Detecting Attomole Amounts of Small RNA

mirVana™ miRNA Detection Kit

mirVana™ miRNA Detection Kit

Sensitive -- Detect miRNA or siRNA in as little as 10 ng total RNA
Specific -- Extremely low background with no need to optimize hybridization or washing conditions
Simple and fast -- Single tube procedure eliminates the need to transfer to membrane for hybridization
Multiple target detection-- Detect small RNAs and mRNAs in the same sample

The new mirVana miRNA Detection Kit provides a faster and more sensitive alternative to Northern analysis for detecting small RNAs. With this patented technology, quantitation of small RNA species can be performed with as little as 10-50 ng of total RNA. The assay is incredibly sensitive and is able to detect attomole (10-18 mol) amounts of target RNA. In addition, the mirVana miRNA Detection Kit can be used to simultaneously detect several small RNAs of the same size or both small RNA and long RNA species in the same sample. This means you can now quantitate an siRNA and its target mRNA in the same sample.

Rapid Procedure Based on Solution Hybridization

The mirVana miRNA Detection Kit is based on a simple solution hybridization principle. The sample RNA containing the target RNA(s) of interest is mixed with one or more high specific activity radiolabeled RNA probes and the included Hybridization Buffer. After heat denaturation, each mixture is incubated at 42°C to hybridize the probe to its complementary RNA molecules. Unhybridized RNA species and excess RNA probe is then removed by a rapid ribonuclease digestion step. The hybridized, protected RNA fragments are recovered in the same tube using Ambion's patented single step technology for simultaneous ribonuclease inactivation and nucleic acid precipitation. RNA samples are then resuspended with the provided Gel Loading Buffer and analyzed on a denaturing polyacrylamide gel. Because the hybridization is performed in solution, the procedure ensures a sensitive and linear detection signal after autoradiography. Furthermore, the included Hybridization Buffer has been specifically developed to provide the optimal sensitivity and specificity of detection with short antisense probes. Thus the procedure is ideal for detecting small RNA molecules such as siRNA or miRNA.

Complete Kit with Included Controls

Each mirVana miRNA Detection Kit provides enough reagents for 100 reactions. The kit also contains a control transcription template and Probe Elution Buffer to prepare and gel purify a 32 nt RNA probe specific for miR-16 miRNA. When used with the provided Mouse Kidney Total RNA, this probe generates a 22 nt protected fragment. The kit also comes with a detailed Instruction Manual and comprehensive information about experimental setup and probe design/preparation.

Figure 1. Differential Recovery of Small RNAs During Total RNA Isolation. Total RNA was isolated from 1x106 HeLa cells using three standard RNA isolation procedures: single phenol/guanidinium extraction (RNAwiz™), glass fiber filter (RNAqueous®) purification, or double phenol extraction (ToTALLY RNA™). Purified RNA samples were analyzed on a 1% denaturing agarose gel and recovery of messenger RNA (ß-actin, GAPDH), small nuclear RNA (U1 snRNA), and miRNA (miR-16, miR-22) were assessed by Northern blot or solution hybridization with the mirVana miRNA Detection Kit.

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RNA Isolation Can Affect miRNA Analysis Recent studies suggest that microRNAs (miRNAs) may play an important regulatory role in gene expression, especially during differentiation and development. Many miRNAs are expressed at differing levels across tissues and developmental stages. When studying miRNAs in tissues or cells, choosing a compatible RNA isolation method is critical. Not all isolation procedures provide quantitative recovery of small RNAs. In fact, one of the most common methods utilization of a silica matrix or glass fiber filter can result in the loss of almost all small RNAs. Figure 1 (below left) shows a comparison of three traditional methods of RNA isolation: a single-phase phenol based method (RNAwiz™), a glass fiber filter method (RNAqueous®), and a guanidinium/double phenol extraction method (ToTALLY RNA™). The data demonstrate that miRNAs are recovered by both phenol-based methods while the standard glass fiber filter method yields virtually no detectable miRNA. This suggests that, while perfectly suitable for isolating messenger RNA, traditional glass fiber filter based methods are not appropriate for the recovery of small RNAs including miRNAs.

Total RNA Does Not Always Contain miRNA
When purchasing commercially available purified RNAs it is also important to consider the isolation method used to produce the RNA. If the RNA was prepared using traditional glass fiber filter methodologies, it most likely does not contain detectable levels of miRNAs. Ambion has developed optimized protocols for isolating RNA from various tissues, and we are currently evaluating each of our FirstChoice® Total RNA products for the presence of miRNAs. This includes RNA from over 80 different human, mouse, and rat tissues.