A real-time polymerase chain reaction-based protocol for low/medium-throughput y-chromosome microdeletions analysis

Ludovica Segat, Lara Padovan, Darja Doc, Vincenzo Petix, Marcello Morgutti, Sergio Crovella, Giuseppe Ricci

Research output: Contribution to journalArticlepeer-review


Purpose: We describe a real-time polymerase chain reaction (PCR) protocol based on the fluorescent molecule SYBR Green chemistry, for a low-to medium-throughput analysis of Y-chromosome microdeletions, optimized according to the European guidelines and aimed at making the protocol faster, avoiding post-PCR processing, and simplifying the results interpretation. Methods: We screened 156 men from the Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Institute for Maternal and Child Health IRCCS Burlo Garofolo (Trieste, Italy), 150 not presenting Y-chromosome microdeletion, and 6 with microdeletions in different azoospermic factor (AZF) regions. For each sample, the Zinc finger Y-chromosomal protein (ZFY), sex-determining region Y (SRY), sY84, sY86, sY127, sY134, sY254, and sY255 loci were analyzed by performing one reaction for each locus. Results: AZF microdeletions were successfully detected in six individuals, confirming the results obtained with commercial kits. Conclusion: Our real-time PCR protocol proved to be a rapid, safe, and relatively cheap method that was suitable for a low-to medium-throughput diagnosis of Y-chromosome microdeletion, which allows an analysis of approximately 10 samples (with the addition of positive and negative controls) in a 96-well plate format, or approximately 46 samples in a 384-well plate for all markers simultaneously, in less than 2h without the need of post-PCR manipulation.

Original languageEnglish
Pages (from-to)1349-1355
Number of pages7
JournalGenetic Testing and Molecular Biomarkers
Issue number12
Publication statusPublished - Dec 1 2012

ASJC Scopus subject areas

  • Genetics(clinical)


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