I am designing qPCR primers for some genes in drosophila melanogaster.
I am currently using NCBI tool which uses Primer3 and BLAST to find the primer pairs from the refSeq records. I am using the thermofynamic secundary structure aligments to "avoid" harpins, dimers and primer-dimers in Primer3 tool, however I am later checking them manually with OligoAnalyzer 3.1 tool (http://eu.idtdna.com/sessionTimeout.aspx) to make sure harpins have at most DG=-3, self-dimer is less than DG=-5, cross-dimer (sense and antisense primers homology) to be less than DG=-5, and so on.
My question are:
1 - If the template we will use for qPCR is RNA, do I have to search primers that span an exon-exon junction?
2 - Since we will amplify from RNA template, do I need to do the reverse complement of the designed primers found from refSeq (mRNA)? See attached file with an example, for a gene in positive strand, the forward primer matches to the gDNA, and the revesere primer is the reverse complement of the gDNA. And the opposite for a gene in the reverse strand. How should I order my primers?