MicroRNAs (miRNAs) are small, highly conserved RNA molecules that act as key regulators of development, cell proliferation, differentiation, and the cell cycle. Now that many miRNA sequences are known, researchers are increasingly analyzing and comparing miRNA expression levels between different tissues, developmental stages, or disease states.

Here we provide an experimental overview for the quantitation of specific miRNA expression levels by qRT-PCR using both Applied Biosystems and Ambion products. Use of these products provides researchers with a validated, reliable, ready-to-use approach for the quantitation of miRNA levels in a variety of sample types, hopefully accelerating the discovery of the biological roles of miRNAs in cells and in human disease. In the included case study, we describe the analysis of miRNA levels from total RNA and RNA samples enriched for small RNA. These samples included both frozen or RNAlater® Solution-treated mouse, brain, liver, and lung tissues.

An Effective Method for miRNA Isolation

A. Sample Acquisition and Storage
Once samples have been obtained, they should be processed immediately. Tissue should be frozen (small pieces in liquid nitrogen is preferable) or placed in RNAlater solution for storage until RNA extraction is performed. RNAlater is an aqueous tissue storage reagent that protects RNA within intact, unfrozen samples.

B. Isolation of miRNA-containing Total RNA
Isolation of miRNA begins when total RNA that includes the small RNA fraction is isolated from the samples of interest. However, not all isolation methods retain the small RNA fraction. Therefore, it is important to use RNA isolation methods specifically adapted to retain it. The mirVana miRNA Isolation Kit was developed to retain these small RNA species either in a background of total RNA or as an enriched fraction of RNA species 200 nucleotide in size or smaller. The initial organic extraction of the mirVana miRNA Isolation Kit provides a robust front-end purification that removes cell debris and most DNA. In addition, reagents and a procedure are provided to enrich the population of RNAs that are 200 bases and smaller, using two sequential filtrations through GFFs with different ethanol concentrations. Although generally not necessary for real-time PCR applications, small RNA enrichment results in lower background and enhanced sensitivity of small RNA detection by solution hybridization, Northern analysis, and other methods, as compared to the same assay using total RNA.

Assessing RNA Yield, Purity, and Quality

Figure 1. Efficient Recovery of MicroRNAs. Total RNA was isolated from brain, liver and lung tissue using the mirVana™ miRNA Isolation Kit. Typically, one can expect to get about 1 µg RNA for every mg of tissue. The mirVana Isolation Kit also provides reagents and a procedure to enrich the population of RNAs that are 200 bases and smaller.  Since tRNA and other small functional RNAs comprise 5-20% of the total RNA population, the gross recovery of enriched RNA by A260 will only be about a tenth that of total, but the amount of miRNA present will be the same.