Technical note
MicroRNA profiling directly from low
amounts of plasma or serum using the
Multiplex Circulating miRNA Assay with
Firefly™ particle technology
Abstract
We introduce a new assay that enables the profiling of up to 68 microRNAs (miRNAs) of choice in the same
well directly from plasma or serum with no need for RNA purification. The Multiplex Circulating miRNA Assay
using Firefly technology allows the parallel processing of 96 samples with readout on standard flow cytometers.
The platform is built on hydrogel particles that maximize miRNA capture capacity and provide a solution-like
environment, making the assay sensitive and specific. We demonstrate how this assay can be used for miRNA
profiling with PCR sensitivity directly from as little as 10 μl of serum or plasma with no need for RNA purification,
or from less than 100 pg of purified RNA. We also introduce easy-to-use bioinformatics tools that are integrated
with the assay software for fast, efficient data analysis.
Technology overview
MicroRNA (miRNA) profiling has tremendous potential
for the diagnosis and prognosis of a broad range of
diseases. Due to their stability in blood, miRNAs are
ideal biomarkers; however, the full promise of miRNA
profiling has yet to be realized, largely due to the limited
throughput of existing validation technologies, errors
introduced during sample purification, and insufficient
methods for analytical normalization and quality control.
We have developed the Multiplex Circulating miRNA
Assay using Firefly technology to enable direct detection
of up to 68 miRNAs of choice with PCR sensitivity from
crude biofluids including serum, plasma and exosomes,
with no need for RNA purification.
Superior sensitivity. While miRNAs in circulation may
represent ideal biomarkers, they are present in very
low abundance. Typically, in order to profile miRNAs
in complex samples such as plasma or serum, it is
necessary to purify RNA. Most protocols require a
relatively large sample volume (200 μl) to obtain a
sufficient RNA yield for downstream profiling. The
Multiplex Circulating miRNA Assay is designed to
accommodate very low input RNA, allowing robust
detection of many miRNAs in single-cell equivalent
amounts (<10 pg). Because of this sensitivity, it is ideal
for profiling miRNAs from small volumes of biofluids of
10 μl or less.
Multiplexing and customization. The Multiplex Circulating
miRNA Assay is capable of measuring up to 70 targets
(68 + 2 built-in controls) simultaneously with no physical
splitting of the sample. This true multiplexing gives a
profile in every well, making the workflow simpler than
existing methods and eliminating the need for inter-well
normalization. The assay is completely customizable –
researchers may choose from pre-defined or custom
miRNA panels to detect any combination of miRNAs from
any species, whether annotated in miRbase or not.
Throughput and protocol. The assay is performed in a
96-well filter plate, allowing the parallel analysis of up
to 96 samples. The Multiplex Circulating miRNA Assay
workflow (Figure 1) involves six main steps:
1.Addition of 40 μl digest buffer to 40 μl plasma or
serum, followed by a 45 minute incubation at 60°C to
lyse cells (not required if starting from purified RNA).
2.Addition of particles, hybridization buffer and sample
to assay wells, followed by a 60 minute hybridization at
37°C. 2X rinse.
3.Addition of labeling buffer, followed by a 60 minute
incubation at room temperature. 3X rinse.
A
C
4.Elution of miRNAs from particles, followed by addition
of PCR mix to eluent and 60 minute amplification
program in a thermocycler.
5.Transfer back to plate, followed by 30 minute recapture
at 37°C. 2X rinse.
6.Incubation with a fluorescent reporter for 45 minutes at
room temperature followed by 2X rinse and scanning
on a standard flow cytometer.
Overall, the miRNA circulating assay takes 4–7 hours
from sample to data, depending on how many samples
are processed in parallel and which cytometer is used for
scanning.
