Hi everybody, recently I have a serious probem with the primers I have designed for qPCR gene expression studies. I already have the GAPDH primers so in the beginning I have started with designing the primers for three different genes. The workflow I did is as follows:
1. I looked up the transcript sequences from NCBI Gene
2. I have designed the primers spanning exon-exon junctions (Tm~60C 20 bases)
3. I have checked the primers from NCBI Primer BLAST according to the RefSeq mRNA database
4. I have checked the stability and Delta G values for hairpin, self dimer and heterodimer from IDT Oligo Analyzer
Finally I ordered them after I saw everything is clear.
However, I only got product from the GAPDH primers when I run them on agarose gel. So I decided to order new primers which belong only to one exon (not spanning another). And the result was again negative, I only got weak bands on agarose gel in 2 of 3 primers (and quite good band of GAPDH product).
Just to mention, I have used random primers for cDNA synthesis.
Here is the question, where is the mistake or misunderstanding that I could be wrong in this experiment?