Park Gyungsoon, Borkovich Katherine A
Plasma Bioscience Research Institute, Kwangwoon University, Wolgaedong, Nowongu, Seoul, Republic of Korea.
Methods Mol Biol. 2012;883:155-64. doi: 10.1007/978-1-61779-839-9_12.
Due to crucial roles in gene regulation, noncoding small RNAs (smRNAs) of 20-30 nucleotides (nt) have been intensively studied in mammals and plants, and are known to be implicated in significant diseases and metabolic disorders. Elucidation of biogenesis mechanisms and functional characterization of smRNAs are often achieved using tools, such as separation of small-sized RNA and high-throughput sequencing. Although RNA interference pathways such as quelling and meiotic silencing have been well described in Neurospora crassa, knowledge of smRNAs in filamentous fungi is still limited compared to other eukaryotes. As a prerequisite for study, isolation and sequence analysis of smRNAs are necessary. We developed a protocol for isolation and library construction of smRNAs of 20-30 nt for Solexa sequencing in two -filamentous fungi, N. crassa and Fusarium oxysporum f.sp. lycopersici. Using 200-300 μg total RNA, smRNA was isolated by size fractionation, ligated with adapters, and amplified by RT-PCR for Solexa sequencing. Sequence analysis of several cDNA clones showed that the cloned smRNAs were not tRNAs and rRNAs and were fungal genome specific.
由于在基因调控中发挥关键作用，20至30个核苷酸（nt）的非编码小RNA（smRNA）已在哺乳动物和植物中得到深入研究，并且已知与重大疾病和代谢紊乱有关。smRNA的生物合成机制阐明和功能表征通常使用诸如小尺寸RNA分离和高通量测序等工具来实现。尽管诸如基因压制和减数分裂沉默等RNA干扰途径已在粗糙脉孢菌中得到充分描述，但与其他真核生物相比，丝状真菌中smRNA的知识仍然有限。作为研究的先决条件，smRNA的分离和序列分析是必要的。我们开发了一种用于在两种丝状真菌——粗糙脉孢菌和番茄枯萎病菌中分离20至30 nt的smRNA并构建文库以进行Solexa测序的方案。使用200至300μg总RNA，通过大小分级分离法分离smRNA，与接头连接，并通过RT-PCR扩增以进行Solexa测序。对几个cDNA克隆的序列分析表明，克隆的smRNA不是tRNA和rRNA，而是真菌基因组特异性的。
