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.

摘要

印迹基因以亲本特异性方式差异表达。等位基因的亲本起源通过基因内DNA多态性来区分。已通过半定量 RT-PCR 分析了亲本等位基因表达的比较。在这里，我们开发了一种用于印迹基因Ube3a等位基因表达的新定量方法，该基因失活和突变导致 Angelman 综合征，主要由神经元组织中的母体等位基因表达。在该方法中，通过液滴数字 PCR (ddPCR) 与等位基因特异性锁定核酸 (LNA) TaqMan 探针结合扩增 cDNA，该探针由 FAM 和 HEX 标记，旨在检测目标区域中的 SNP。ddPCR 分析表明 Ube3a 的有义转录本除神经元组织外，在成人组织中的两个亲本等位基因中均等表达，其中来自父本等位基因的Ube3a表达约占成人大脑中Ube3a总表达的 10% 至 14%，在脊髓中为 20%。Ube3a的反义转录本在成人大脑中的表达量为Ube3a有义转录本的60% 至 70% 。ddPCR还评估了出生后大脑发育过程中Ube3a转录物的变化。ddPCR 方法比半定量 PCR 分析印迹基因的偏斜或微弱等位基因表达更可靠、更简单。