High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many ant...High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lnc RNA（long non-coding RNA）, can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix（Xist antisense RNA）, chicken LXN（latexin） and GFM1（Gelongation factor, mitochondrial 1）, were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.展开更多
Allele specific expression is essential for cellular programming and development and the diversity of cellular phenotypes. Traditional analysis methods utilize RNA and depend on single nucleotide polymorphisms,thus to...Allele specific expression is essential for cellular programming and development and the diversity of cellular phenotypes. Traditional analysis methods utilize RNA and depend on single nucleotide polymorphisms,thus to suffer from limited amount of materials for analysis. The rapid development of next-generation sequencing technologies provides more comprehensive and powerful approaches to analyze the genomic, epigenetic, and transcriptomic data, and further to detect and measure allele specific expressions. It will potentially enhance the understanding of the allele specific expressions, their complexities, and the effect on biological processes. In this paper, we extensively review the state-of-art enabling technologies and tools to analyze, detect, and measure allele specific expressions, compare their features, and point out the future trend of the methods.展开更多
基金supported by the National Natural Science Foundation of China(31301958)the Chinese Postdoctoral Science Foundation(2013T60808)
文摘High-throughput sequencing has identified a large number of sense-antisense transcriptional pairs, which indicates that these genes were transcribed from both directions. Recent reports have demonstrated that many antisense RNAs, especially lnc RNA（long non-coding RNA）, can interact with the sense RNA by forming an RNA duplex. Many methods, such as RNA-sequencing, Northern blotting, RNase protection assays and strand-specific PCR, can be used to detect the antisense transcript and gene transcriptional orientation. However, the applications of these methods have been constrained, to some extent, because of the high cost, difficult operation or inaccuracy, especially regarding the analysis of substantial amounts of data. Thus, we developed an easy method to detect and validate these complicated RNAs. We primarily took advantage of the strand specificity of RT-PCR and the single-strand specificity of S1 endonuclease to analyze sense and antisense transcripts. Four known genes, including mouse β-actin and Tsix（Xist antisense RNA）, chicken LXN（latexin） and GFM1（Gelongation factor, mitochondrial 1）, were used to establish the method. These four genes were well studied and transcribed from positive strand, negative strand or both strands of DNA, respectively, which represented all possible cases. The results indicated that the method can easily distinguish sense, antisense and sense-antisense transcriptional pairs. In addition, it can be used to verify the results of high-throughput sequencing, as well as to analyze the regulatory mechanisms between RNAs. This method can improve the accuracy of detection and can be mainly used in analyzing single gene and was low cost.
文摘Allele specific expression is essential for cellular programming and development and the diversity of cellular phenotypes. Traditional analysis methods utilize RNA and depend on single nucleotide polymorphisms,thus to suffer from limited amount of materials for analysis. The rapid development of next-generation sequencing technologies provides more comprehensive and powerful approaches to analyze the genomic, epigenetic, and transcriptomic data, and further to detect and measure allele specific expressions. It will potentially enhance the understanding of the allele specific expressions, their complexities, and the effect on biological processes. In this paper, we extensively review the state-of-art enabling technologies and tools to analyze, detect, and measure allele specific expressions, compare their features, and point out the future trend of the methods.
