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 (19 24 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
The mirVana™ System for Small RNA Analysis
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
"miRNA Certified" FirstChoice RNAs
Sensitive Detection of Small RNAs
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. 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 µg of total RNA (3). In contrast, only 1 µg 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 µg 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
Preparation of RNA Probes
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 µg 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 µg 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.