Complex Loci in Human and Mouse Genomes (original) (raw)
Figure 2
Validation of the Expression of Randomly Selected _cis–_Antisense Pairs by RT-PCR
To confirm the expression of complementary transcripts, we performed orientation-specific RT-PCR as described previously [12,24]. Primers were designed to amplify regions of exon overlap. For each candidate or control, four RT-PCR reactions (corresponding to the four lanes in each gel image) were carried out using adult mouse brain RNA as template. Orientation specificity was achieved by restricting which primers were present during reverse transcription single-strand synthesis: no primer (first lane), only sense primer (second lane), only antisense primer (third lane), and both sense and antisense primers (fourth lane). In all reactions, both primers were present during the subsequent PCR reactions. For candidates, sense and antisense primers were designed with respect to the genomic plus strand. For controls, primers were designed with respect to the control transcript. Out of five highly expressed control genes with no evidence of antisense transcription in sequence databases, we detected antisense transcription for one (Rps27). We reproducibly observed evidence of anti-Rps27 transcripts using two different primer pairs (unpublished data). We tested 20 _cis–_antisense pairs from our computationally constructed dataset and detected expression of both strands for 16 (underlined). For one additional _cis–_antisense pair (number 11), the result was ambiguous because of the presence of many bands of unexpected size. The 20 _cis–_antisense pairs were selected at random from the mouse dataset, with the requirements that exon overlaps be at least 200 bp (to allow amplicons of at least 100 bp) and that there be at least one cDNA or EST from adult brain supporting the exon overlap on each strand.