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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 methods for the detection of small RNAs have not been previously available. RT-PCR, while being very sensitive, cannot be used to amplify small RNAs since in standard RT-PCR, the primers are about the same size as the small RNAs. As a result, siRNAs and miRNAs have been primarily detected by Northern analysis, a technique that is time-consuming and relatively insensitive.


The mirVana System for Small RNA Analysis
To facilitate the analysis of small RNA expression, Ambion has developed the mirVana system, a complete solution for the isolation and detection of small RNAs (Figure 1). The mirVana products include:

The mirVana miRNA Isolation Kit--for isolation of total RNA containing small RNAs, or preparation of fractions enriched in small RNAs (<200 nt).

"miRNA certified" FirstChoice RNAs--human, mouse, and rat total RNAs that have been verified to contain miRNAs and other small RNA molecules

The mirVana miRNA Detection Kit--for the sensitive detection and quantitation of miRNA and other small RNA

The mirVana miRNA Probe Construction Kit--for rapid synthesis of template and RNA probes by in vitro transcription

The mirVana miRNA Probe & Marker Kit--for the 5' end-labeling and purification of RNA or DNA probes, and preparation of small RNA markers

Figure 1. A Fully Integrated System for Small RNA Analysis.


Isolation of Small RNAs
There are two widely used methods for isolating total RNAs: 1) phenol/chaotropic reagent extraction, and 2) binding to and elution from glass fiber filters (GFF). Ambion has determined that many small RNAs are not retained when total RNA is extracted by traditional GFF methods which are commonly used in most laboratories (see poster for more information). We have, however, developed a modified GFF-based procedure--through the use of optimized binding and wash solutions--that provides quantitative yield of miRNAs and other small RNAs from virtually any tissue or cell line. A modification of this procedure can also be used to enrich small RNAs from a total RNA population. This technology is now available as the mirVana miRNA Isolation Kit (patent pending).


"miRNA Certified" FirstChoice RNAs
When purchasing commercially available purified RNAs, it is also important to consider the RNA isolation method used to isolate the RNA. Most of Ambion's FirstChoice Total RNAs have been validated for the presence of small RNA molecules--and we are constantly working on optimized isolation procedures for these and other tissue and cell line samples. Visit our web catalog for an up-to-date list of "miRNA certified" FirstChoice RNAs.


Sensitive Detection of Small RNAs
Although Northern analysis can be used to detect and quantitate small RNA, the technique is labor-intensive and, more importantly, not very sensitive. Ambion scientists have developed a sensitive assay that has been optimized for small RNA quantitation--the patented mirVana miRNA Detection Kit--which is based on solution hybridization and subsequent RNase protection. Short radiolabeled RNA probe is simply mixed with total RNA in an optimized hybridization buffer and all single-stranded RNAs (e.g. unhybridized probe, mRNAs, rRNA) are then digested by a mixture of RNase A and T1. Probe:target RNA hybrids are quickly recovered using a patented single-step RNase inactivation/precipitation procedure, run on a denaturing polyacrylamide gel, and visualized by autoradiography. In this assay, full-length and protected probes are easily distinguished by the presence of a short sequence at the 5' or 3' end of probes that is not complementary to the RNA target (Figure 2).

Figure 2. The mirVana miRNA Detection Kit for small RNA Detection


The mirVana miRNA Detection Kit has been used to detect miRNAs and siRNAs in a variety of tissues and cells (see poster for more information). The sensitivity and specificity of the assay is illustrated in Figure 3 where miR-124 miRNA was detected only in human brain total RNA. This experiment confirms the tissue-specific expression of miR-124, previously determined by Northern blot analysis using 30 g of total RNA (3). In contrast, only 1 g of total RNA was used in the solution hybridization assay. Moreover, miR-124 expression could be detected in as little as 10 ng of total brain RNA after overnight exposure of the gel (data not shown).

Figure 3. Tissue-specific expression of miR-124 miRNA. miR-124 was detected in 1 g of the indicated FirstChoice Total RNA from 14 different human tissues using the mirVana miRNA Detection Kit and a 5' end-labeled RNA probe prepared with the mirVana Probe & Marker kit. Gel was exposed for 5 hours.


