An initial aim of this study was to characterise GHRLOS in a range of tissues. As we have previously demonstrated the expression of GHRLOS mRNA transcripts in the human stomach 8, we performed 5' RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends) on this tissue. Unexpectedly, we identified a number of new exons (exon I-III) [GenBank:EU789528, EU789529, and EU789530] that are 2.5 to 4.8 kb upstream of the previously reported GHRLOS transcription start sites 8 in exon 4* (4*a-c). To simplify the numbering of GHRLOS exons, the previously reported 8 exons 4*, 2**, 2* and -1* have been renamed exon 1 to 4, respectively; while the exons upstream of the reported start sites in exon 1 are denoted by Roman numerals (I-III). Importantly, in the GHRLOS variants demonstrated via 5' RLM-RACE, exon 1 is extended, with a 106 nt region (exon 1d, hereafter termed exon 1) overlapping exon 4 of the ghrelin gene (see [Additional file 1]). The novel first exon, exon I, is 51 bp in size (Fig. 1) and overlaps the 3' untranslated region of the adjacent gene, TATDN2 (TatD DNase domain containing 2, also known as hypothetical protein KIAA0218) 13. This gene initiates on the same DNA strand as GHRLOS, approximately 32,000 base pairs upstream of the 51 bp exon I of GHRLOS.

In order to determine the polyadenylation site(s) of GHRLOS transcripts, 3' RACE and inverse PCR were conducted using normal stomach and prostate tissues, the RWPE-1 prostate cell line and the PC3 prostate cancer cell line (which is derived from prostate cancer cells that have metastasised to the bone). We obtained 3' RACE clones from the PC3 prostate cancer cell line, which demonstrated a 1.4 kb exon 4a and a 1.1 kb exon 4b, with the putative polyadenylation signal AAATTA [GenBank:EU789531, and EU789532] (Fig. 1). The 3' RACE data matches a number of Expressed Sequence Tags (ESTs) from subchondral bone [GenBank:BM991802], foetal liver [GenBank:AI056187], pooled liver and spleen [GenBank:BX118778; R01027], Wilms' tumour (a paediatric cancer of the kidney) [GenBank:AI174574], pooled brain cancers [GenBank:AI863058] and high grade serous carcinoma of the uterus [GenBank:AI289483]. Moreover, we performed uncapped inverse PCR, and sequencing of amplicons from the PC3 cell line revealed a 90 bp exon I and exon 4 sequence with a 3' end differing by less than 20 base pairs when compared to the 3' RACE and reported ESTs [GenBank:EU789533]. Interestingly, the putative 3' end of exon 4 corresponds to publicly available Affymetrix Human Exon 1.0 ST array data (Exon Cluster ID 183613). Furthermore, all of the amplicons that we obtained were followed by a stretch of adenosines at the 3' ends that are not present in the genomic sequence. We, therefore, concluded that we had reached a genuine 3' end of GHRLOS.

In order to examine the size and tissue distribution of GHRLOS transcripts, we performed Northern blot analysis of mRNA from human stomach and 12 normal, human tissues. With a riboprobe designed to span exons I, II, 1 and 2 of GHRLOS, a weak, smeared signal ranging from approximately 1.0 to 2.0 kb in size was observed in the human stomach upon a lengthened exposure time (data not shown). A riboprobe spanning exon 1 alone (a region common to all GHRLOS RNA isoforms) resulted in signals ranging from 1.0 to 5.5 kb in size in the pancreas, prostate, salivary gland and thymus (Fig. 2). Upon longer exposure times, a smear in the 1.0 to 5.5 kb range was seen in all tissues, except for peripheral blood leukocytes and urinary bladder (data not shown). This suggests that GHRLOS is a fully processed transcript consisting of many mRNA isoforms.

The RACE, inverse RT-PCR and Northern blotting experiments indicate that GHRLOS is extensively spliced and that isoforms range greatly in size (from approximately 1.0–5.5 kb). To examine the alternative splicing pattern of GHRLOS in greater detail, we performed RT-PCR using a range of human tissues and cell lines. We used a forward primer common to exon Ia (identified via 5' RLM-RACE) and a reverse primer in a region common to exons 4a and b. Sequence analysis indicated that, with the exception of exons 1 and 4, which are common to all known GHRLOS isoforms, GHRLOS exons are highly polymorphic in size and exon skipping occurs frequently. A representative amplicon banding pattern is shown in [Additional File 2]. The highly variable splicing pattern revealed by RT-PCR is consistent with the diffuse and broad signal which we detected by Northern blotting. In total we obtained 13 different GHRLOS splice variants (Fig. 3). Analysis using GMAP, a genomic mapping and alignment program for mRNA and EST sequences 14, indicated that all GHRLOS exons are flanked by canonical splice donor and acceptor sites (GT/AG), except for exons 3a and 3c where the splice junction is the common non-canonical splice pair GC/AG 15. GHRLOS exons and introns identified are listed in [Additional File 3].

