No abstract

Studying the molecular basis of Non-Obstructive Azoospermia (NOA), a type of male infertility with failed spermatogenesis at various stages, can also help in exploring molecular basis of human spermatogenesis and possibly pave way to identify new targets for male contraceptive development. Hence, we initiated a functional genomics study by applying RNA-seq. Testicular biopsies collected from donors with Non-Obstructive Azoospermia (NOA), Obstructive Azoospermia (OA), Congenital Bilateral Absence of the Vas Deferens (CBAVD), and Varicocele (VA) conditions. Strong association of 100+ genes with human spermatogenesis and NOA has been detected via NGS-based transcriptomic analysis. In addition, 20 RNA molecules have been short-listed for potential diagnostic applications (non-obstructive azoospermia vs. obstructive azoospermia, varicocele or normal). A hierarchical list of several genes and alternatively spliced mRNAs, transcribed differentially in NOA, is reported -based on a 'strength of association'. Such association with NOA, spermatogenesis or both is a new finding for many genes as revealed by a comparison with a newly prepared comprehensive list of genes having such association with human spermatogenesis/NOA. Many top-ranking genes involved in viral gene expression were up-regulated in testes from NOA-patients, while those associated with an antiviral mechanism were down-regulated. A tangential finding: while most well-established control mRNAs did not qualify, two new ones worked best in RT-qPCR experiments. Needle-aspiration of testicular biopsies, followed by the use of short-listed promising candidate biomarkers (i.e., 16 mRNA & 4 chimeric transcripts) and control mRNAs in RT-qPCR-based diagnostic assays, may help to avoid open surgeries in future.

BackgroundStandard 3′ Affymetrix gene expression arrays have contributed a significantly higher volume of existing gene expression data than other microarray platforms. These arrays were designed to identify differentially expressed genes, but not their alternatively spliced transcript forms. No resource can currently identify expression pattern of specific mRNA forms using these microarray data, even though it is possible to do this.ResultsWe report a web server for expression profiling of alternatively spliced transcripts using microarray data sets from 31 standard 3′ Affymetrix arrays for human, mouse and rat species. The tool has been experimentally validated for mRNAs transcribed or not-detected in a human disease condition (non-obstructive azoospermia, a male infertility condition). About 4000 gene expression datasets were downloaded from a public repository. ‘Good probes’ with complete coverage and identity to latest reference transcript sequences were first identified. Using them, ‘Transcript specific probe-clusters’ were derived for each platform and used to identify expression status of possible transcripts. The web server can lead the user to datasets corresponding to specific tissues, conditions via identifiers of the microarray studies or hybridizations, keywords, official gene symbols or reference transcript identifiers. It can identify, in the tissues and conditions of interest, about 40% of known transcripts as ‘transcribed’, ‘not-detected’ or ‘differentially regulated’. Corresponding additional information for probes, genes, transcripts and proteins can be viewed too. We identified the expression of transcripts in a specific clinical condition and validated a few of these transcripts by experiments (using reverse transcription followed by polymerase chain reaction). The experimental observations indicated higher agreements with the web server results, than contradictions. The tool is accessible at http://resource.ibab.ac.in/TIPMaP.ConclusionThe newly developed online tool forms a reliable means for identification of alternatively spliced transcript-isoforms that may be differentially expressed in various tissues, cell types or physiological conditions. Thus, by making better use of existing data, TIPMaP avoids the dependence on precious tissue-samples, in experiments with a goal to establish expression profiles of alternative splice forms – at least in some cases.