Guria Ashirbad, Sharma Priyanka, Srikakulam Nagesh, Baby Akhil, Natesan Sankar, Pandi Gopal
Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, India.
Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, India.
Front Mol Biosci. 2022 May 13;9:886366. doi: 10.3389/fmolb.2022.886366. eCollection 2022.
Covalently closed circular RNAs are neoteric to the eukaryotic family of long non-coding RNAs emerging as a result of 5'-3' backsplicing from exonic, intronic, or intergenic regions spanning the parental gene. Owing to their unique structure and stability, circular RNAs have a multitude of functional properties such as micro-RNA and protein sponges, direct and indirect modulators of gene expression, protein translation, and many unproven activities apart from being potential biomarkers. However, due to their low abundance, most of the global circular RNA identification is carried out by high-throughput NGS-based approaches requiring millions of sequencing reads. This lag in methodological advancements demands for newer, more refined, and efficient identification techniques. Here, we aim to show an improved version of our previously reported template-dependent multiple displacement amplification (tdMDA)-NGS method by superimposing the ribosomal depletion step and use of H minus reverse transcriptase and RNase H. Implication of tdMDA using highly replicative Phi29 DNA polymerase after minimizing the linear and ribosomal RNA content further intensifies its detection limit toward even the abysmally expressing circular RNA at a low NGS depth, thereby decreasing the cost of identifying a single circular RNA. A >11-fold and >6-fold increase in total circular RNA was identified from the improved-tdMDA-NGS method over the traditional method of circRNA sequencing using DCC and CIRI2 pipelines, respectively, from subsp. Furthermore, the reliability of the improved-tdMDA-NGS method was also asserted in HeLa cell lines, showing a significant fold difference in comparison with the existing traditional method of circRNA sequencing. Among the identified circular RNAs, a significant percentage from both rice (∼58%) and HeLa cell lines (∼84%) is found to be matched with the previously reported circular RNAs, suggesting that the improved-tdMDA-NGS method can be adapted to detect and characterize the circular RNAs from different biological systems.
共价闭合环状RNA是真核生物长链非编码RNA家族中的新成员，它是由跨越亲本基因的外显子、内含子或基因间区域进行5'-3'反向剪接产生的。由于其独特的结构和稳定性，环状RNA具有多种功能特性，如作为微小RNA和蛋白质海绵、基因表达的直接和间接调节因子、蛋白质翻译，以及除了作为潜在生物标志物之外的许多未经证实的活性。然而，由于它们的丰度较低，大多数全球环状RNA的鉴定是通过基于高通量NGS的方法进行的，这需要数百万次测序读数。方法学进展的这种滞后需要更新、更精细和高效的鉴定技术。在这里，我们旨在通过叠加核糖体去除步骤以及使用H减逆转录酶和核糖核酸酶H来展示我们之前报道的模板依赖性多重置换扩增（tdMDA）-NGS方法的改进版本。在最小化线性和核糖体RNA含量后，使用高度复制的Phi29 DNA聚合酶进行tdMDA，进一步提高了其对低NGS深度下极低表达环状RNA的检测限，从而降低了鉴定单个环状RNA的成本。与使用DCC和CIRI2管道的传统环状RNA测序方法相比，改进的tdMDA-NGS方法分别从亚种中鉴定出的总环状RNA增加了>11倍和>6倍。此外，改进的tdMDA-NGS方法在HeLa细胞系中的可靠性也得到了证实，与现有的传统环状RNA测序方法相比显示出显著的倍数差异。在鉴定出的环状RNA中，发现水稻（约58%）和HeLa细胞系（约84%）中有很大比例与之前报道的环状RNA匹配，这表明改进的tdMDA-NGS方法可用于检测和表征来自不同生物系统的环状RNA。