Northern blotting demonstrated GHRLOS transcripts 1–5.5 kb in size, however, further RT-PCR and 3' RACE analysis with a number of different primer combinations failed to reveal GHRLOS transcripts larger than 2 kb (data not shown). For this reason, we hypothesised that the ghrelin antisense gene may extend further upstream. Capped RLM-RACE of stomach tissue and uncapped inverse PCR of the PC3 prostate cancer cell line (Fig. 1) indicated that a GHRLOS promoter (exon I) was present at the very end of the 2 kb 3' untranslated exon 8 of TATDN2. Moreover, we discovered that multiple Cap Analysis of Gene Expression (CAGE) tags are clustered approximately 1.5 kb upstream of the GHRLOS transcription start sites found via 5' RLM-RACE. CAGE tags are an average of 20–21 nucleotides and are produced by large-scale sequencing of concatemers derived from the 5' ends of capped mRNA 1617. The CAGE method, therefore, detects the most 5' site of the mRNA transcripts (the transcription start site) and gives an unbiased and comprehensive picture of the positions and usage of transcription start sites 18. To confirm if this region belongs to GHRLOS, we employed nested RT-PCR using thymus tissue, foetal brain tissue and Hep G2 hepatocarcinoma cell line cDNA (with RNA reverse transcribed using oligo(dT) primers). The forward primers were present immediately downstream of the CAGE tag cluster (which is 1.5 kb upstream of the 51 bp exon I of GHRLOS) and the reverse primers spanned exon 1 (which is common to all known GHRLOS variants). Sequence analysis revealed several novel exon I variants, which were approximately 1601 bp, 994 bp and 526 bp in length. All of these variants spliced into the expected acceptor site of the 106 bp exon 1 (Fig. 1 and [Additional File 4]). The end of the 1601 bp exon I (termed exon Ib) spanned the 51 bp exon I (exon Ia) identified via capped RLM-RACE of the stomach, while the other exons (Ic-d) employed different upstream donor sites, suggesting that they are alternative first exons. The data demonstrate that there are at least two first GHRLOS exon regions in the untranslated region of TATDN2. This suggests that the GHRLOS promoter is a broad-type, TATA-less promoter that initiates transcription at many sites 19.

The length of GHRLOS transcripts initiating in exon I (present in the 3' UTR of TATDN2) is 1.3–3.6 kb, corresponding in size to the Northern data. However, the potential identity of the approximately 5.5 kb transcript seen in the Northern blot with a full-length, 106 nt exon 1 probe (Fig. 2) was not determined. After prolonged exposure of the Northern blot, the 5.5 kb transcript was observed in all tissues except the urinary bladder and uterus (data not shown). TATDN2 is only approximately 1 kb upstream of exon II and 4.5 kb from exon 1 of GHRLOS, suggesting that transcription-induced chimaeras (TICs) may be generated 20 and give rise to large GHRLOS transcripts. Because TICs must contain a first exon of the upstream gene 20, we employed primers in exon 2 (immediately after the start codon) of TATDN2 and exon 1 of GHRLOS (which was also the sequence of our Northern riboprobe). Using nested RT-PCR (on cDNA reverse transcribed using oligo(dT) primers), we isolated a 2831 bp TATDN2-GHRLOS amplicon from the thymus [GenBank:EU789553] (Fig. 4). This transcript has canonical splice donor and acceptor sites (GT/AG) and splices into the expected acceptor site of the 106 bp exon 1. This variant harbour significant open reading frames corresponding to the TATDN2 protein, but contains alternative exons and a premature termination codon more than 50 bp upstream of the final coding exon 7 of TATDN2 (Fig. 4). This is likely to result in degradation of the mRNA by nonsense-mediated RNA decay (NMD), a surveillance mechanism that detects and degrades mRNA that may encode truncated proteins with dominant-negative or deleterious gain-of-function activities 21.

We have identified several novel GHRLOS transcripts. This includes overlapping GHRLOS transcripts initiating in the TATDN2 3' UTR and putative transcription-induced chimaeras of TATDN2 and GHRLOS. These findings extend the previously reported length of GHRLOS by ~37 kb. We propose that GHRLOS harbour several promoters with start sites in exon 1 (of GHRLOS), and in the 3' UTR and the first exon of TATDN2.

