BMC Genomics 2007, 8:42 http://www.biomedcentral.com/1471-2164/8/42 for determining exact TSSs, such as 5' RACE or primer extension are laborious and are not selective for the com- plete transcript. Consequently, many mRNA sequences stored in public databases lack information about their genuine 5' ends, mainly due to the difficulties in obtain- ing full-length cDNA. Several bioinformatic and experi- mental approaches have been developed to explore full- length cDNAs and the human transcriptome [5]. Compu- tational predictions may represent a powerful tool to localize first exons and TSSs on an averaged genome-wide scale [6,7], however they may fail at the level of individual genes or in genes with complex regulatory patterns (e.g. multiple or tissue-specific TSS). Recently a number of experimental approaches to com- pile TSSs on a genome-wide scale have been established including the Database of human Transcriptional Start Sites (DBTSS) [8], whole genome tilling array analysis [9], and the exploration of mouse and human CAGE tag libraries [10]. To enable future progress we need to com- plete and revise these catalogues with an accurate annota- tion of the 5' and 3'end, and include splice isoforms of the transcripts. In addition to genome wide approaches, there is a need for more specific studies, which cover tissue spe- cific genes, expressed in a restricted manner. Identification of potential transcription signals that are tissue specific relies on the correct determination of transcriptional start sites. In this work we describe an experimental approach to identify the TSSs of a selected group of genes, which are predominantly expressed in retina. We focused our atten- tion on the human retina, due to its unique and special- ised function. This complex tissue, composed of multiple, highly differentiated and specific cell types (e.g. rod and cone photoreceptors, amacrine cells, Mueller glial cells), expresses a large number of specific genes. Mutations in many of these genes result in blinding disorders. The sub- set of genes expressed in human retina has been partially elucidated [11,12], with a number of studies defining genes that are either highly expressed in retina or which pose a crucial target of transcription factors in this tissue [13-16]. We selected a pool of retina expressed genes and employed Cap selective RACE to ensure amplification and subsequent cloning of genuine TSSs. We describe herein the results of this analysis, reporting the correct TSSs within this group of retinal transcripts. Results Genes Selection and in silico assembly 76 annotated genes were selected for analysis. The selec- tion was done based on the following criteria: (i) specific or high levels of expression in retina, (ii) a role in retina specific physiological processes or retinal development, and (iii) involvement in retinal disease. A compilation of all tested genes including gene symbol, definition, chro- mosomal location and tissue/cell type of expression is shown in Additional file 1. cDNA and transcript sequences available in public data- bases (RefSeq, NCBI and Ensemble gene predictions cov- ered by at least one EST, Unigene ESTs database) were downloaded and new assemblies generated using Seq- Man. We found that 5' transcript termini represented in public datasets can be readily identified by clusters of cDNA ends in the assemblies. Additionally, the information about putative TSSs was assessed in The Eukaryotic Promoter Database [17] and Database of Transcriptional Start Sites (DBTSS) [8]. These data were compiled to create a prelim- inary gene model which was used to design primers for the subsequent Cap-finder RACE experiments. Experimental examination of TSSs Cap-finder RACE cDNA fragments were cloned and a var- iable number of clones were sequenced for each gene, depending on the number and the sizes of the colony PCR products detected on the gel. We obtained products for 54 genes out of the 76 genes analysed. A summary of the results obtained with Cap-finder RACE is shown in Tables 1. Genes for which the promoter and TSS were already known (RHO and OPN1SW) served as internal positive controls. For each gene we detected at least one splice var- iant that agreed with one or more RefSeq annotated exons. Our strategy relies on the location of gene specific primers within internal exons. We obtained those cDNA products that covered at least one exon-exon junction and thus ruled out the possibility of amplification of genomic con- tamination. This strategy has enabled us to identify alter- natively spliced 5' ends that arise from tissue specific gene expression and regulation. Table 1 lists the results of the Cap-finder RACE experi- ments for 54 retinal expressed genes and the correspond- ing RefSeq entry (database release 18). These results can be grouped into five categories with reference to RefSeq; (i) new TSSs within novel exons (8 genes), (ii) alternative splice form of the second exon (2 genes: IMPG1, SAG), (iii) extension of the annotated first exon (27 genes), (iv) length shortening of the annotated first exon (4 genes), (v) confirmation of previously annotated TSSs (13 genes). Table 1 provides the exact nucleotide positions of 5' ter- mini of the Cap-finder RACE cDNA clones referring to the UCSC Human Genome Browser (March 2006 assembly). In defining the interval where TSSs are located we report the start, and when present, the internal frequent start and end nucleotide position of each TSS. Sequences from this Page 2 of 14 (page number not for citation purposes)