Why are the RT² qPCR Primers not designed to cross exon-intron junctions or boundaries?
For SYBR Green-based qPCR detection, the most important parameter for primer design is the generation of only a single gene-specific amplicon with high amplification efficiency, without the production of primer dimers. Primer assays amplifying short products contained within a single exon meet this parameter most optimally. Primers that cross exon-intron junctions may still detect processed pseudogenes, heteronuclear RNA (hnRNA), as well as unannotated alternative transcripts and splice variants, thus complicating SYBR Green-based qPCR detection.
