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flashPAGE™ Electrophoretic
Fractionator
Purifying Small RNAs: Using Mature MicroRNAs
for Microarray Analysis
Small RNA analysis methods (e.g., hybridization
assays, expression profiling) require various RNA input amounts
and sizes. Small RNA fractions often include tRNA, 5S rRNA, 5.8S
rRNA, small mRNA fragments, and precursor miRNAs. The presence
of these RNA species does not affect Northern analysis
and solution hybridization; however, they can obscure results
of some experiments, particularly microarray studies. To obtain
mature miRNA, use the flashPAGE™ Electrophoretic Fractionator,
which purifies small RNA species (15–35 nt range) from
total RNA samples. Here we show how an RNA fraction that contains
only mature miRNA can provide better miRNA profiling results
than an enriched small RNA (or total RNA) sample. This is just
one example of the increased sensitivity gained by further removing
unwanted RNA species from the sample to be analyzed. These sensitive
methods may be required for detection and identification of less
highly expressed miRNAs.
In microarray experiments, unwanted small RNA
species not only compete for reagents used in labeling reactions
but also add to background signals and potentially hybridize
to probes. Figure 1 shows two microarrays hybridized with RNA
that was from the same RNA source but purified by two different
means, so that the enriched small RNA fraction contains RNA <200
nt and the mature miRNA fraction, isolated with the flashPAGE
Fractionator, contains mature miRNA (15–35 nt). Duplicate bladder
and lung samples (15 µg total RNA input that was enriched
or fractionated) were compared to each other to provide a two-color
readout and provided consistent results. The higher signal intensity
and lower background from the samples that contain only mature
miRNA (flashPAGE-purified RNA) are immediately noticeable. The
patterns are recognizable between the two arrays, but some information
is more apparent, primarily with lower-strength signals. This
difference is quantified by assessing the correlation with a
standard for the tissues studied (Figure 2). The standard represents
the average of many assays from the same tissues, and deviation
is minimal from samples using the same (non-diseased) tissues.
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Figure
1. Representative Arrays Using
Enriched or flashPAGE™-purified Samples. Target
RNA samples from human bladder and lung samples prepared
from the same parent total RNA (A) RNA
enriched small RNA (<200 nt) and (B) RNA
fractionated with the flashPAGE Fractionator were labeled
using the mirVana™ miRNA Labeling Kit
and compared on identical arrays (custom-printed
arrays using the mirVana miRNA Probe Set): Red indicates higher expression in bladder,
green indicates higher expression in lung.
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Figure 2. Correlation of Bladder
and Lung Data with Standard Profiles Using Enriched or
flashPAGE™-purified Samples. Data such
as that shown in Figure 1 were compared to consensus
data from many trials of relative expression between
the two tissues. The percent of correlation was compared
for 10 µg, 5 µg, and 1 µg of input
total RNA (prior to either enrichment or flashPAGE purification).
The first set of columns in each figure shows the correlation
if all spots on the test arrays above background are
used (Basic Filters), while the second and third sets
show a similar comparison if only array signals with
a Sum Intensity >3000 or >4500 are compared.
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The correlation stays high as the amount of
input is decreased from 10 to 1 µg with samples purified
with the flashPAGE Fractionator (Figure 2B), but drops radically
for those samples merely enriched for small RNA (Figure 2A).
This drop is due to low-intensity signals as indicated by the
higher correlation from the enriched samples (Figure 2A) when
only those signals with very high intensities over 4500 quanta
are compared.
As more microRNAs (miRNAs) are discovered,
it is becoming clear that the first wave of miRNAs that were
identified are expressed at a higher level than the ones now
being discovered. This means that determining expression levels
of many (perhaps the vast majority) of miRNAs will require a
highly-sensitive method. The flashPAGE Fractionator, especially
in conjunction with the mirVana miRNA Microarray System, is designed for that purpose.
Scientific Contributors
Kerri Keiger, Patricia Powers, Rick Conrad • Ambion,
Inc.
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