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Assays Specific to the miRNAs of
Interest
Until now, small RNAs like microRNAs (miRNAs) could not be
analyzed by traditional PCR. TaqMan MicroRNA Assays have been
validated to amplify specific small RNAs, enabling accurate
and specific quantitation of miRNA expression levels by real-time PCR. A large
collection of pre-designed, off-the-shelf TaqMan MicroRNA Assays
for human, mouse, rat, A. thaliania, C. elegans, and D.
melanogaster are available from Applied Biosystems. Each one is functionally
validated, convenient, and easy to use.
Real-Time Analytical Instruments
and Reagents Validated for miRNA Detection Protocols
Applied Biosystems Real-Time PCR Systems make real-time PCR
more accessible than ever before by providing powerful solutions
to fit the needs of any laboratory. By taking advantage of
gold-standard TaqMan reagent-based technology with universal
thermal cycling conditions, TaqMan Gene Expression Master Mix
is the ideal reagent solution for TaqMan assays using DNA or
cDNA as the target. Both the Master Mix and the Applied Biosystems 7900HT Fast Real-Time PCR System have been validated for use with TaqMan
MicroRNA Assays.
Case Study
In our study, the following TaqMan MicroRNA Assays and controls were used: hsa-miR-1, hsa-miR-24, hsa-miR-16, hsa-miR-133a, hsa-miR-145, RNU6B (U6 Control), and RNU19 (U19 Control). The miRNAs to which these assays were designed have been shown to exhibit differential expression patterns in cancerous tissues as compared to normal tissues and may play a role in oncogenesis [1–3].
Real-time PCR was performed by adding 1.34 μL
(a 458 ng tissue equivalent) of each completed RT reaction
to a target TaqMan MicroRNA Assay reaction using TaqMan Universal
PCR Master Mix (final reaction volume equal 20 μL) (TaqMan Gene Expression Master Mix was not yet available when this study was conducted). Samples
were tested in triplicate and run on the Applied Biosystems
7900HT Fast Real-Time PCR System. Assay results
were collected and analyzed using SDS 2.2.2 software.
Results and Conclusions: Figure 4 shows a ~3.3 Ct difference between miRNA levels in
the total RNA samples and the samples enriched for miRNA,
which indicates about a tenfold enrichment. This is consistent
with our expectations given that the data were normalized
to input mass of RNA. Of note, the differential miRNA expression
level trends between the different tissues were similar in
both sample types. This experiment, as well as others done
in our labs, demonstrates that miRNA enrichment preserves
miRNA differential expression patterns as compared to total
RNA. Although we find that total RNA isolated with the mirVana
miRNA Isolation Kit yields more than sufficient signal with
TaqMan MicroRNA Assays, other techniques such as microarray
and Northern analysis require this enrichment to yield sufficient
signal for analysis.
Figure 4 also indicates that the frozen and RNAlater Solution-treated samples yielded Cts that were roughly equivalent. This
experiment demonstrates that there is no significant difference
in miRNA expression profiles from frozen and RNAlater Solution-treated
tissues when RNA is isolated using the mirVana miRNA Isolation
Kit.
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| Figure 4. Real-time
PCR Results in Frozen Versus RNAlater® Solution-Treated Sample. The
frozen and RNAlater Solution-treated samples are roughly
equivalent, and the enriched samples show ~3.3 Ct’s
increase in signal, consistent with about a tenfold enrichment.
U6 and U19 are TaqMan® MicroRNA Assay Controls, which
have been designed to aid in relative quantitation.
*hsa-miR-133a (Panel A) and hsa-miR-1 (Panel B) were spiked into the samples as controls. |
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