Sequence analysis of GHRLOS (excluding the putative TATDN2-GHRLOS transcription-induced chimaeras that are likely to result in nonsense mediated decay) reveals that GHRLOS transcripts do not harbour protein coding potential, but rather have several features of non-coding RNA genes. GMAP analysis showed that GHRLOS spans approximately 44 kb on 3p25.3 (data not shown). The Mulan sequence conservation profile for the human and putative vertebrate GHRLOS orthologues indicated that most GHRLOS exons (I, II, III, 2, 3) are not highly conserved compared to the putative mouse and rat orthologues (Fig. 5). The 106 bp exon 1 (which spans exon 4 of the ghrelin gene) and approximately 90 bp of the 1.4 kb exon 4 (which spans exon -1 of the ghrelin gene) are conserved, however 8. RT-PCR experiments revealed no evidence for antisense transcription from exon I to exon 1 (of GHRLOS) in the mouse (data not shown), suggesting that ghrelin antisense transcription (at these specific locations) may not be conserved. Furthermore, there is very low sequence similarity between the chicken, frog, opossum and human GHRLOS sequences (Fig. 5). Interestingly, exons II, 2 and 3 appear to be highly conserved between human and dog [Additional File 5], with 70–75% homology for exons 2 and 3. However, no significant open reading frames (ORFs) are conserved between dog and human GHRLOS sequence (data not shown). Taken together, this suggests that GHRLOS has evolved rapidly and may be unique to primates.

Our study suggests that GHRLOS is a non-coding RNA. In silico translation revealed that the heterogeneous GHRLOS RNAs contain multiple stop codons, resulting in lack of extensive reading frames, and the putative ORFs do not span conserved regions (data not shown). Moreover, no significant sequence similarity to any known proteins was observed (data not shown). Finally, screening of GHRLOS sequence against a reference collection of repeats (RepbasE) using CENSOR 22 identified a 203 bp overlap of exon 4 with the extinct 224 bp MIR3 SINE element, which is present in all vertebrates 23 (Fig. 5). Interestingly, the presence of repeat elements in exons of non-coding RNAs has been reported previously 24252627

We have discovered that exon 4 of GHRLOS is also on the opposite strand of a novel terminal exon of the neighbouring SEC13 gene in a tail-to-tail, 3' to 3', fashion (Fig. 6A) [Additional File 6]. SEC13 plays an important role in protein transport and is a component of the COPII complex 2829. BLAST analysis identified a brain tumour EST [GenBank:BF931280] 30, which contains exon 7 and 8 of SEC13, as well as 206 bp sequence corresponding to a novel exon 9b of SEC13 10.7 kb upstream of exon 8. We verified the expression of this EST by RT-PCR (using cDNAs reverse transcribed with oligo(dT) primers) (Fig. 6B) and by sequencing [GenBank:EU789555]. We have named this SEC13 variant, SEC13-tentative (SEC13-T). SEC13-T may encode a 372 amino acid protein. The first 285 amino acids are identical to SEC13, while the normal C-terminal 37 amino acids have been replaced by 87 amino acids encoded by the alternative terminal exon 9b (Fig. 6C and 6D). The putative C-terminal-coding exon of the SEC13-T isoform appears to be conserved only in primates (data not shown). Therefore SEC13-T alternative splicing is likely to be human-specific or primate-specific.

RT-PCR analysis (with primers spanning GHRLOS terminal exons) and Northern blotting (which is only suitable for high copy number transcripts) demonstrated that the size of GHRLOS transcripts is highly variable, resulting in significant transcript heterogeneity. We, therefore, employed a quantitative, real-time RT-PCR approach in order to more precisely gauge the expression of GHRLOS in a range of tissues and cell lines. As the number of alternatively spliced GHRLOS transcripts makes it impossible to generate real-time RT-PCR primers that are unique to each splice variant (data not shown), a strand-specific quantitative RT-PCR assay with primers in exon 4 (which is common to all GHRLOS variants) was designed to detect total GHRLOS RNA expression (Fig. 7A).

The level of total GHRLOS transcript expression varied greatly in different human tissues, with high levels in the thymus, testis, foetal brain, uterus, cerebellum, ovary, thyroid, and whole brain. Very low levels of total GHRLOS RNA expression were detected in the stomach, foetal liver and pancreas (Fig. 7B). In the thymus the level of expression was approximately 133 fold higher than in the stomach (P < 0.01) and 110-fold higher than in the foetal liver (P < 0.01). Furthermore, the level of expression in the adult liver was 9-fold higher than in the foetal liver (P < 0.05), indicating differential expression according to developmental stage in this tissue. The level of GHRLOS in the foetal brain was two-fold higher than the adult brain, but this was not statistically significant (P > 0.05).

