title: A simple multiplex real-time PCR methodology for the SMN1 gene copy number quantification.
abstract: Spinal muscular atrophy (SMA) is an autosomal recessive disease caused, in about 95% of SMA cases, by homozygous deletion of the survival motor neuron 1 (SMN1) gene or its conversion to the highly homologous SMN2 gene. The molecular diagnosis of SMA is usually carried out by a PCR-Restriction fragment length polymorphism (RFLP) approach. However, this approach is not useful for identification of healthy deletion carriers. TaqMan technology is one of the most reliable and widely adopted techniques for the SMN1 copy number evaluation. However, several limitations of this technique have been described. Particularly, DNA extraction methods and accurate template quantification have been shown to be critical for reliable results. In this work, we set up a reliable, highly reproducible, and easy-to-perform TaqMan technology-based protocol to obtain the SMN1 gene copy number assessment. We demonstrate that PCR amplification of both target gene and reference gene in the same reaction mix, instead of separated mixes, greatly reduces reported criticisms of simplex TaqMan technology. The multiplex real-time PCR we describe allows interlaboratory samples and data exchange, without the need to equalize the DNA isolation technique. Further, the protocol described below requires fewer replica tests than the simplex methodology does, leading to reduced overall cost for the diagnostic assay.
authors.affiliation: Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi, Udine, Italy.
authors.forename: Nadia
authors.initials: N
authors.lastname: Passon
authors.forename: Federico
authors.initials: F
authors.lastname: Pozzo
authors.forename: Cristiano
authors.initials: C
authors.lastname: Molinis
authors.forename: Elisa
authors.initials: E
authors.lastname: Bregant
authors.forename: Cinzia
authors.initials: C
authors.lastname: Gellera
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Damante
authors.forename: Renata I
authors.initials: RI
authors.lastname: Lonigro
mesh_terms: D001483:Base Sequence; D002648:Child; D004252:DNA Mutational Analysis / Q000379:methods / Q000706:statistics & numerical data; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D005260:Female; D017353:Gene Deletion; D018628:Gene Dosage; D006580:Genetic Carrier Screening / Q000379:methods; D005820:Genetic Testing / Q000379:methods / Q000706:statistics & numerical data; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D015203:Reproducibility of Results; D055532:SMN Complex Proteins / Q000235:genetics; D014897:Spinal Muscular Atrophies of Childhood / Q000175:diagnosis / Q000235:genetics*; D055533:Survival of Motor Neuron 1 Protein / Q000235:genetics*; D055540:Survival of Motor Neuron 2 Protein
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D055532:SMN Complex Proteins; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein