Small RNA Isolation and Library Construction for Expression Profiling of Small RNAs from Neurospora and Fusarium Using Illumina High-Throughput Deep Sequencing

Abstract

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.