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New Tools for Small RNA Analysis


Non-coding small RNAs such as transfer RNAs (tRNA), ribosomal RNAs (rRNA), small nucleolar RNAs (snoRNA), and small nuclear RNAs (snRNA) play critical roles in a variety of cellular processes, from regulation of mRNA translation, pre-mRNA splicing or transcription to chromosome maintenance and gene imprinting. In addition, two novel classes of small non-coding RNAs (1924 nt)--referred to as small interfering RNAs (siRNAs) and microRNAs (miRNAs)--have recently emerged as powerful regulators of gene expression in a variety of organisms (1,2). siRNAs are now widely used as tools for gene silencing experiments in mammalian cells and organisms. Here we describe methods for small RNA isolation, labeling, and analysis.


Analyzing the Expression of Small RNAs
Currently, the analysis of miRNA and siRNA expression suffers from two technological drawbacks. Firstly, most standard RNA isolation protocols were not designed to efficiently recover small RNAs species. For example, glass fiber filters, which are widely used to prepare total RNAs, do not quantitatively recover various RNAs that are smaller than 200 nt (such as 5S rRNA, snRNAs, snoRNAs, tRNAs, miRNAs, and siRNAs). Commercially available total RNA preparations do not always contain representative amounts of small RNA species because the RNA isolation protocols have not been optimized to recover small RNAs. A second technological challenge is that sensitive
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