IsoPrimer: a pipeline for designing isoform-aware primer pairs for comprehensive gene expression quantification.

Motivation: Eukaryotic genes can perform different functions by generating multiple transcripts through the alternative splicing mechanism. The accurate quantification of gene expression in specific conditions is important for functional assessment and requires an accurate PCR primer pair design to target all expressed alternative transcripts, a complex and prone-to-error task if performed manually. Results: To efficiently address this task, we developed a pipeline, called IsoPrimer, to design PCR primer pairs targeting the specific set of expressed splicing variants of the genes of interest, to be used in quantitative PCR, e.g. in RNA-seq validation experiments. IsoPrimer, according to the level of expression of the splicing variants derived from an RNA-seq dataset, can: (i) identify the most expressed gene isoforms; (ii) design primer pairs overlapping exon-exon junctions common to the expressed variants; (iii) verify the specificity of the designed primer pairs. Availability and implementation: IsoPrimer is available for download from https://github.com/BioinfoUNIBA/IsoPrimer.