microRNAs (miRNAs) are endogenous, 21-24 nt RNAs that mediate post-transcriptional gene regulation by pairing with the 3' untranslated region of messenger RNAs and acting as translational repressors. The study of miRNA is an emerging and exciting area of research with applications for basic, applied, and therapeutic science. Recent reports suggest a role for miRNAs in development, cell differentiation, apoptosis, and cancer. Ambion has recently developed several new tools for isolation and detection of miRNAs, which were used to investigate miRNA expression in white blood cells.

Introduction

In the past few years, several discoveries have indicated that miRNAs play a role in various cellular functions. Early studies of miRNAs suggest that they could become a valuable tool for elucidating how gene expression can direct development, differentiation, and proliferation. This area of research could provide more insight into the causes of certain diseases, leading to the identification of novel therapeutic or diagnostic targets. Interest in the identification, detection, and use of small RNA molecules has exploded in response to these discoveries. However, standard RNA isolation and quantitation techniques have been optimized for larger RNAs and are not always favorable for recovering and analyzing smaller RNA species.

Analysis of Small RNA Expression

The size, and in some cases the low expression levels, of small RNAs can make their analysis difficult. The small size of these molecules alone precludes the use of RT-PCR as a detection method, since they are too short to be specifically primed for PCR-mediated amplification. Furthermore, most RNA isolation procedures have been optimized to recover long (>500 nt) messenger RNAs, while ignoring smaller molecules. As a result, conventional RNA extraction methods can result in the loss of substantial amounts of small RNAs from samples. To address the issues of small RNA recovery and detection, Ambion scientists have developed the mirVana™ line of products, a group of kits designed to provide a complete solution for small RNA analysis.

The mirVana™ miRNA Isolation Kit (patent pending) was codeveloped with the mirVana™ miRNA Detection Kit to provide quantitative yields of small RNAs (including miRNA, siRNA, tRNA, and rRNAs) from virtually any biological sample. The patented mirVana miRNA Detection Kit is a set of reagents for small-volume solution hybridization assays that is far more efficient than membrane-based hybridization (e.g. Northern blots). The assay can detect relatively abundant miRNA species, such as miR-124 in brain, in as little as 10 ng of total RNA.

An example of small RNA analysis using these tools is shown in Figure 1. The data show that the same amount of 5.8S and 5S rRNA was recovered from mouse brain, kidney, liver and thymus. In contrast, significant variations in miRNA levels were observed in these tissues. For example, let-7 was more abundant in brain and kidney, and miR-16 was highly expressed in thymus only. As expected, based on published reports [1, 2], miR-124 was detected only in the brain sample.


Figure 1. Analysis of miRNA Expression Across Mouse Tissues. Total RNA was isolated from ~100 mg of the indicated tissues with the mirVana™ miRNA Isolation Kit. Purified RNA (1 µg) was analyzed on a 15% denaturing polyacrylamide gel stained with ethidium bromide (left panel). let-7 miRNA, and 5.8S and 5S rRNA were detected by Northern blot. miR-16 and miR-124 miRNA were detected by solution hybridization with the mirVana™ miRNA Detection Kit. All probes were labeled and purified with the mirVana™ Probe & Marker Kit.

Expression of miRNA in Blood Cells

Although miRNAs have been found in all mammalian tissues examined so far, there is little if any published information about miRNA expression in blood. This may be due to the inherent difficulty in isolating high quality RNA from human blood. To examine the efficiency of the mirVana system for use in analysis of miRNAs in white blood cells, blood was collected from 14 healthy volunteers. Leukocytes were fractionated from whole blood and the total RNA population, including miRNA, was extracted using the mirVana miRNA Isolation Kit. The RNA recovered from the fractionated white blood cells was quantitated by UV absorbance and analyzed by ethidium bromide staining on a 15% denaturing polyacrylamide gel. RNA yields ranged from about 20-40 µg per 10 ml blood sample. No obvious degradation of the ribosomal RNA and tRNA bands was observed. Four representative samples are presented in Figure 2.


Figure 2. Analysis of miRNA Expression in White Blood Cells. Leukocytes were collected from 10 ml EDTA-anticoagulated blood samples from four different donors, and total RNA was isolated with the mirVana™ miRNA Isolation Kit. Samples were analyzed as described in Figure 1 except that a probe specific for miR-22 miRNA was used in the solution hybridization assay.


The RNA samples were then analyzed for the presence of several small RNAs using Northern blot or the mirVana miRNA Detection Kit. Interestingly, several miRNAs--let-7, miR-16, and miR-22--were detected in total RNA from white blood cells (Figure 2). miR-16 was the most abundant miRNA in all of the 14 samples. A direct comparison of total RNA samples from other human tissues showed that miR-16 is expressed at a higher level in white blood cells than in any other tissues we have examined (data not shown). While different expression levels were observed across donors for the few miRNA tested, there was no clear donor-specific difference in the global miRNA expression pattern. Experiments are in progress to determine whether other miRNAs may have donor-specific expression patterns and whether these patterns vary over time.

Outlook

As the study of small RNA molecules continues, the need for fast, robust, and sensitive miRNA analysis tools is increasing. Further investigation of the biological role of miRNA will be facilitated by methods such as those incorporated in the mirVana system. High throughput detection of miRNA will undoubtedly help to unravel their complex spatial and temporal expression patterns, as well as determine variations in miRNA levels during tissue development and in disease states.