Figure 1.

Schematic representation of the Capping-RACE procedure. (A) Total RNA is treated with vaccinia capping enzyme (VCE), which caps only the 5′ triphosphorylated RNA. (B) First-strand cDNA synthesis is performed using M-MLV reverse transcriptase with a gene-specific primer (GSP1). (C) A template switching oligonucleotide (TSO) with a 3′ end composed of three ribonucleic acid guanines hybridizes to the cDNA 3′ poly(C) and is used as a template for reverse transcriptase. (D) Hybrid RNA is removed by RNase H. (E) Nest-PCR is performed using two sets of primers. The PCR products are cloned into pMD18-T vectors, which are sent out for commercial sequencing.