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

ABSTRACT

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 gene Ube3a, 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 of Ube3a was equally expressed from both parental alleles in adult tissues except neuronal tissues, where Ube3a expression from the paternal allele was about 10 to 14% of total Ube3a expression in adult brain, and 20% in spinal cord. The antisense transcript of Ube3a was expressed at 60% to 70% of the sense transcript of Ube3a in adult brain. Changes in the Ube3a transcripts 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.

KEYWORDS:

• Genomic imprinting
• droplet digital PCR
• locked nucleic acid
• TaqMan probe
• Ube3a gene
• Angelman syndrome

Acknowledgments

The authors thank Ayumi Nakagaki for technical support of ddPCR assay and Katsunori Hironaka (Bio- Rad Laboratories, Tokyo, Japan) for helpful suggestions regarding the ddPCR analysis and we would like to thank Editage (www.editage.com) for English language editing.

Author contributions

M.M. and T.K. carried out cDNA synthesis, ddPCR and qPCR assays, and S.H. maintained the mouse strains. T.K designed the study, analysed results and drafted the manuscript. All authors reviewed the manuscript.

Disclosure statement

The authors declare that they have no competing interests.

Supplementary material

Supplemental data for this article can be accessed here.