High sensitivity in quantitative, competitive RT-PCR using QIAGEN ® enzymes*

Two oligonucleotides were synthesized, a 53mer and 70mer with a 25-base overlap. The oligo sequences corresponded to bases 434 to 559 of the mouse VEGF 164 A mRNA, with a 26-base deletion of bases 457 to 482. The oligonucleotides were annealed and the non-annealed regions were filled in using QIAGEN Taq DNA Polymerase. The resulting 97 bp DNA fragment was cloned into a plasmid containing a T7 promoter, and RNA was transcribed from the plasmid in vitro using T7 RNA polymerase (Figure 1).