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mirVana™ miRNA
Isolation Kit
mirVana™ miRNA Probe Set
Your Data: Involvement of miRNAs in the Regulation of Innate Immune Responses
microRNAs (miRNAs) have been implicated in the
control of innate mammalian immune responses as negative regulators.
Dr. Taganov and colleagues (California Institute of Technology,
Pasadena, CA USA) assessed the expression profile of 200 miRNAs
in human monocytes, which revealed that several miRNAs are endotoxin
responsive genes. The results suggested that miR-146a/b functions
as a novel negative regulator that helps to fine-tune the immune
response. This study was performed using several techniques—microarrays,
Northern blots, and quantitative PCR.
Konstantin D Taganov, Mark P Boldin, Kuang-Jung
Chang, and David Baltimore.
Division of Biology, California Institute of Technology, Pasadena,
CA 91125
MicroRNA Expression Profiling Research
Microarray analysis was performed to identify
miRNA genes that may regulate innate immunity receptors. One
of the first steps in this study was to isolate high quality
total RNA from THP-1 human monocytic cells after stimulation
with lipopolysaccharide (LPS) (E. coli strain 055:B5). Total
RNA (40 µg)
was enriched for small RNA species using the mirVana™ miRNA Isolation
Kit, tailed with the mirVana miRNA Labeling Kit, and fluorescently
labeled with Cy™3 and Cy5 dyes (Amersham, NJ). Microarrays
were prepared by robotic spotting of the mirVana miRNA Probe
Set--DNA oligonucleotide probes complementary to 200 miRNA sequences
of human, mouse, and rat origins. The labeled RNA sample was
then hybridized to the resulting slide arrays.
The microarray study revealed up-regulation
of miR-146, miR-155, and miR-132 expression, and this result
was validated by quantitative RT-PCR (qRT-PCR). For qRT-PCR analysis,
total RNA was isolated using the mirVana miRNA Isolation Kit,
and miRNA expression was quantified and measured by using Applied Biosystems TaqMan® MicroRNA
Assays or the Ambion mirVana qRT-PCR miRNA Detection Kit. The kinetics
of miR-146a/b suggested that it is an immediate, early-response
gene.
Because the human genome contains two miR-146
genes, Northern blot analysis was performed using DNA probes
complementary to the mature sequences. The mirVana miRNA Isolation
Kit was again used to isolate total RNA and probe hybridizations
were performed using ULTRAhyb®-Oligo Ultrasensitive Hybridization
Buffer. Results suggested that miR-146a was strongly expressed,
and, by further promoter analysis, miR-146a was found to be an
NFk-B-dependent gene. This groundbreaking study highlights the
importance of miRNAs in the immune response and how a variety
of Applied Biosystems and Ambion reagents can facilitate the
discovery of miRNA roles in critical biological processes.
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