|
miRNA Expression in White Blood Cells
Emmanuel Labourier, Marianna Goldrick,
Ambion, Inc.
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.
back
to top
|
