![]() Next, the amounts of RNA were quantified using a NanoDrop (Thermoscientific). The results showed that the RNAs were suitable for further analysis, such as northern blotting (Figure 1a,b). To detect the integrity of the RNA, we loaded the RNA onto 15% PAGE gels under denaturing conditions (7 M urea) or onto 1.2% agarose gels with EtBr for staining. To test the method of RNA preparation in this study, total RNA and small RNAs were extracted with two different reagents. In a eukaryotic cell, the abundance of rRNA and tRNA impairs the detection of small RNAs. Preparation of total RNA and small RNA for Northern blotting Taken together, these data suggest that biotin-labeled probes can be widely used to investigate small RNAs and even mRNAs in the future. Furthermore, storage of biotin-labeled probes is convenient, and the probes are stable therefore, northern blots can be carried out with ease. When we applied this improved method to Arabidopsis thaliana and Oryza sativa for validation, the results suggested that this method was sensitive and efficient, as it was capable of detecting as little as 1 to 5 μg of total RNA. In this study, we designed biotin-labeled oligonucleotide probes to investigate the expression of small RNAs. Researchers have also modified probes with locked nucleic acid (LNA) to improve sensitivity (Gao & Peng, Lopez-Gomollon, ).īiotin is another molecule that can increase the sensitivity of small RNA detection. In recent years, researchers have been continuously improving the method for northern blotting with digoxigenin (DIG)-labeled modified probes (Kim et al, ). Several improvements have been made to traditional Northern blotting protocols, and non-isotopic-labeling methods using digoxigenin (DIG) represent a safe alternative for the detection of small RNAs (Ramkissoon et al, ). Although isotope labeling is often inconvenient, hazardous and restricted by many institutions, this classic method is still the most popular method for investigating the expression of small RNAs. Because the low abundance of small RNAs can be problematic for detection via northern blotting, some researchers have enriched small RNAs using LiCl or other methods (Song et al, ). RRNAs and tRNAs account for over 90% of the total RNA in a eukaryotic cell, whereas small non-coding RNAs account for only approximately 2%, and detection of these RNAs by some methods is difficult (Zhuang et al, ). To gain further insight, 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. To date, thousands of small non-coding RNAs and their target mRNAs or genes have been computationally predicted however, few of them have been experimentally confirmed or properly characterized. Recently, high-throughput sequencing technology has facilitated the exploration of small non-coding RNAs (Creighton et al, Studholme, ). elegans two decades ago, remarkable advances in the characterization of this gene family have been achieved (He & Hannon, ). Although the mechanisms and pathways by which miRNAs and siRNAs regulate their target genes are largely obscure, the initial findings have demonstrated that miRNAs and siRNAs have a broad impact on epigenetics. In plants, miRNAs and siRNAs are the two major classes of endogenous small RNAs and are important for development, genome stability, gene expression and stress responses (Chen, Wei et al, ). The three major types of small non-coding RNAs-miRNAs, siRNAs, and piRNAs-are distinguished by their different modes of biogenesis (Chen, Huang et al, ). Small non-coding RNAs are 20-30 nt long and function as sequence-specific negative regulators of gene expression at the transcriptional and/or posttranscriptional levels (He & Hannon, Voinnet, ). In eukaryotic cells, small non-coding RNAs have been demonstrated to play fundamental roles in gene expression modification during development (Zamore & Haley, ).
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