大鼠乳腺ACBD6环形转录本的克隆与验证
毕业论文关键词：大鼠乳腺；ACBD6；环形RNA；验证
The Cloning and Validation of ACBD6 Circular Transcription From Rat Mammary Gland
Abstract：The purpose of this experiment is to clone circular ACBD6 transcript from rat mammary gland tissue, and use different methods to verify the circular structure. This study collected the rat breast tissue at first, and extracting total RNA from the mammary gland of rats, by PCR we cloned suspected ACBD6 circular transcript.Using the method of real-time PCR with circular and linear primers, we cloned the RNase R digesting cDNA and cDNA reverse transcribed by Oligo (dT) and random primers. Compared and analyzed the Ct value from real-time PCR results under different conditions, we can have a preliminary judgment for ACBD6 circular transcript. After sequencing and comparison of its sequence with ACBD6 sequence alignment, we confirmed that it is circular ACBD6 transcript. We used RNAfold software for secondary structure prediction. By comparison and analysis between cACBD6 mRNA and ACBD6 mRNA secondary structure of the minimum free energy, we speculate ACBD6 circular transcription might have important function. This experiment laid a solid foundation on further study of the function of ACBD6 circular transcription and their different expression of organization.
Key words：Rat mammary gland; ACBD6; Circular RNA; validation
目录
引言 1
1.材料与方法 2
1.1 材料 2
1.2方法 2
1.2.1提取RNA 2
1.2.2 RNase R消化处理 2
1.2.3 Oligo (dT)和随机引物的反转录 3
1.2.4 荧光定量PCR 3
1.2.5. 反向剪接位点的测序 3
1.2.6. 数据统计 4
2.结果与分析 4
2.1 RNase R消化处理 4
2.2 Oligo (dT)和随机引物的对比 4
2.4二级结构预测 6
3.讨论 8
致谢 9
参考文献 9
引言
环形RNA（circular RNA, circRNA）是一类不具有5'帽子和3'poly(A)尾巴、以共价键形成环形结构的非编码RNA分子，主要包括了外显子来源的环形RNA分子(exonic circRNAs)，内含子来源的环形RNA分子(circular intronic RNAs，ciRNAs)以及由外显子和内含子共同组成的环形RNA分子(retained-intron circRNAs)三类 [1]。20世纪70年代对RNA病毒的观察研究首次发现了circRNA的存在[2,3]，这同时也开启了该类RNA的研究历程。1979年，Hsu 和Coca-Prados等报道利用电子显微镜在真核生物细胞质中发现了环形的RNA[4]，进一步说明circRNA可能是一类广泛存在于真核生物和病毒之中的物质。在1980年，Arnberg等在酵母的线粒体中发现了非编码的环形RNA（circRNAs）[5]；紧接着在1990年，circRNA也被证实存在于酿酒酵母中，且发现其特性与肝炎病毒有一定的相似性[6]。随后在1993年，Cocquerelle等又在人体细胞中发现了circRNA[7]。20世纪90年代初内源性circRNA在人体DCC细胞的转录中被发现[8]。随后仅在个别基因中报道了circRNA。随着时间的推移，circRNA在人类细胞、小鼠、线虫等生物中存在的事实也得以验证。