By incorporation of 32P-dCTP as described [42].Direct testis cDNA selection and sequencingConclusions The identification of lineage-specific Y genes recapitulates the diversity of Y gene content and signifies the importance of comparative studies of the mammalian Y-chr. This study provides a base for further research on Y-chr evolution and male-fertility. As the expression of the bovine ZNF280B/ZNF280BY and ZNF280A/ ZNF280AY is predominant in the testis, their autosomal orthologs in other eutherians may play roles in spermatogenesis. We believe that additional functional analysis of ZNF280B/ZNF280BY and ZNF280A/ZNF280AY will offer insights into gene regulation in spermatogenesis, the evolution of the mammalian Y-chr and advance the assembly of the bovine Y-chr sequence. MethodsRNA extraction and cDNA synthesisTotal RNA was extracted from bovine testicular tissue at 4 days, 20 days, 3 months, 8 months and 2 years of age with Trizol?reagent (Invitrogen, Carlsbad, CA, USA). Equal amounts of total RNA from different age groupsDirect testis cDNA selection was carried out as described [42]. After a pre-hybridization step with bovine Cot-1 DNA (Applied Genetics Laboratories, Melbourne, FL, USA) to block the repetitive elements, the adaptor-ligated cDNA was PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25105516 hybridized with the biotinylated BTAY probe for 50 hr in 0.75 mM NaCl, 20 mM sodium phosphate (pH 7.2), 5 mM EDTA, 5?Denhardt’s solution and 0.1 SDS. Hybridized cDNA was isolated with streptavidin paramagnetic Dynabeads M-280 (Invitrogen, Carlsbad, CA, USA) per the manufacturer’s instructions. After washing twice with wash solution I (1 ?SSC, 0.1 SDS) for 15 min at room temperature to remove the unbound cDNAs, and washing with wash solution II (0.1 ?SSC, 0.1 SDS) 4 times, 20 min each at 65 to remove the non-specifically bound cDNAs, the selected cDNA was eluted from the beads by 1 ?SSC at 95 for 5 min, and then amplified by PCR with the adaptor oligo 1 as the primer. Selection efficiency was assessed by qPCR with Y-linked genes, SRY and DDX3Y, as positive controls and, b-Actin and CDYL, as negative controls. PCR products were clonedYang et al. BMC Genomics 2011, 12:13 http://www.biomedcentral.com/1471-2164/12/Page 11 ofusing a TOPO-TA cloning kit (Invitrogen, Carlsbad, CA, USA). A total of 2,208 random clones were grown overnight at 37 in 2 ml, 96-deep-well culture plates. Dotblotting the clones with BTAY fragments yielded 753 (most likely) non-redundant clones. Plasmid DNA was purified by alkaline lysis (Qiagen, Valencia, CA, USA), and sequenced on an ABI-3730XL DNA analyzer at the Pennsylvania State University Genomics Core Facility.RT-PCRTotal RNA was extracted from eight different tissues (including testis, liver, kidney, spleen, cerebellum, adrenal gland, longissimus muscle, and lymph node) of a two years old bull and ovarian tissue from a mature cow, treated with DNase I (Ambion, Austin, TX, USA) and reverse transcribed using SuperScriptTM III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed with gene-specific primers (GSPs) (Additional file 7) in a 20 l volume containing 10 ng cDNA, 200 M dNTPs, 1.5 mM MgCl 2 , 2.5 M of each primer and 1 unit Taq DNA polymerase (Bioline, Taunton, MA, USA). The PCR conditions were: 94 for 7 min followed by 35 Conduritol B epoxide cycles each of 95 for 40 sec, 55-65 for 40 sec, 72 for 40 sec, with a final extension at PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27363707 72 for 7 min. Products were resolved on 1.5 agarose gels with ethidium bromide in 1 ?TAE buffer.RACEselected 1.
