Use of real-time RT-PCR for the detection of allelic expression of an imprinted gene.
Measurement of the relative amounts of transcripts from two alleles is important in the study of imprinted genes, since quantitative differences that vary among tissues or individuals, and subtle differences in the ratio of allelic expression can have pathobiological significance. Discrimination of alleles is commonly based on PCR, followed by restriction endonuclease digestion to recognize a polymorphic site. However, the use of restriction enzymes misses most of the available single nucleotide polymorphisms. Practically, it requires substantial post-PCR analyses including the restriction enzyme digestion and gel electrophoresis, all of which increase turn around time. Taking advantage of our previous study identifying lung adenocarcinomas displaying biallelic expression of the imprinted gene MEST, we investigated the validity of a method of allelic discrimination in a real-time PCR assay using allele-specific probes. Allelic expression of the MEST gene in the range of 4-fold differences was detected. This new method should enhance our ability to rapidly and accurately assess allelic expression of imprinted genes in a number of samples.
Suda
Tetsuji
T
Division of Molecular and Cell Genetics, Department of Molecular and Cellular Biology, School of Life Sciences, Faculty of Medicine, Tottori University, Tottori 683-8503, Japan.
Katoh
Motonobu
M
Hiratsuka
Masaharu
M
Fujiwara
Mutsunori
M
Irizawa
Yoshito
Y
Oshimura
Mitsuo
M
D000230:Adenocarcinoma / Q000235:genetics*; D000483:Alleles; D015870:Gene Expression; D018392:Genomic Imprinting; D006801:Humans; D019656:Loss of Heterozygosity; D008175:Lung Neoplasms / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D011506:Proteins; C103637:mesoderm specific transcript protein