Expression of GHRLOS in a number of continuous cell lines was also examined using real-time RT-PCR (Fig. 7C). Levels of GHRLOS expression in the U-937 (Non-Hodgkin's lymphoma) and SW1353 (chondrosarcoma) cell lines were similar to the cerebellum, uterus, foetal brain, testis and thymus. High levels of expression were observed in the HEK293 human embryonic kidney cell line and in the CaCo-2 (colon adenocarcinoma), SAOS-2 (osteosarcoma), U-87 MG and U251-MG (glioblastoma), OVCAR-3 (ovarian adenocarcinoma) and Hep G2 (hepatocarcinoma) cancer cell lines. It is interesting to note that multiple Hep G2 CAGE tags have been identified in the exon I region of GHRLOS (see [Additional File 4]). GHRLOS RNA appears to be significantly upregulated in the Hep G2 cell line compared to adult and foetal liver (P < 0.05) (Fig. 7B and 7C). This observation could indicate that GHRLOS may be a specific target in the development of liver and other cancers.

We examined the expression levels of the GHRL-GHRLOS cis-natural antisense transcript (cis-NAT) pair via quantitative real-time RT-PCR assays detecting total transcription from the ghrelin gene (GHRL). As expected 31, the highest level of GHRL expression was found in the stomach (data not shown), followed by the testis and pancreas (Fig. 8). When comparing total ghrelin and GHRLOS RNA expression in the stomach, GHRL was expressed at 2300 fold higher levels than GHRLOS, with GHRLOS expression almost undetectable (P < 0.001). The levels of GHRLOS expression were higher than GHRL in the thymus, whole brain, the SW1353 chondrosarcoma cell line, uterus and prostate. However, total GHRL RNA levels were higher than GHRLOS in the pancreas and the OVCAR-3 ovarian cancer cell line.

Non-coding RNAs are frequently not conserved between species, suggesting that they are either biological noise (non-functional transcription), or that they have species specific-functions. Species-specific non-coding transcripts have been observed and there is strong evidence that non-coding transcripts are functionally significant 4849505152. Interestingly it has recently been observed, using in silico analysis, that even between closely related Drosophila species non-coding RNAs are not conserved 53. While the number of protein coding genes in distant eukaryotes (such as worms, mice, and humans) is approximately equal, the relative amount of non-coding DNA increases in proportion to eukaryotic complexity 5455. Mattick and colleagues explain this paradox by hypothesising that non-coding RNAs have evolved to enable the emergence of organisms with increasingly complex higher levels functions 5556. Our in silico analysis suggests that GHRLOS exons show very low sequence conservation in vertebrate species. Furthermore, exon 4 of GHRLOS contains a transposable element, a feature observed in many non-coding RNAs 252627.

Our bioinformatic studies indicate that GHRLOS does not encode a protein and, therefore, is a non-coding RNA. Taking into account the full-length sequence of GHRLOS, the length of the open reading frames is very short. Moreover, GHRLOS RNAs are numerous (due to extensive splicing), contain a large number of stop codons and there is little nucleotide and putative amino acid conservation between vertebrates, making it less likely that this gene encodes proteins. Although it has been suggested that many small peptides may be translated 57, the majority of small peptides are processed from larger precursor proteins, as is ghrelin itself which is processed from preproghrelin. Therefore, while it cannot be excluded that GHRLOS encodes short ORFs (that are not conserved in the mouse) it appears unlikely that GHRLOS encodes biologically active peptides.

We examined the GHRLOS expression profile to strengthen the hypothesis that it is a candidate non-coding RNA. It has been demonstrated that mammalian long non-coding RNAs are expressed in a tissue-specific manner, indicating that they are biologically significant 5058596061. In both humans and mice, the major tissues of non-coding expression are the complex organs; the brain, testis, and thymus 5061. This also holds true in Drosophila, where the majority of the candidate non-coding RNAs are expressed in the central nervous system 53. Indeed, non-coding RNAs are emerging as important regulators of complex systems, such as the central nervous system (brain) and intricate processes, including spermatogenesis in the testis 62636465. We demonstrated high levels of GHRLOS in the thymus, brain, testis, uterus, ovary and thymus, while the expression levels in the stomach, where GHRL is highly expressed 1, were almost undetectable. Our data demonstrate that GHRLOS is predominantly expressed in a limited number of tissues and cell types, suggesting that these transcripts have physiological functions in distinct cell types and tissues. Moreover, real-time RT-PCR showed extremely low levels of GHRLOS in the foetal and adult liver and high levels in the Hep G2 hepatocarcinoma cell line, suggesting that GHRLOS expression may be altered in liver cancer. Indeed, it has recently been reported that non-coding RNA expression is frequently altered in cancer 5866. This indicates that non-coding RNAs may have specific functions in normal cells. Much like protein-coding transcripts, ncRNAs may act as tumour suppressors, or be upregulated in cancer and act as oncogenes. An examination of GHRLOS expression in cancer would, therefore, be of great interest.