Another application of the mirVana miRNA Detection Kit, is the detection of two or more RNAs in the same sample. For instance, an expressed siRNA and its target mRNA can be monitored simultaneously. See Application: Analysis of miRNA Recovery After Gel Purification (sidebar) or the Ambion miRNA Resource for additional applications of this versatile kit.


Preparation of RNA Probes
Small RNA probes for the solution hybridization assay can be readily prepared using one of two different methods: in vitro transcription (IVT) or 5' end labeling of an existing RNA oligonucleotide. Depending on the probe sequence and the radiolabeled nucleotide used, IVT probes typically result in ~2-fold greater sensitivity than 5' end labeled probes due to their higher specific activity (Figure 4). In contrast, 5' labeled RNA probes are more easily generated as they do not require gel purification. Ambion has developed and optimized kits for both probe-labeling methods -- the mirVana miRNA Probe Construction Kit for IVT and the mirVana Probe & Marker Kit for 5' end labeling.

Figure 4. Comparison Between In Vitro Transcribed (IVT) and 5' End Labeled Probes. The expression of miR-16 miRNA and 5S rRNA was analyzed in 0.5, 1, 2 or 4 g of FirstChoice Total RNA from mouse kidney included in the mirVana miRNA Detection Kit using the indicated DNA or RNA probes included in the mirVana miRNA Probe Construction Kit (IVT probes) or mirVana Probe & Marker Kit (5' end labeled probes).


The mirVana miRNA Probe Construction Kit includes reagents for preparation of IVT templates as well as reagents for the IVT reaction. All you need to provide is an inexpensive, desalted, target-specific DNA oligonucleotide. A short sequence cleavable by RNase A and T1 is added to the 5' end of probe during the procedure. Probes prepared with the kit are therefore suitable not only for solution hybridization assays, but also for Northern blot or in situ hybridization analysis (using nonisotopically labeled probes).

The new mirVana Probe & Marker Kit provides reagents for the rapid preparation and purification of short, high specific activity 5' end-labeled RNA probes. Such probes are widely used in a variety of assays, including RNA structure/function studies, Northern blot and primer extension analysis. For solution hybridization assays, the antisense probe should carry only a few nontarget-specific nucleotides at its 3' end. The kit can also be used to label and purify DNA probes (see Northern Blot in Figure 4) and to prepare small Decade RNA markers (150, 100, 90, 80, 70, 60, 50, 40, 30, 20, and 10 nt; see Figure 5), which are included in the kit.

Figure 5. miRNA Detection after Gel Purification. miRNAs were gel purified on a 15% denaturing polyacrylamide gel from 150 g of mouse lung or thymus total RNA using standard procedures. Recovery was analyzed with the mirVana miRNA Detection Kit and autoradiography (2 hours exposure) by comparing the amount of miR-16 miRNA in the sample before (1% or 0.5% of the starting total RNA) and after purification (2% or 1% of the miRNA fraction). Percentage of recovery (59 and 53% respectively) was then determined by quantification with a phosphorimager. mir-16 probe and Decade Markers were prepared with the reagents included in the mirVana Probe & Marker Kit.


A Complete Solution
Ambion's mirVana system provides the first complete solution for small RNA analysis. The system allows for the isolation of total RNA samples that contain small RNAs--or the preparation of RNA samples that are enriched for small RNAs. Alternatively, "miRNA certified" total RNAs from over 80 different human, mouse, and rat tissues can simply be purchased. Once the RNA samples are obtained, high specific activity radiolabeled probes can be prepared by IVT or by 5' end-labeling, and target small RNAs be detected using the sensitive solution hybridization assay. The mirVana system is ideal for the detection of small RNAs molecules in a variety of experimental settings (see sidebar: Analysis of miRNA Recovery after Gel Purification), deciphering miRNA expression patterns across tissues and species, or determining siRNA expression levels in cell lines and organisms.


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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 1560 mirVana miRNA Isolation Kit up to 40 purifications
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