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Genome-wide Discovery and Profiling of Small RNA
Sponsored,vendor-submitted protocol    Sponsored by Applied Biosystems, part of Life Technologies    Published in November 2009 (p.59) DOI: 10.2144/000113024

The SOLiD™ System provides researchers with a highly sensitive method for genome-wide analysis of RNAs without the probe bias of microarrays. Using the SOLiD™ Small RNA Expression Kit (SREK), researchers can now generate small RNA libraries in a single day with a simple, easy-to-use protocol that preserves the strand information of the original molecule.

Introduction

The impact of small RNA expression on biological systems is a burgeoning area of scientific research. In response to the growing demand for robust and time-efficient methods for small RNA expression analysis, Applied Biosystems has developed a highly sensitive new method for small RNA expression analysis. Advantages of this method include significant reduction in the time, labor, and starting material required compared to current methods. The SOLiD™ Small RNA Expression Kit (SREK), coupled with the SOLiD™ System, provides researchers with a simplified method for hypothesis-neutral whole-genome expression analysis of small RNAs that preserves the strand information of the original molecule. Figure 1 shows this workflow with the Applied Biosystems’ solutions for each step.


Figure 1. Whole-genome expression analysis of small RNA. (Click to enlarge)


Step 1: RNA Isolation

The Ambion mirVana™ miRNA Isolation and Ambion mirVana PARIS™ Kits provide fast and efficient protocols for isolating total RNA ranging in size from 10-mers to kilobases from cells and tissue. In addition, these kits include reagents for enriching small RNA species <200 bases. Alternatively, the Ambion RNAqueous®-Micro RNA Isolation Kit allows recovery of RNA containing both large and small RNA species from small samples, including LCM samples. Total RNA can be used in subsequent steps with little loss in data quality. However, since total RNA samples can vary greatly in small RNA content, we recommend further enrichment of the small RNA fraction by size selection using the Ambion flashPAGE™ Fractionator and flashPAGE Reaction Clean-Up Kit. This flashPAGE System provides a fast and reliable means of purifying the 10–40 nt nucleic acid fraction (mature miRNAs).

Step 2: Small RNA Library Preparation

The SOLiD™ Small RNA Expression Kit converts total RNA into a cDNA library suitable for emulsion PCR and can be completed in a single day. Small RNA in the sample is hybridized to specialized adaptor oligonucleotides that contain a defined sequence required for SOLiD™ sequencing. The adaptors are then ligated directionally and simultaneously to the small RNA molecules in as little as 2 h. Following ligation, samples are reverse transcribed to generate cDNA, and treated with RNase H to digest the small RNA from the RNA/cDNA duplex. The cDNA is PCR-amplified for 12–15 cycles, yielding duplex DNA libraries suitable for SOLiD™ sequencing.

Step 3: SOLiD Sequencing

The purified small RNA libraries are compatible with the standard SOLiD™ sequencing workflow. First, libraries are titrated into a water-in-oil emulsion PCR, where each PCR mini-reactor clonally amplifies a single molecule of library template onto an individual 1-µm bead. The beads that are densely covered with template are enriched and covalently bound to a glass slide. Ligationbased sequencing is then performed on a SOLiD™ Analyzer.

Step 4: Analysis

Tags generated by the SOLiD™ Analyzer are then mapped back to known small RNAs for tissue-specific profiling applications, or to a reference sequence for discovery of novel RNAs. The number of tags that map uniquely to a given target sequence provides a relative expression level for each small RNA target.

Step 5: Validation

TaqMan® MicroRNA standard and custom assays may be used to validate results from the SOLiD™ Analyzer. TaqMan is the gold standard for RNA quantitation and provides a complementary technology platform for validation of novel discoveries. Expanding Next Generation Sequencing to RNA Discovery and Profiling

The SOLiD™ Small RNA Expression Kit provides a streamlined workflow that greatly reduces the time, cost, and experimental variability associated with library preparation. This approach, coupled with the SOLiD™ System, provides a highly sensitive method for digital gene expression that enables the discovery and profiling of novel RNAs without the probe-bias of microarrays.


Figure 2. (Click to enlarge)


Correspondence

Address correspondence to Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA.

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Applied Biosystems, part of Life Technologies
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