The tissue expression profile of TSGA10 mRNA throughout various organs was studied by quantitative PCR performed on cDNA from human tissue. Primers were designed with Beacon Designer® version 5.11 software (Premier Biosoft, Palo Alto, CA, USA) with one primer flanking an intron–exon junction to avoid amplification of genomic DNA. Quantitative PCR was carried out on human normalized multiple-tissue cDNA panels (BD Bio Sciences, Palo Alto, CA, USA) as well as pituitary, aorta (Stratagene
Cloning Systems) and adrenal cortex cDNA prepared from normal adrenal tissue removed during adrenal adenoma surgery. Reactions were performed on a MyiQ iCycler (Bio-Rad, Hercules, CA, USA) in a volume of 25 μl, with 200 nm of each primer using iQ™ SYBR®Green Selleckchem MG132 supermix (Bio-Rad) as per the manufacturer’s instructions. All samples were run in triplicate. Thermal cycles consisted of an initial denaturation step of 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. Standard curves were then established from the serial dilution of TSGA10 and control glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) PCR templates. TSGA10 mRNA levels were deduced from the standard curve and normalized to the endogenous GAPDH tissue content. A total of 27 cDNA clones were isolated and identified from immunoscreening of a human pituitary cDNA expression library with the sera from two APS1 patients, one with clinical GH deficiency and one with no reported pituitary manifestations. These clones represented 11 different proteins of AZD1208 ic50 which one was TPH isoform 1, a well-known APS1 autoantigen . Recombinant proteins Chlormezanone from the remaining 10 cDNA clones were produced by ITT and immunoprecipitation was performed against a test panel of sera from six APS1 patients and five healthy blood donors to determine the possible antigenicity. Most of these recombinant products were recognized solely by the screening serum, by both APS1 sera and control sera or by none of the sera. A single clone TDRD6, isolated from the patient with pituitary manifestation was further analysed
and found in 49% of APS1 patients as reported previously . An additional cDNA clone isolated from the patient without any pituitary deficits encoded testis specific, 10 (TSGA10), a gene located on chromosome 2q11.2. ITT of two of the TSGA10 clones resulted in good quantities of recombinant proteins that were used for immunoprecipitation with the test panel of sera. Both TSGA10 recombinant proteins were efficiently immunoprecipitated by the screening serum but not by any of the healthy controls; one of the corresponding TSGA10 clones was therefore selected for further studies. The TSGA10 gene consists of 19 exons spanning over 80 kb of genomic DNA. Two transcript variants have been reported, differing in the 5′ UTR. Both variants are transcribed from exon 6 to exon 21 and encode a 698 amino acid protein.