Firefly
Multimix
B
Add
Labeling Mix
Add
PCR mix
Transfer back
to Plate
Hybridize
Label
Amplify
60 minutes
37º Shaking
60 minutes
RT Shaking
60 minutes
Thermocycler
Analyze
Scan
Add
Reporter
Recapture
30 minutes
37º Shaking
Report
Samples
30 minutes
RT Shaking
Figure 1: Multiplex Circulating miRNA Assay workflow. True multiplexing gives you a profile in every well, making the workflow simpler and more
efficient than other methods. (a) brightfield and (b) fluorescence images of encoded hydrogel particles. (c) Multiplex Circulating miRNA Assay
workflow. After capture, labeling and amplification of target miRNAs, assay readout is performed using a standard flow cytometer. Data files from the
cytometers are interpreted with the Firefly Analysis Workbench software for analysis and export.
Molecular workflow. The unique combination of hydrogel
particles with a post-hybridization labeling assay enables
miRNA profiling with enhanced performance across a
broad range of sample types. The poly(ethylene glycol)
hydrogel provides a high-capacity, bio-inert substrate
with superior thermodynamics relative to solid surfaces.
miRNAs are effectively captured in a three-dimensional
volume where they are subsequently labeled and
amplified. Our post-hybridization labeling method is ideal
for the detection of miRNA targets directly from crude
samples, regardless of purity (Figure 2).
With the Firefly technology, unlike other approaches,
targets are labeled after they have been captured by
miRNA-specific probes embedded in the hydrogel
particles. Probes are designed to have three binding
sites: one for a specific miRNA and two for universal
adapter sequences used for subsequent amplification
(Figure 2). After the target miRNAs are captured on
their corresponding probes, universal adapters are
attached to the miRNAs via ligation and are amplified
and labeled by PCR amplification with labeled primers,
then re-hybridized to the probes and detected via
fluorescence. The level of fluorescence is quantitative,
providing an accurate indication of target level in a given
sample.
Importantly, because the miRNA hybridization also acts
as a purification step, this approach is not affected by
PCR inhibitors, including heparin, that reduce sensitivity
in other detection systems.
DNA Probes
universal
label region
Sample
Labeling Mix
PCR with
Universal
Primers
Rinse and
Meltoff
3’
Re-hybe
onto
Particles
Report
target-specific
region
universal
label region
5’
Unligated
Adapter
Unbound
RNA
PEG
miRNA Capture
End-Labeling
Universal
Amplification
Melt-Off
Re-Capture
Report
Figure 2: Multiplex Circulating miRNA Assay labeling and amplification scheme. Probes embedded throughout the particle hydrogel have sites for
a specific miRNA and two adjacent sites for universal amplification. The assay workflow involves direct miRNA capture, end-labeling with adapters
followed by universal amplification, re-capture and reporting with fluorescence.
Assay sensitivity and specificity
A
Sensitivity
targets detected
We assessed the sensitivity of the Multiplex Circulating
miRNA Assay using a mixture of total RNA isolated
from human brain, lung and liver (Figure 3a). We
demonstrate the high sensitivity of the assay by
measuring the number of miRNAs detected from varying
input amounts. The detection limit for each miRNA was
determined as a multiple of the background noise
across negative control wells for each target. Out of a
total of 45 target miRNAs, 91% were detected down to
39 pg of input RNA (Figure 3a) and 51% were detected
down to 2 pg. Error bars indicate standard deviation
between technical replicates (n=3).
50
45
40
35
30
25
20
15
10
5
0
B
We also assessed specificity of the assay using miRNAs
let-7a, 7b, 7c and 7d individually spiked into a panel
containing probes for 7a, 7b, 7c, 7d, 7e, 7g and 7i, which
differ by one or two nucleotides (Figure 3b, top). We
observed low cross-reactivity for all off-target probes,
typically 2–8%. We obtained similar results for the
mir-30 family (Figure 3b, bottom). Figure 3c shows the
reproducibility of the assay across four different samples
for two replicates with 5 ng of starting total RNA. All
replicates show Pearson correlations >0.99 (Figure 3c).
