miRNA Expression in White Blood Cells
|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.|
Analysis of Small RNA Expression
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
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.