基于PCR 技术的人脑基因可变剪接差异性研究

目的:对人脑的基因可变剪接位点差异性进行分析。方法:通过RNA-Seq得到的转录组数据,分析推测出在不同年龄的7对样本的大脑前额叶皮层(prefrontal cortex,PFC)表达的某些基因具有可变剪接位点的差异性;为验证此推测,设计7对引物,提取不同年龄样本的RNA,通过反转录合成cDNA,采用PCR技术扩增目的序列。结果:在已剪接和(或)未剪接产物预期片段处有明显的条带。结论:通过Quantity One软件分析后,确定在不同年龄间存在可变剪接差异。基于PCR技术的基因可变剪接分析对RNA-seq数据集的预测结果进行验证是一种有效新颖的PCR新应用。     

一个肝癌相关长链非编码RNA的克隆及序列分析

最近我们利用新一代测序(next generation sequencing,NGS)技术对肝细胞癌(hepatocellular carcinoma,HCC)患者活检标本及正常对照肝组织样品进行高通量RNA测序(RNA-sequencing,RNA-Seq),在肝癌样品中染色体11q13.1区域检测到几个相邻的RNA-Seq信号峰,而在正常对照组织中没有检测到,且该染色体区域目前尚无已知基因登录,提示这几个RNA-Seq峰可能代表一个或多个未知的新基因.以此为线索,证实这几个RNA-Seq峰来自同一个新基因,并克隆了该基因全长序列,在克隆该基因全长序列时,发现该基因编码的RNA存在多种剪接形式,最长的转录本为3 562 bp.将该基因编码的12条代表性RNA转录本序列递交到美国国立生物技术信息中心(National Center for Biotechnology Information,NCBI)的GenBank数据库中,GenBank ID号分别为KC136297~KC136308.该基因编码的RNA没有发现明显的开放阅读框(open reading fragment,ORF),提示该基因可能编码长链非编码RNA(long non-coding RNA,lncRNA).为了探讨该lncRNA基因可能的转录调控机制,我们用生物信息学方法预测了该lncRNA基因潜在启动子区域,发现在其转录起始位点上游-719~-469 bp处有一个潜在的启动子,其中包含7个Sp1、1个STAT5和1个EGR1转录因子结合位点.该lncRNA在肝细胞癌发生发展过程中的作用机制值得进一步深入研究.     

Identification of alternatively spliced dab1 isoforms in zebrafish

We have investigated the genomic organization, the occurrence of alternative splicing and the differential expression of the zebrafish disabled1 (dab1) gene. Dab1 is a key effector of the Reelin pathway, which regulates neuronal migration during brain development in vertebrates. The coding region of the zebrafish dab1 gene spans over 600 kb of genomic DNA and is composed of 15 exons. Alternative splicing in a region enriched for tyrosine residues generates at least three different isoforms. These isoforms are developmentally regulated and show differential tissue expression. Comparison with mouse and human data shows an overall conservation of the genomic organization with different alternative splicing events generating species-specific isoforms. Because these alternative splicing events give rise to isoforms with different numbers of phosphorylateable tyrosines, we speculate that alternative splicing of the dab1 gene in zebrafish and in other vertebrates regulates the nature of the cellular response to the Reelin signal.Electronic supplementary material Supplementary material is available for this article at and accessible for authorised users.