I am currently exploring changes in the serotonergic and glutamatergic system (rat brains) in respons to a treatment. I have validated primers for almost all receptors and transporters of these two neurotransmitter, except the gene for the serotonin transporter (SCL6A4). I'm using NCBI's primer blast tool to ensure specificity and netprimer to exclude primer pairs with a tendency to form primer-products (hairpins, primer-dimers and cross-dimers). I have so far tried four different primer pairs but none of them seems to produce anything near a decent amplification signal or standard curve, even though these primer pairs are specific with no primer-products and designed with the exact same guidelines in mind as all my other (functional) primers. My setup includes:
- SYBR green
- The cDNA I am using is from hippocampus and frontal cortex
Could this lack of amplification be caused by lack of SCL6A4 mRNA in these brain areas? Or does anyone have a alternative theory or experiences with this gene?
Any help is very much appreciated!