selecting in stress conditions. These plants are PCR positive and cDNA prepared is also perfect, as housekeeping genes are being amplified.
First thing you should confirm that it is truly transformed. A simple PCR is not sufficient. How many copies of the transgenes are inserted in the host genome? Is it primary transformants (TO) or later generations?
These are T1 generations. Yes we have to do southern for copy number analysis. But these progeny lines are surviving in stress conditions for which the controls didn't survived at all. They were then acclimatised and gradually revived from stress conditions and finally established in transgenic containment facility.
10 years ago I have met the same problem. Inserted gene was PCR amplified only in homozygous brassica genotypes of the late generations. No explanation has been found.
I agree with Guiseppe. Southern blots (from later generation, not T0) to assess the T-DNA copy number and whether the copies are intact are needed to confirm the presence/absence of the transgene. If there is just a little bit of contamination (from using the same lab equipment that was used to make transgene constructs) in the RT-PCR it might yield a false positive.
Are you sure your primers match the cDNA you have used for transformation? Usually, only the coding region is used to generate the overexpression construct. If your primers match the 5' or 3' UTR they will not work in that case. Have you tested your primers on the plasmid you have used to generate the transgenics?
How do you make your cDNA? Oligo dT or gene-specific?
Hope lots of good answers above gave you several options to look into. Did you have RT-PCR assay validated with positive controls, like the plasmid DNA? If not, I recommend doing this before trying anything else.
Another possibility is truncation of your T-DNA. Do you have confidence that the region selected for amplification by RT-PCR is necessary for the stress survival? Does the PCR you mentioned in the question (where you get positives) include the region selected for RT-PCR?
If the gene is expressed at very low levels you may not get enough amplification in the expected number of cycles.
Guess truncated T-DNA transfer or co-suppression (particularly if the inserted gene is identical to an endogenous ones, although cosuppression may occur with any multiple T-DNA insert). Be sure to identify single-insertion transformants.
If you designed an experiment where a silenced gene would induce stress resistance, it could probably happen to you that: a) you have multiple T-DNA insertion, and your transgenic lines are PCR-positive; b) you have silencing of your gene, and subsequent resistance to stress; c) because of silencing, RT-PCR does not detect your transcript. Might this be the case?
It sounds like your gene it nor expressed, otherwise you would get the complementary DNA. Look if plants have a phenotype and remember that by checking the present of the transegene by PCR does not mean that it is transcribed into an mRNA...
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