Abstract
BackgroundSmall non-coding RNAs are essential regulators of gene expression at the transcriptional and posttranscriptional levels. High-throughput sequencing has revealed thousands of predicted small RNAs; however, only a few of these have been well characterized. Northern blotting is the most convincing method for small RNA validation.FindingsIn this study, we improved the Northern blot method by using biotin-labeled probes. miRNAs and siRNAs derived from both Arabidopsis thaliana and Oryza sativa were investigated. The results suggest that this improved method is sensitive and efficient, with approximately 5 μg of total RNA being sufficient for detection. Furthermore, long-term storage of probes labeled in this manner is more convenient, less contaminative and degradative compared with traditional probes.ConclusionsThis protocol is an alternative strategy for small RNA detection and represents an efficient means of researching small RNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-014-0026-1) contains supplementary material, which is available to authorized users.
Highlights
Small non-coding RNAs are essential regulators of gene expression at the transcriptional and posttranscriptional levels
This protocol is an alternative strategy for small RNA detection and represents an efficient means of researching small RNAs
Several methods have been developed to investigate the expression of target small non-coding RNAs, such as Northern blotting, quantitative reverse-transcription PCR (RT-PCR), and in situ hybridization
Summary
Probe Preparation Some of the miRNAs and ta-siRNAs selected for validation are listed in the small RNA database (http://bioinformatics. cau.edu.cn/PMRD/), and some have been reported in previous studies (Allen et al, 2005; Xie et al, 2006; Zhan et al, 2012). Probes modified with biotin on the 5’ or 3’ terminus were synthesized and purified via HPLC by the GenScript Company (Nanjing, China). These probes were designed to be completely complementary to the miRNA or tasiRNA nucleotide sequence and are listed in Additional file 1: Table S1. Total RNA was extracted from leaves using TRIzol reagent (Invitrogen, USA) following a previously described protocol (Hu et al, 2012). Small RNAs were extracted from leaves using RNAiso for Small RNA (TaKaRa, Dalian, China). The aqueous phase was extracted with chloroform by vortexing for 2 minutes and centrifuging the mixture at 12,000 g for 5 minutes at 4°C. Total RNA and small RNAs were quantified using a NanoDrop 2000c Spectrophotometer (Thermo Scientific, USA) and visually assessed on a 15% denaturing PAGE gel containing 7 M urea or on a 1.2% agarose gel
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