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| An efficient and rapid method to detect and verify natural antisense transcripts of animal genes |
| Zhang Li1, Zhao Rui1, Xiao Mei1, Lin Shu-dai2, Li Bi-xiao2, Qiu Feng-fang2, Ma Jing-e2, Zhang De-xiang2, Nie Qing-hua2, An Li-long1, Zhang Xi-quan2 |
1 Agricultural College, Guangdong Ocean University, Zhanjiang 524088, P.R.China
2 Guangdong Provincial Key Lab of Agro-Animal Genomics and Molecular Breeding/Key Lab of Chicken Genetics, Breeding and Reproduction, Ministry of Agriculture/South China Agricultural University, Guangzhou 510642, P.R.China |
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Abstract 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 lncRNA (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 (G elongation 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.
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Received: 30 June 2015
Accepted:
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| Fund: This work was supported by the National Natural Science Foundation of China (31301958) and the Chinese Postdoctoral Science Foundation (2013T60808). |
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Corresponding Authors:
AN Li-long, Tel/Fax: +86-759-2383247,
E-mail: anlilong@126.com; ZHANG Xi-quan, Tel/Fax: +86-20- 85285703, E-mail: xqzhang@scau.edu.cn
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| About author: ZHANG Li, E-mail: zhangli761101@163.com; |
Cite this article:
Zhang Li, Zhao Rui, Xiao Mei, Lin Shu-dai, Li Bi-xiao, Qiu Feng-fang, Ma Jing-e, Zhang Dexiang, Nie Qing-hua, An Li-long, Zhang Xi-quan.
2016.
An efficient and rapid method to detect and verify natural antisense transcripts of animal genes. Journal of Integrative Agriculture, 15(9): 2070-2076.
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