Here we have characterised the structure and organisation of GHRLOS, a ghrelin antisense gene, suggesting that GHRLOS has multiple first exons. Therefore, it is possible that GHRLOS could be a part of very large, continuous ncRNA species in the 3p25 chromosomal region and beyond. In the absence of hallmarks, such as large open reading frames, mapping complex non-coding RNA genes remains a complex task. For example, two RNAs in the FMR1 locus, the cis-NAT ASFMR1 67 and the non-coding RNA gene FMR4 (found just upstream of FMR1 51) may be one continuous RNA isoform 51.

It is currently difficult and costly to determine the mechanism of function of long non-coding RNAs. The roles of non-coding RNAs are likely to be diverse, and there is strong evidence that they play a role in regulating important pathways. Many ncRNAs are expressed during development, neural differentiation, during macrophage activation and in cancer, indicating that they have key functions in these processes 126368. Non-coding RNAs have been found to play a role in the silencing of overlapping genes in cis 69, in the silencing of distant chromosome regions in trans 70, in nuclear trafficking 71, apoptosis 5172, promoter repression 73, and can act as tumour suppressors 74. Moreover, ncRNAs are emerging as markers for complex human disease, including lung cancer 75, heart disease 76, and a range of other pathologies 6877. GHRLOS may also serve as a host gene for snoRNA (small nucleolar RNAs) genes 78 or GHRLOS RNA transcripts may be precursors for short RNAs, such as micoRNAs 77, endogenous siRNAs 7980, piRNAs 81 and other novel, short non-coding RNA species 8283.

Although the understanding of natural antisense transcripts (NATs) remains in its infancy, they have been associated with a range of regulatory mechanisms that are not necessarily mutually exclusive. This includes transcriptional interference, RNA masking and dsRNA mediated gene-silencing via direct interaction between the sense and antisense transcripts 98485.

While it is difficult to predict GHRLOS function, the fact that all spliced GHRLOS variants share exon 1, which overlaps the 3' untranslated exon 4 of GHRL is striking. Our findings suggest that GHRLOS functions as a non-coding RNA. There are a few examples that suggest that antisense transcripts are important in the regulation of endocrine hormone receptors, including a thyroid hormone receptor 8687, and the luteinising hormone/choriogonadotropin receptor 88 gene and in the regulation of growth factors 89. Interestingly, it has been recently suggested that the invertebrate (insect) polypeptide hormone allatostatin may be regulated by cis-NATs 90. To date, however the physiological and pathophysiological roles of natural sense/antisense pairs have not been elucidated for any vertebrate endocrine hormone. Further studies are necessary to reveal the function and molecular mechanisms regulating the candidate non-coding RNA gene GHRLOS.

Multiple sequence alignments were generated using the MUltiple sequence Local AligNment and conservation visualization tool (Mulan) 91. Human [UCSC hg18], chicken [UCSC galGal3], frog [UCSC xenTro2], opossum [UCSC monDom4], mouse [UCSC mm9], rat [UCSC rn4], dog [UCSC canFam2], rhesus macaque [UCSC rheMac2], and chimpanzee [UCSC PanTro2] genomic sequences were obtained via the Evolutionary Conserved Regions (ECRs) Browser 92 and forwarded to Mulan to generate a full local alignment of the GHRLOS locus. GHRLOS was annotated based on the exons sequenced in this study.

To identify putative antisense exons and transcripts, we examined the publicly available Affymetrix Human Exon 1.0 ST Array tissue panel dataset consisting of 11 tissues (breast, cerebellum, heart, kidney, liver, muscle, pancreas, prostate, spleen, testes, and thyroid) using the Affymetrix All Exon track in the UCSC Genome Browser 93 and the Integrated Genome Browser (IGB) from Affymetrix 94.

To locate transcription start sites in the putative first exons of GHRLOS, CAGE (Cap Analysis of Gene Expression) tags (deposited by the RIKEN consortium and its collaborators) were obtained via the Genome Network Platform Viewer 95. We then recovered the RNA library information for each CAGE tag starting site. Briefly, each CAGE tag was individually queried against the 1.4 GB CAGE tag sequencing file, (release date 13.11.2006) available on the Genome Network Platform website using the UNIX grep command 96.

The exon-intron-structure of ESTs and mRNA entries identified from BLAST searches, as well as sequenced PCR amplicons obtained in this study, were analysed against the human genome (NCBI release 35) using GMAP 14. Presence of open reading frames was analysed by NCBI ORF Finder 97, Fickett's TestCode 9899 and ESTScan2 100101. The presence of transposable elements in GHRLOS sequence was examined using CENSOR v4.2.8 22. Protein domain analysis was performed using the SMART database 102.

