Amplified RNA degradation in T7-amplification methods results in biased microarray hybridizations

BACKGROUND: The amplification of RNA with the T7-System is a widely used technique for obtaining increased amounts of RNA starting from limited material. The amplified RNA (aRNA) can subsequently be used for microarray hybridizations, warranting sufficient signal for image analysis. We describe here an amplification-time dependent degradation of aRNA in prolonged standard T7 amplification protocols, that results in lower average size aRNA and decreased yields. RESULTS: A time-dependent degradation of amplified RNA (aRNA) could be observed when using the classical "Eberwine" T7-Amplification method. When the amplification was conducted for more than 4 hours, the resulting aRNA showed a significantly smaller size distribution on gel electrophoresis and a concomitant reduction of aRNA yield. The degradation of aRNA could be correlated to the presence of the T7 RNA Polymerase in the amplification cocktail. The aRNA degradation resulted in a strong bias in microarray hybridizations with a high coefficient of variation and a significant reduction of signals of certain transcripts, that seem to be susceptible to this RNA degrading activity. The time-dependent degradation of these transcripts was verified by a real-time PCR approach. CONCLUSIONS: It is important to perform amplifications not longer than 4 hours as there is a characteristic 'quality vs. yield' situation for longer amplification times. When conducting microarray hybridizations it is important not to compare results obtained with aRNA from different amplification times

Cross-platform gene expression signature of human spermatogenic failure reveals inflammatory-like response

BACKGROUND The molecular basis of human testicular dysfunction is largely unknown. Global gene expression profiling of testicular biopsies might reveal an expression signature of spermatogenic failure in azoospermic men. METHODS Sixty-nine individual testicular biopsy samples were analysed on two microarray platforms; selected genes were validated by quantitative real-time PCR and immunohistochemistry. RESULTS A minimum of 188 transcripts were significantly increased on both platforms. Their levels increased with the severity of spermatogenic damage and reached maximum levels in samples with Sertoli-cell-only appearance, pointing to genes expressed in somatic testicular cells. Over-represented functional annotation terms were steroid metabolism, innate defence and immune response, focal adhesion, antigen processing and presentation and mitogen-activated protein kinase K signalling pathway. For a considerable proportion of genes included in the expression signature, individual transcript levels were in keeping with the individual mast cell numbers of the biopsies. When tested on three disparate microarray data sets, the gene expression signature was able to clearly distinguish normal from defective spermatogenesis. More than 90% of biopsy samples clustered correctly into the corresponding category, emphasizing the robustness of our data. CONCLUSIONS A gene expression signature of human spermatogenic failure was revealed which comprised well-studied examples of inflammation-related genes also increased in other pathologies, including autoimmune disease

A One-Step Real-Time Multiplex PCR for Screening Y-Chromosomal Microdeletions without Downstream Amplicon Size Analysis

BACKGROUND: Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. PRINCIPAL FINDINGS: The widely established "EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)" were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. SIGNIFICANCE: With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection

Human spermatogonial markers

In this review, we provide an up-to-date compilation of published human spermatogonial markers, with focus on the three nuclear subtypes Adark, Apale and B. In addition, we have extended our recently published list of putative spermatogonial markers with protein expression and RNA-sequencing data from the Human Protein Atlas and supported these by literature evidence. Most importantly, we have put substantial effort in acquiring a comprehensive list of new and potentially interesting markers by refiltering the raw data of 15 published germ cell expression datasets (four human, eleven rodent) and subsequent building of intersections to acquire a robust, cross-species set of spermatogonia-enriched or -specific transcripts