title: A real-time polymerase chain reaction-based protocol for low/medium-throughput Y-chromosome microdeletions analysis.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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 2 h without the need of post-PCR manipulation.
authors.affiliation: Institute for Maternal and Child Health-IRCCS Burlo Garofolo, Trieste, Italy. segat@burlo.trieste.it
authors.forename: Ludovica
authors.initials: L
authors.lastname: Segat
authors.forename: Lara
authors.initials: L
authors.lastname: Padovan
authors.forename: Darja
authors.initials: D
authors.lastname: Doc
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Petix
authors.forename: Marcello
authors.initials: M
authors.lastname: Morgutti
authors.forename: Sergio
authors.initials: S
authors.lastname: Crovella
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ricci
mesh_terms: D053713:Azoospermia / Q000235:genetics; D002872:Chromosome Deletion; D041322:Chromosomes, Human, Y / Q000235:genetics; D005260:Female; D006801:Humans; D007248:Infertility, Male; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D008297:Male; D060888:Real-Time Polymerase Chain Reaction; D012729:Sex Chromosome Aberrations; D058533:Sex Chromosome Disorders of Sex Development / Q000175:diagnosis* / Q000235:genetics
chemical_list: D051741:Kruppel-Like Transcription Factors; C054430:ZFY protein, human