Figure 9. Discriminating VEGF isoform-specific RT-PCR using human cDNA extracts further highlights the importance of primer design.

A, Human cDNAs from colon, prostate and kidney (Primer Design Ltd) were amplified in RT-PCR reactions using an exon 4 forward primer together with either DB165b/189b-1 (lane 1), h165b/189b-2 (lane 2), h165b/189b-3 (lane 3), h165b/189b-4 (lane 4) or h165b/189b-5 (lane 5). Primer sequences are highlighted in Table 1 & Fig 2. A putative VEGF165b (∼212 bp) product is evident in lanes 2 & 3, but absent in lane 1. All three reverse primers spanned 5 bases across the 8b/7 junction, however differed in their melting temperatures (see main text), with h165b/189b-2 & 3 exhibiting more compatible Tms with the forward primer. A similar 165b product was more abundantly amplified in lanes 4 & 5 using reverse primers spanning more bases (13 or 14 respectively) across the 8b/7 splice junction. In addition, these latter reverse primers also amplified a product corresponding to 188b (∼283 bp). However DNA sequencing revealed that the product amplified in lanes 2 & 3 was not VEGF but LIM domain only, protein isoform 7, confirming that detection of apparent VEGFxxxb species was only evident using 8b/7 reverse primers with increasing complementary sequence to exon7. B, Similar results were obtained in RT-PCR reactions using cDNA extracted from a normal kidney patient biopsy and amplified with the exon 4 forward primer and the DB165b/189b-1 (lane1), h165b/189b-2 (lane 2) and h165b/189b-4 (lane 4) reverse primers. C, RT-PCR using the DBexon7/DB3'UTR forward/reverse primers capable of simultaneously amplifying VEGFxxx & VEGFxxxb, confirmed only VEGFxxx (194 bp) amplification in the human samples tested.