Characterisation of ghrelin natural antisense transcripts in the normal human stomach. A. Ethidium bromide stained agarose gel showing the verification of the candidate antisense gene ghrelinOS by orientation-specific RT-PCR in the human stomach. Antisense transcripts were amplified using a primer in exon 4* as the reverse transcription (RT) primer (R), while for the detection of sense transcripts, a primer in exon 2* was used in the RT reaction (F). The RT reactions were subjected to PCR using both the exon 2* and 4* primers (for 30, 35 or 40 cycles). NTC = no template control (water). M = MassRuler Express DNA ladder (Fermentas). B. Alignment of sequences derived from two 5' RLM-RACE products (TSS86 and TSS63) and a CAGE tag starting site corresponding to a 28 bp exon 4* (TSS28, T03F009D342E) with exon 4* and flanking genomic sequence. The lower case letters indicate upstream genomic or downstream intron 4* sequences. The position and sequence of the nested gene-specific 5' RACE primer (5'OS-in-R) is indicated with an arrow and underlined, respectively. For comparison, the (sense) ghrelin gene exon 4 sequence is shaded in grey.