Imprinting analysis by droplet digital PCR coupled with locked nucleic acid TaqMan probes

Imprinted genes are differentially expressed in a parent-of-origin-specific manner. Parental origin of the alleles is discriminated by intragenic DNA polymorphisms. Comparisons of parental allelic expression have been analysed by semiquantitative RT-PCR. Here, we developed a novel quantitative method for allelic expression of the imprinted geneUbe3a, which inactivation and mutations cause Angelman syndrome and predominantly expressed by the maternal allele in neuronal tissues. In this method, cDNA was amplified by droplet digital PCR (ddPCR) coupled with allele-specific locked nucleic acid (LNA) TaqMan probes, which labelled by FAM and HEX were designed to detect the SNPs in the target regions. ddPCR assay demonstrated that the sense transcript ofUbe3awas equally expressed from both parental alleles in adult tissues except neuronal tissues, whereUbe3aexpression from the paternal allele was about 10 to 14% of totalUbe3aexpression in adult brain, and 20% in spinal cord. The antisense transcript ofUbe3awas expressed at 60% to 70% of the sense transcript ofUbe3ain adult brain. Changes in theUbe3atranscripts during postnatal brain development were also evaluated by ddPCR. The ddPCR method is far more reliable and simpler to use than semiquantitative PCR to analyse skewed or faint allelic expression of imprinted genes.

Automated summary

Imprinted genes are expressed in a parent-of-origin-specific manner, discriminated by intragenic DNA polymorphisms. A novel quantitative method using ddPCR and LNA TaqMan probes was developed to analyze allelic expression of the imprinted gene Ube3a, revealing maternal allele predominance in neuronal tissues. Changes in Ube3a transcripts during postnatal brain development were also evaluated, demonstrating the reliability and simplicity of ddPCR in analyzing skewed or faint allelic expression.