The following cell lines (originally obtained from the American Type Culture Collection/ATCC, Rockville, MD unless specified) were cultured in their recommended media: Prostate and/or prostate cancer derived cell lines DU145 (ATCC HTB-81), RWPE-1 (ATCC CRL-11609), RWPE-2 (ATCC CRL-11610), LNCaP (ATCC CRL-1740), 22Rv1 (ATCC CRL-2505) and PC3 (ATCC CRL-1435), HEK293 human embryonic kidney (ATCC CRL-1573), SAOS-2 osteosarcoma (ATCC HTB-85), Hep G2 hepatocarcinoma (ATCC HB-8065), U-87 MG and U-251 MG glioblastoma (ATCC HTB-14 and JCRB Cell Bank # IFO50288, respectively), CaCo-2 colorectal adenocarcinoma (ATCC HTB-37), SW1353 chondrosarcoma (ATCC HTB-94), and OVCAR-3 ovarian cancer (ATCC HTB-161). All cells were grown in T80 or T175 flasks (Nagle Nunc International, Roskilde, Denmark) in 95% CO₂ in a Sanyo incubator at 37°C. Total RNA was harvested from cultured cells at 70% confluence using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions.

Tissue Total RNA was obtained from the stomach, prostate (FirstChoice, Ambion, Austin, TX), foetal liver, adrenal gland, liver, trachea, salivary gland, spinal cord, skeletal muscle, lung, placenta, bone marrow, kidney, heart, whole brain, thyroid, cerebellum, uterus, foetal brain, testis, thymus (Human total RNA Master Panel II, Clontech, Mountain View, CA) and pancreas (Clontech).

To further characterise the 5' end of the putative ghrelin antisense RNAs, 5' RACE was undertaken using FirstChoice RLM-RACE-Ready human stomach cDNA (Ambion) according to the manufacturer's instructions. The first round PCR was performed with an adapter-specific sense primer (5'adapter-out-F, Table 1) and an exon 2-specific antisense primer (5'OS-out-R in Table 1). PCR product (1 μl) was used in a secondary, nested PCR with a gene specific primer in exon 2 (5'adapter-in-F and 5'OS-in-R, Table 1). PCRs were performed in a total reaction volume of 50 μl using 1 U of Platinum Taq Polymerase High Fidelity (Invitrogen) according to the manufacturer's instructions.

For GHRLOS 3' RACE, human stomach and PC3 prostate cancer cell line total RNA was reverse transcribed using Transcriptor reverse transcriptase (Roche Applied Science Applied Science, Penzberg, Germany) and 10 μM adapter primer (3'-RACE-adapter, Table 1) from the FirstChoice RLM-RACE Kit (Ambion). 3' RACE was performed with 2 μl of this cDNA. Two 3' RACE reactions were performed – one combined an exon 4 GHRLOS-specific forward primer and an adapter-specific reverse primer (3'4F and 3'2OR, Table 1), and the other used an adapter-specific reverse primer and an exon 2-specific forward primer 3'2OR/F, Table 1). PCR products were then diluted and used in a secondary, nested PCR with a gene-specific forward and a reverse adapter primer (3'2IF/R, Table 1). PCR products were purified using a High Pure PCR purification kit (Roche Applied Science), cloned into pCR-XL-TOPO (Invitrogen) or pGEM-T Easy (Promega, Madison, WI), transformed into DH5α chemical competent cells (Invitrogen) and sequenced at the Australian Genome Research Facility (AGRF, Brisbane, Australia) using BigDye III (Applied Biosystems, AB, Foster City, CA).

