Assessment of transcriptional directionality by RT-PCR. Sample results from (a) a control and (b) a sense-antisense candidate. PCR primers were designed to amplify predicted regions of bidirectional transcription. Control primers were designed to amplify either non-overlapping regions of candidate transcripts or randomly selected regions of non-candidate transcripts. For each candidate or control, four RT-PCR reactions were carried out using total human RNA from a single tissue as template. Orientation of transcripts was assessed by restricting which primer was present during RT single-strand synthesis. 1, Both primers present during RT single-strand synthesis (positive control); 2, only antisense orientation-specific primer present during RT single-strand synthesis; 3, only sense-orientation-specific primer present during RT single-strand synthesis; 4, neither primer present during RT single-strand synthesis (negative control for genomic contamination). L, 100 bp DNA ladder (Gibco-BRL). In all four reactions, both primers were present during the subsequent PCR reactions. In these examples, the control primers in (a) targeted a 127 bp region of 'chromosome condensation-related SMC-associated protein 1' (NM_014865; Hs.5719) over which we did not observe bidirectional transcription, and the candidate primers in (b) targeted a 113 bp region of mannose-6-phosphate receptor (cation dependent) (NM_002355; Hs.75709) which our results suggested was shared by an overlapping RNA species. The template in both cases is total human placental RNA (Clontech). In the control (a) only sense transcription is detected over the queried region (the appropriately sized band in lane 3). In the candidate (b) both antisense and sense transcription are detected (appropriately sized bands in lanes 2 and 3, respectively).