奶牛乳腺ACBD6环形RNA的克隆与验证
毕业论文关键词：奶牛乳腺；环形RNA；逆转录；实时荧光定量PCR
The cloning and validation of cow mammary gland ACBD6 circular RNA
Abstract:Objective :Nowadays with the developmenting of bioinformatics technology many experimental method can used in RNA detection. So more and more circular RNAs come under observation. The structure and function of the circular RNA has caused our attention. Some circular RNAs play an important role in regulation. The structure of circular RNA is more stable than linear RNA. Real-time fluorescent quantitative PCR method is used to test the CT values of cow mammary gland ACBD6 RNA which deal with the oligo (dt) and random primers or under linear RNase R processing. So we can validation the ACBD6 RNA whether it is circular RNA. Methods: Collecting the ACBD6 RNA in cow mammary gland organization, and design primers with oligo (dt) and random primers or linear RNaseR Enrichment processing. The following in reverse transcription and in the real-time fluorescent quantitative PCR .The last analysis of the result. Results: The results of the real-time fluorescence quantitative PCR ACBD6 RNA are different, The CT value with the oligo (dt) is bigger and with the RNase R is smaller.The results of the two methods is different.Ring sequence is very consistent with Gene pool sequence.Next predict the secondary structure of the circular and linear RNA.
Key words: The cow mammary gland; Circular RNA ; real-time quantitative PCR
目 录
引言 1
1 材料与方法 2
1.1试验样品 2
1.1.1试验样品的采集 2
1.1.2实验试剂 2
1.1.3实验仪器 2
1.2 实验方法 3
1.2.1提取组织的总RNA 3
1.2.2所提取的总RNA的检测 3
1.2.3实时荧光定量PCR 4
2 结果与分析 7
2.1 RNAse R处理/不处理的对比 7
2.2 Oligo (dT)和随机引物的对比 8
2.3测序结果 8
2.4二级结构预测 10
3 讨论 11
致谢 12
参考文献 12
引言
环形RNA是一类通过反向插接方式产生的非编码RNA，其不具有5'帽子和3'poly(A)尾巴，目前因其剪接来源不同主要分为外显子来源环形RNA（exonic circRNAs）和内含子来源环形RNA（ciRNAs）。环形RNA很早就存在了而且很早就被发现了，但长期以来由于知识和技术方面的限制，所以很难对其进行全面且具体的研究。环状RNA（circRNAs）不仅存在于各种生物细胞中还存在各类病毒中，它的结构比一般线性的RNA稳定、丰度非常高和组织特异性表达等特征。最近的研究表明，一些circRNA作为竞争性内源RNA（ceRNA）对基因表达调控起着至关重要的作用。近年来人们借助高通量测序技术，结合其它现代分子生物学手段对其进行了研究，使得对环形RNA的研究有了非常重要的进展。目前已发现，环形RNA结构对比于线状RNA其结构更加稳定，并且circRNA起着重要的调控作用，包括抑制miRNA、促进转录等。