To simultaneously obtain the 5' and 3' ends of GHRLOS transcripts, we employed Rolling Circle Amplification-RACE (Rapid Amplification of cDNA Ends 103, an improved inverse PCR approach. Briefly, 3 μg stomach, prostate, RWPE-1 cell line and PC3 prostate cancer cell line total RNA were reverse transcribed using 10 U of Transcriptor reverse transcriptase (Roche Applied Science) and 100 μM HPLC-purified 5'-end phosphorylated oligo d(T)-adapter primer (Phospo-dT, Table 1) (Proligo, Boulder, CO) according to the manufacturer's instructions. The single-stranded cDNA was purified using a High Pure PCR purification kit (Roche Applied Science) and eluted in 50 μl elution buffer (10 mM Tris-HCl, pH 8.5). Next, 25 μl purified linear cDNA was circularised using 100 U of CircLigase (EPICENTRE Biotechnologies, Madison, WI) and purified as before. After self-ligation, 15 μl circular cDNA was added to a rolling circle amplification reaction with 10 U of φ 29 DNA polymerase (NEB) and 10 μM HPLC-purified random hexamer primers with two phosphothioate linkages on their 3'ends (Pthioate-hex, Table 1) (Proligo). Following a 21 h incubation at 30°C in a waterbath, RCA-products were subjected to two rounds of inverse PCR with 1 U of Platinum Taq HIFI polymerase (Invitrogen), as per manufacturer's instructions. PCRs were performed in a reaction volume of 50 μl using a PTC-200 thermocycler (MJ Research, Waltham, MA). For the first round amplification, the RCA reaction was diluted 1/100 in water, and 1 μl used in an outer PCR with a forward primer at the start of exon 4 and a reverse primer in exon I (IPCR-out-F and IPCR-ALL-R, Table 1). After 35 cycles at a 60°C annealing temperature, the outer PCR product was diluted 100 times in water and 1 μl was used in a hemi-nested PCR of 20 cycles, with annealing at 60°C (IPCR-in-F and IPCR-ALL-R, Table 1). Amplification products were eluted from agarose gels in 50 μl water overnight, reamplified, cloned into pCR-XL-TOPO (Invitrogen), transformed into One Shot MAX Efficiency DH5α-T1R chemically competent cells (Invitrogen) and sequenced at the Australian Genome Research Facility (AGRF, Brisbane, Australia).

Initially, a cRNA probe spanning exon I, II, 1 and 2 of GHRLOS was employed. Briefly, a 5' RACE clone in pGEM-T Easy was linearised with SalI restriction enzyme and a cRNA probe was synthesised using T7 RNA polymerase and a digoxigenin (DIG) RNA labelling kit (Roche Applied Science). Probe concentration was estimated by dot blot comparison with digoxigenin-labelled standards. 500 ng stomach poly(A)⁺ RNA (FirstChoice, Ambion) was separated on a 1.2% formaldehyde gel and blotted, as described previously 103. Samples were electrophoresed with 50 ng RNA Molecular Weight Marker II (Roche Applied Science). The blot was hybridised to 50 ng/mL DIG-labelled cRNA probe overnight. Prehybridisation and hybridisation was performed with DIG-Easy Hyb (Roche Applied Science) at 65°C. The membranes were washed twice for 5 min at room temperature with 1 × Saline-Sodium Citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) and then washed three times for 10 min at 65°C with 0.1 × SSC, 0.1% SDS. The membrane was then reacted with an alkaline phosphatase (AP)-conjugated anti-DIG antibody (Roche Applied Science). AP activity was detected using a chemiluminescence method using CDP-Star (Roche Applied Science).

A second cRNA probe, which spanned exon 1 (which is common to all known GHRLOS mRNA isoforms) was synthesised from 100 ng human stomach genomic DNA (BioChain, Hayward, CA) using the PCR method 104 (Ex1-cRNA-F/R, Table 1). The PCR product was purified using a High Pure PCR Product Purification Kit (Roche Applied Science) and the DIG-labelled cRNA probe synthesised and quantified as detailed above. A multi-tissue membrane containing poly(A)⁺ RNA from 12 human tissues (brain, duodenum, oesophagus, pancreas, PBL/leukocytes, prostate, salivary gland, testis, thymus, thyroid, urinary bladder and uterus) was purchased from OriGene (Rockville, MD). Prehybridisation and hybridisation were performed as described above, except that ULTRAhyb Ultrasensitive Hybridization Buffer (Ambion) was used instead of DIG-Easy Hyb (Roche Applied Science). Equivalent loading between tissues on the blot was determined by rehybridising with 20 ng/mL DIG-labelled β-actin cRNA probe (Roche Applied Science).

For non-quantitative RT-PCR analysis of GHRLOS splicing, RT-PCRs were performed with a forward primer in a region common to the 5' terminal exon Ia/b and a reverse primer in the 3' terminal exon 4 of GHRLOS (Ito4-F/R, Table 1). cDNA was synthesised in a final volume of 20 μl from 3 μg total RNA from tissues and cell lines using 10 U of Transcriptor reverse transcriptase (Roche Applied Science), 20 U of RNasin Plus RNase Inhibitor (Promega) and a 3' RACE adapter primer (3'-RACE-adapter, Table 1) at 55°C according to the manufacturer's instructions. PCR amplicons from the stomach, prostate, foetal brain, heart, thymus, testis, and pancreas were purified, sub-cloned and sequenced as described above.

To detect long, chimaeric transcripts, we employed RT-PCR with a forward primer in exon 2 of TATDN2 (ChiOut-F, Table 1) and a reverse primer in exon 1 of GHRLOS (ChiOut-R, Table 1). PCR was carried out with 1 U of Platinum Taq HIFI polymerase (Invitrogen) as per manufacturer's instructions, extending at 68°C for 2.5 minutes per cycle. cDNA was synthesised as above in a final volume of 20 μl from 2 μg total RNA, from the Hep G2 hepatocarcinoma cell line, CaCo-2 colorectal adenocarcinoma cell line, OVCAR-3 ovarian cancer cell line, and from a range of normal tissues (testis, prostate, pancreas, thymus, and foetal brain). RT-PCR products were sub-cloned and sequenced as described above.

