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Isolation to Functional Analyses

While the microRNA (miRNA) field is rapidly expanding, these small, non-poly(A) tailed molecules still pose challenges for research. Most RNA analysis techniques have been optimized for longer molecules, not small 21-23 nt RNAs. Their small size precludes standard RT-PCR analysis, and purification and amplification methods that make use of a poly(A) tail, present on mRNAs, won't work.

Here and inside this issue we describe research tools developed specifically for miRNA analysis, including isolation and enrichment of the small RNA fraction, internal high specific activity labeling as well as end labeling of miRNA molecules, up and down modulation of expression, quantitative detection, and profiling via array analysis.

MicroRNAs (miRNAs) are an important class of small RNA molecules that are expressed in eukaryotes. Although the first miRNA was identified in a genetic screen in 1993 [1], it was not until 2001 that the breadth of the miRNA gene class was recognized with the cloning and sequencing of more than one hundred miRNAs from worms, humans, and mice [1-4]. These evolutionarily conserved, noncoding RNA molecules regulate translation of mRNAs through base pair interactions [reviewed in 5]. With some exceptions, worm and human miRNAs inhibit translation of specific mRNAs, while plant miRNAs induce mRNA degradation.

In cells, the ~22 nt mature, functional miRNAs are produced by a recently descr ibed process (Figure 1). A primary transcript (pri-mRNA) that can be more than 1,000 nt in length is first produced in the nucleus. An RNA hairpin (precursor miRNA, pre-miR) comprising ~80 nt that includes the mature miRNA results from digestion of pri-miRNA by the double-strand-specific ribonuclease, Drosha [6]. The resulting pre-miRNA is transported to the cytoplasm via a process that involves Exportin-5 [7,8]. The pre-miRNA is further digested by Dicer [6,8] to generate a short, partially double-stranded RNA wherein one strand is the mature miRNA. The mature miRNA is taken up by a protein complex that is similar to, if not identical to, the RNA-Induced Silencing Complex (RISC) that supports RNA interference (RNAi) [9,10] and the bound complex funtions to regulate translation. For more information on miRNA processing and function, see Reagents for Up and Down- Regulation of miRNA Activity in Mammalian Cells, and MicroRNA Profiling by Array Analysis Reveals Critical BioMarkers.

Figure 1. miRNA and siRNA Processing Pathway.

Obtaining Samples Containing miRNAs, siRNAs, and Other Small RNAs

Because of their small size, isolation and detection of miRNA, siRNA, and other small RNA molecules is not trivial. For instance, methods featuring glass fiber filter (GFF) based binding are used routinely for total RNA sample preparation, but most of these methods do not result in quantitativ e recovery of RNA species smaller than ~200 nt. Likewise, current RT-PCR methods developed for mRNA quantitation are incompatible with miRNA and siRNA analyses because these small RNAs are only about the size of the primers used for reverse transcription.

Ambion has developed methods specifically designed for small RNA isolation and quantitation to facilitate miRNA expression studies. These methods are also useful for the quantitation of siRNAs expressed by or introduced into cells and tissues. The mirVana miRNA Isolation Kit uses a rapid, modified GFF-based procedure to recover all RNA, including small RNA species, from cultured cell and tissue samples. With this kit, RNA samples can also be specifically enriched for RNAs smaller than ~200 nt. Such enrichment can dramatically increase the sensitivity of downstream assays. For examples of experimental data, see New Tools for Small RNA Analysis.

Obtaining Samples Containing miRNAs, siRNAs, and Other Small RNAs

The new mirVana PARIS Kit goes one step further. With this kit you can isolate protein and small RNA-containing total RNA from the same sample. The mirVana PARIS procedure also includes a protocol to enrich the population of small RNAs to increase sensitivity of downstream experiments. These features make this kit ideal for correlating mRNA, miRNA, or siRNA levels with protein levels. For examples of experimental data, see Isolate Total RNA and Protein From the Same Sample.

Preisolated Small RNA

Depending on the isolation method used, commercially prepared RNA may lack some or all of the small RNA fraction. To address this problem, Ambion scientists have optimized isolation procedures used to generate FirstChoice Total RNA from normal human, mouse, and rat tissues, and certify these RNAs to contain small RNAs including miRNA, siRNA, and small nuclear RNA.

Small RNA Analyses

Ambion has developed a series of kits to simplify the detection of small RNAs by solution hybridization, Northern analysis, or in situ hybridization. The mirVana miRNA Detection Kit uses a sensitive solution hybridization assay to detect small RNA species in total RNA populations. Solution hybridization allows detection of multiple miRNAs as well as siRNAs and their mRNA targets in the same assay (for examples of experimental data generated with this kit, see Detecting Attomole Amounts of Small RNA [12]. To simplify probe synthesis for small RNA analysis, Ambion has also introduced the mirVana Probe & Marker Kit to prepare 5' end labeled probes and t he mirVana miRNA Probe Construction Kit to prepare in vitro transcribed probes. Other reagents useful for small RNA analysis include Decade Markers, a set of small RNA markers for polyacrylamide gel electrophoresis, and ULTRAhyb-Oligo, an ultrasensitive hybridization buffer optimized for Northern analysis using small RNA probes.

Like the study of protein-encoding genes, miRNA expression analysis could potentially identify miRNAs involved in many important processes (see MicroRNA Profiling by Array Analysis Reveals Critical BioMarkers). The early results of miRNA studies suggest that these molecules play key roles in many biological processes, including differentiation and oncogenesis. Functional studies featuring reagents for over- and under-expressing miRNAs will then enable a better understanding of miRNAs just as cDNA expression vectors and siRNAs have done for other genes. For example, synthetic pre-miRNAs (Pre-miR miRNA) and antisense miRNAs (Anti-miR miRNA Inhibitors) have been developed to study gene expression and other phenotypic changes in transfection assays (see Reagents for Up- and Down-Regulation of miRNA Activity in Mammalian Cells). Also, the pSilencer 4.1-CMV series of vectors now enables researchers to conduct long-term ectopic miRNA expression and gene silencing exper iments in stably transfected cells (see pSilencer 4.1-CMV: Versatile Vectors for Expression of siRNA, miRNA, and mRNA).

Ambion, the industry leader in providing solutions for working with RNA, is the first company to provide tools specifically for the analysis of miRNAs and other small RNAs. Please visit for more information.

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Ordering Information
Cat# Product Name Size 1550 mirVana miRNA Probe Construction Kit 30 rxns 1552 mirVana miRNA Detection Kit 100 rxns 1554 mirVana Probe & Marker Kit 30 rxns + 10 marker rxns 1556 mirVana PARIS Kit up to 40 purifications 1560 mirVana miRNA Isolation Kit up to 40 purifications 1562 mirVana miRNA Labeling Kit 20 rxns 1564V1 mirVana miRNA Probe Set 1 set 7778 Decade Markers 10 rxns 8663 ULTRAhyb-Oligo 125 ml 17000 Anti-miR miRNA Inhibitor 5 nmol 17001 Anti-miR miRNA Inhibitor 20 nmol (4 x 5 nmol) 17003 Customer-defined Anti-miR miRNA Inhibitor 20 nmol 17100 Pre-miR miRNA Precursor Molecule 5 nmol 17101 Pre-miR miRNA Precursor Molecule 20 nmol (4 x 5 nmol) 17103 Customer-defined Pre-miR miRNA Precursor Molecule 20 nmol


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