Quantitative real-time polymerase chain reaction for the verification of genomic imbalances detected by microarray-based comparative genomic hybridization.
The American College of Medical Genetics guidelines for microarray analysis for constitutional cytogenetic abnormalities require abnormal or ambiguous results from microarray-based comparative genomic hybridization (aCGH) analysis be confirmed by an alternative method. We employed quantitative real-time polymerase chain reaction (qPCR) technology using SYBR Green I reagents for confirmation of 93 abnormal aCGH results (50 deletions and 43 duplications) and 54 parental samples. A novel qPCR protocol using DNA sequences coding for X-linked lethal diseases in males for designing reference primers was established. Of the 81 sets of test primers used for confirmation of 93 abnormal copy number variants (CNVs) in 80 patients, 71 sets worked after the initial primer design (88%), 9 sets were redesigned once, and 1 set twice because of poor amplification. Fifty-four parental samples were tested using 33 sets of test primers to follow up 34 CNVs in 30 patients. Nineteen CNVs were confirmed as inherited, 13 were negative in both parents, and 2 were inconclusive due to a negative result in a single parent. The qPCR assessment clarified aCGH results in two cases and corrected a fluorescence in situ hybridization result in one case. Our data illustrate that qPCR methodology using SYBR Green I reagents is accurate, highly sensitive, specific, rapid, and cost-effective for verification of chromosomal imbalances detected by aCGH in the clinical setting.
Yu
Shihui
S
Department of Pathology, Children's Mercy Hospitals and Clinics, and University of Missouri-Kansas City School of Medicine, Kansas City, Missouri, USA.
Kielt
Matthew
M
Stegner
Andrew L
AL
Kibiryeva
Nataliya
N
Bittel
Douglas C
DC
Cooley
Linda D
LD
D052160:Benzothiazoles; D002675:Child, Preschool; D055028:Comparative Genomic Hybridization / Q000592:standards*; D003959:Diamines; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry; D005820:Genetic Testing / Q000592:standards*; D042822:Genomic Instability / Q000235:genetics*; D006801:Humans; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis / Q000592:standards*; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D013997:Time Factors
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I