Detecting Allelic Expression Imbalance at Candidate Genes Using 5' Exonuclease Genotyping Technology.
Genetic variation along the length of a chromosome can influence the transcription of a gene. In a heterozygous individual, this may lead to one chromosome producing different levels of RNA, compared to its paired chromosome, for a given gene. Allelic differences in gene expression can offer insight into the role of variation in transcription, and subsequently infer a route to conferring disease risk. This phenomenon is known as allele expression imbalance or AEI, which may be assayed using a PCR-based method that includes the quantification of the relative dosage of each allele (e.g., 5' exonuclease assays, TaqMan™). Importantly, in heterozygous individuals the resolution of expression imbalance is performed within a controlled system; the comparison of the alternate allele is reported relative to the wild-type, as the experiment can be performed within a single sample, controlled for background genetic information. Alternative methods for the detection of AEI include Primer-extension MALDI-TOF (Sequenom MassARRAY(®)), Next-Generation Sequencing, and SNP genotyping arrays. Here we present the methods used for the TaqMan™ approach and include a description of the SNP identification, allele-specific PCR, and analytic methods to convert allele amplification metrics to relative allele dosage.
Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland.|Institute of Molecular Medicine, Trinity Centre for Health Sciences, St James's Hospital, Dublin 8, Ireland.
Jillian M
JM
Gahan
Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland.|Institute of Molecular Medicine, Trinity Centre for Health Sciences, St James's Hospital, Dublin 8, Ireland.
Mikaela M
MM
Byrne
Neurosciences and Mental Health Research Institute, Cardiff University School of Medicine, Hadyn Ellis Building, Cathays, Cardiff, UK.
Matthew
M
Hill
Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland.|Institute of Molecular Medicine, Trinity Centre for Health Sciences, St James's Hospital, Dublin 8, Ireland.
Emma M
EM
Quinn
Department of Cardiology, St James's Hospital, Dublin, Ireland.|Institute of Cardiovascular Science, St James's Hospital, Dublin 8, Ireland.
Ross T
RT
Murphy
Institute of Molecular Medicine, Trinity Centre for Health Sciences, St James's Hospital, Dublin 8, Ireland.|Department of Psychiatry, Trinity College Dublin, Dublin, Ireland.
Richard J L
RJ
Anney
Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland. aryan12@tcd.ie.|Institute of Molecular Medicine, Trinity Centre for Health Sciences, St James's Hospital, Dublin 8, Ireland. aryan12@tcd.ie.|Sunny Hill Health Centre for Children, Vancouver, Canada. aryan12@tcd.ie.
Anthony W
AW
Ryan
D000483:Alleles; D005838:Genotype; D043264:Phosphodiesterase I / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide; D060888:Real-Time Polymerase Chain Reaction
D043264:Phosphodiesterase I
5′ exonuclease assay; Allelic expression imbalance; CEPH cell lines; cDNA