 Solution

A practical design uses one common exon-4 forward PCR primer with an exon-specific reverse PCR primer for each transcript isoform. Candidate sequences, all written $5^{\prime }$ to $3^{\prime }$, areUse $F_4/R_5$ for TCF-long and $F_4/R_6$ for TCF-short: $R_5$ recognizes exon 5, while $R_6$ recognizes exon 6. The displayed mature reference sequences predict approximately $130$- and $67$-base-pair products, respectively. Check actual splice products because a transcript with unexpected exon combinations could defeat the assumed specificity. The simple melting-temperature estimates are approximately $62$, $60$ and $60{}^{\circ }\mathrm{C}$.

Prepare matched RNA, remove contaminating DNA, carry out reverse transcription and validate each RT-qPCR assay with known isoform standards, a single-product check, a no-template control and a no-reverse-transcriptase control. The primers span an intron in the genomic arrangement but do not themselves cross a splice junction, so the DNA control remains important. Calibrate amplification efficiency and fluorescence response before comparing molecule numbers between the two assays. A redesigned junction-specific assay is another option once additional suitable flanking sequence is available.