BMC Genomics 2007, 8:42 http://www.biomedcentral.com/1471-2164/8/42 CAN, implemented in the UCSC Genome Browser) and gene annotation that are confirm by at least one EST [43]. To evaluate first exons conservation we used BioEdit pair- wise and multiple alignments [44] Primer design Gene specific reverse primers were selected within exons other than the first exon to obtain spliced products. Two primers were chosen for each gene, in order to perform a nested PCR, which allows to enhance specificity, and to obtain a sufficient amplification product for rare tran- scripts. Primers were designed using the Primer3 software [45], and checked for uniqueness by querying against the human genome. RACE protocol We applied the RNA-ligase-mediated RACE (RLM-RACE) system from Ambion. RNA sample from adult human ret- ina was treated with DNAse I. After DNase treatment and inactivation, 10 μg of total RNA was dephosphorylated for 60 min at 37°C with 10 U Calf Intestinal Phosphatase to remove the 5'-phosphate from all RNA species except those that have a cap structure (present on all Pol II tran- scripts). RNA was then phenol/chloroform extracted, pre- cipitated and re-suspended in water. Dephosphorylated RNA was then digested for 60 min at 37°C in a 10-μL reac- tion with 10 U tobacco acid pyrophosphatase. Subse- quently the RNA was incubated for 60 min at 37°C with 1 U T4 RNA ligase and 0.3 μg of an RNA adapter (5' RACE Adapter 5'-GCU-GAU-GGC-GAU-GAA-UGA-ACA-CUG- CGU-UUG-CUG-GCU-UUG-AUG-AAA-3'). After liga- tion, 180 ng of RNA was incubated for 2 min at 75°C in the presence of 5 μM random decamers in RT buffer. Sin- gle-stranded cDNA was generated by the addition of 100 U M-MLV RT and incubation at 42°C for 60 min. Amplification of 5' RACE cDNA was carried out using nested gene-specific primers and adapter specific primers and with 1 μl of the first-strand cDNA reaction. PCR reac- tions were done in 50 μl volume including 5 μl 10× PCR Buffer supplied in the RLM kit, 4 μl dNTP Mix 10 mM, 2 μl 5' RACE gene-specific outer primer (10 μM), 2 μl 5' RACE Outer or Inner Primer (10 μM), and 1 U thermosta- ble DNA polymerase. Cycling conditions were: 5 min ini- tial denaturation at 94°C PCR followed by 35 cycles of 95°C for 30 s, 60-55°C (empirically determined) for 30 s (annealing), 72°C for 30 s (extension) and a final exten- sion at 72°C for 7 mins. Amplified products were ana- lysed on a 3% agarose gel and visualised by ethidium bromide staining. Cloning and sequencing of RACE products 5' RACE products were cloned into pCR-2.1 vector (Invit- rogen). 2 μL of PCR reaction were incubated with 0.1 ng of vector at 16°C over-night. Aliquots of the ligation were used to transform library efficiency chemically competent E. coli DH5α (Invitrogen). 8 to 48 clones of each transfor- mation were subjected to colony PCR and the inserts sequenced with standard M13 forward and reverse prim- ers applying Big Dye Terminator v3.0 chemistry (Applied Biosystems). Sequencing products were separated and analysed on an ABI 3100 DNA sequencer. Sequence analysis Gene sequences and cDNA sequences, obtained using the RACE, were aligned to the human genome using BLAT. cDNA sequence was considered informative only if the following criteria were met: (I) the spliced sequence mapped to the same region of the genome as the gene sequence; (II) the product could be mapped uniquely to the genome with >95% identity and (III) presence of the gene specific primer sequence and the 5' RACE Adapter primer sequence. Primer extension A fluorescein phosphoramidites (FAM)-labelled reverse primer was added to 10 μg of DNAseI-treated total RNA to a final concentration of 5 nM. Samples were heated at 70°C for 5 min followed by 20 min incubation at 58°C and then allowed to cool for 15 min to room temperature. First strand cDNA synthesis was performed using Avian Myeloblastoma Virus (AMV) reverse transcriptase and 5× AMV-RT buffer (Promega) according to the manufac- turer's instructions in a final volume of 60 μl. The primer extension reaction was done in two repeated reaction cycles. After an initial reverse transcription step (60 min at 42°C in a total volume of 50 μl), enzyme was replenished and the samples underwent a second extension reaction (60 min at 42°C, adjusting the buffer to a total volume of 60 μl). Finally 3 μl of 5 M NaOH was added to each cDNA sample, the reaction incubated at 37°C for 15 min and then neutralised with 16 μl of 2 M HEPES free acid. Exten- sion products were purified using the columns AutoSeq G-50 (Amersham Pharmacia Biotech). Samples were sep- arated on 50-cm capillary columns in the POP4 acryl amide polymer (Applied Biosystems) on an ABI PRISM 3100 Genetic Analyser (Applied-Biosystems) Sequencer with GENESCAN 500-ROX added as size standard (Applied-Biosystems). The sequences of the gene specific reverse primers were: RDH12 5' tgagcagcagcctgactctgag- caga gcccaga 3', CNGA3 5'atcttctcggtttgtcacatttagc 3', with 5' ends modified with the fluorescent molecule 6-FAM. Authors' contributions VR contributed to acquisition, analysis, interpretation of data and drafting the manuscript; RC has been involved in acquisition, analysis and interpretation of data and draft- ing the manuscript; BW conceived and coordinated the study and contributed materials and resources. All authors read and approved the final manuscript. Page 11 of 14 (page number not for citation purposes)