To determine if the identified upstream CAGE tag starting sites transcribe exons that belong to GHRLOS, we employed RT-PCR using a forward primer designed to the region immediately after a CAGE cluster in the ~2 kb 3' untranslated region of the adjacent gene TATDN2 (TSS ID T03F009D1927) and a reverse primer in exon 1, an exon which is common to all known GHRLOS variants (F_CAGE and R_CAGEout in Table 1, respectively). cDNA was synthesised in a final volume of 20 μl from 2 μg total RNA, from the Hep G2 hepatocarcinoma cell line and the thymus and foetal brain, using 10 units Transcriptor reverse transcriptase (Roche Applied Science), 20 U of RNasin Plus RNase Inhibitor (Promega) and oligo(dT)₁₈ primers (Proligo) according to the manufacturer's instructions. The cDNAs were subjected to 35 cycles of PCR with a two-minute extension time per cycle, and then diluted 1/100 in water and subjected to a hemi-nested 30-cycle PCR with a nested primer in exon 1 (R_CAGEin, Table 1). PCRs were performed in a total reaction volume of 50 μl using 10 U of Platinum Taq Polymerase High Fidelity (Invitrogen) according to the manufacturer's instructions. Entire PCR products were purified using the High Pure PCR Product Purification Kit (Roche Applied Science), subcloned into pCR-XL-TOPO (Invitrogen) and transformed into One Shot MAX Efficiency DH5α-T1R chemical competent cells (Invitrogen). Insert-positive, purified clones were sequenced by the Australian Genome Research Facility (AGRF, Brisbane, Australia) using the AB PRISM BigDye Terminator Cycle Sequencing Kit v3.1 protocol (AB).

To verify the presence of a novel SEC13 exon identified in a brain tumour EST [GenBank:BF931280], cDNAs reverse transcribed with an oligo(dT) primer (as described above) were challenged by RT-PCR with primers in exon 8 of SEC13 and a reverse primer in the novel exon (231-F/R, Table 1, respectively).

To allow strand-specific and RNA-specific amplification 105106107 of GHRLOS transcripts, reverse transcription was performed using a gene-specific primer in exon 4 with a linker (LK) 107 sequence attached to the 5' end of the primer (GHRLOS-Real-RT-LK, Table 1). cDNA was generated from 1 μg total RNA using 40 U of AMV reverse transcriptase (Roche Applied Science) at 42°C, according to the manufacturer's instructions. The strand-specific, real-time RT-PCR was performed with an exon 4 specific forward primer, a reverse primer with the LK sequence only (GHRLOS-Real-F and LK, Table 1) and a TaqMan probe (Ex4-TaqMan, Table 1). To detect sense GHRL transcripts, we employed a strand-specific RT-PCR approach, with a reverse transcription primer spanning the 3' terminal exon 4 of the ghrelin gene (GHRLex4_RT_LK, Table 1) followed by PCR with an exon 4 specific forward primer (GHRLex4_F, Table 1) and a linker-specific reverse primer. (LK, Table 1). The relative quantification of GHRLOS and GHRL transcripts was estimated by direct normalisation to the threshold cycle (C_T) of the housekeeping gene, 18S ribosomal RNA (18S-Real-F/R, Table 1). 18S PCRs were used to normalise real-time data. As reported for GAPDH 107, 18S RNAs self-primes efficiently in reverse transcription reactions without the addition of random or gene-specific primers. All primers were designed using the Primer Express version 2.0 software (AB).

PCRs were performed in a total reaction volume of 20 μl using Platinum Quantitative PCR SuperMix-UDG w/ROX (Invitrogen) for GHRLOS, while GHRL and the housekeeping gene 18S ribosomal RNA were amplified using 2 × SYBR green master mix (AB). Controls included the use of cDNA, which was reverse transcribed using random hexamers as primers, as well as the reverse transcription of RNA in the absence of primer. Real-time RT-PCR was performed using the AB 7000 sequence detection system (AB) and data analysed using the absolute standard curve method (User Bulletin #2, AB) to determine expression levels in a range of tissues and cell lines. Briefly, we calculated values from duplicate reactions for each sample from standards, which were constructed from PCR products. Statistical significance was determined using the Student's t-test and, where applicable, one-way analysis of variance (ANOVA) with Tukey post-hoc analysis. P-values of < 0.05 were considered to be statistically significant. Data are represented as mean ± standard deviation (S.D.).