Specificity
C
Reproducibility
100%
9.0%
7.9%
3.9%
5.8%
5.3%
2.8%
3.7%
let-7b
5.2%
100% 19.5%
2.9%
3.5%
3.2%
3.4%
4.2%
let-7c
14.4% 18.6% 100%
7.2%
7.9%
6.9%
6.9%
7.6%
let-7d
4.6%
100%
2.0%
2.2%
2.1%
1.6%
3.5%
2.9%
mir-302a mir-302b mir-302c mir-302d mir-502h mir-106b
2500
625
156
39
assay input (pg)
10
2
1
mir-302a
100%
1.9%
1.9%
2.5%
1.3%
5.6%
mir-302b
5.1%
100%
7.9%
6.6%
5.6%
24.8%
mir-302c
1.5%
2.0%
100%
2.6%
0.9%
8.6%
mir-302d
2.2%
2.3%
2.4%
100%
1.3%
8.0%
replicate #2
let-7a let-7b let-7c let-7d let-7e let-7f let-7g let-7i
let-7a
replicate #1
Figure 3: Sensitivity, specificity and reproducibility of the Multiplex Circulating miRNA Assay. (a) Number of target miRNAs detected, out of a total
of 45, versus input amount, (b) assay specificity for families of highly similar miRNAs, (c) data correlation across four different samples at low input
amounts of miRNA.
miRNA profiling from plasma/serum with no purification needed
Robust miRNA profiles can be obtained with the Multiplex
Circulating miRNA Assay directly from crude biofluids
such as serum, plasma, and exosomes with no need for
RNA purification. This approach dramatically simplifies the
workflow (Figure 4a) and eliminates an unreliable step in
sample preparation. This approach reduces the absolute
amount of starting material needed, and also eliminates
the need for phase-separation based RNA extraction.
We compared miRNA expression profiles obtained from
purified RNA with those obtained from crude serum
(Figure 4b). For purified RNA samples, TRIzol® LS was
used with the recommended protocol starting from an
input amount of 250 μl of serum. For the crude samples,
40 μl of serum was used in the digest. In both cases,
an equivalent of 12.5 μl serum was used as input to
the Multiplex Circulating miRNA Assay. As shown in
Figure 4, there is a close correlation between data
from crude serum and purified RNA. The elimination
of RNA purification from miRNA profiling workflow
makes the Multiplex Circulating miRNA Assay ideal
for high-throughput applications where reduction of
pre-analytical variability is crucial.
B
RNA Purification (traditional approach)
Digestion
Phase
Separation
Precipitation
Resuspension
Assay
Detection in Crude (Firefly approach)
Digestion
Purified RNA (log2)
A
Assay
Crude (log2)
Figure 4: miRNA profiling in crude digests vs. extracted RNA. (a) Assay workflows using purified miRNA and crude serum, (b) correlation between
miRNA profiles obtained using the two approaches.
Streamlined expression analysis with an integrated software suite
Proper interpretation of profiling data is a critical
component of biomarker discovery and validation.
Strategies for target normalization and cohort
comparison must be considered carefully during data
analysis for results to be reliable. The Firefly Analysis
Workbench provides the means to interpret, visualize,
normalize and compare data, as demonstrated in the
following study.
To illustrate this, we profiled crude digests of plasma
and serum obtained during a blood draw from a single
patient using a variety of collection methods including
potassium EDTA, lithium heparin, sodium citrate and
sodium heparin. Data were analyzed using the Firefly
Analysis Workbench. After geNorm-based normalization,
expression profiles were compared on a heat map
(Figure 5a), showing consistent profiles for all plasma
samples, including heparin samples, but a slightly varied
profile for the serum sample.
We used the Firefly Analysis Workbench to perform
ANOVA across the various sample types to determine
statistically which miRNAs were differentially expressed
across the sample types. In order to account for
multiple comparisons, we used a Bonferroni correction.
This yielded nineteen miRNAs that were differentially
expressed with statistical confidence (p-value <0.05).
Those with the lowest p-values are shown in Figure 5b.
Interestingly, we observed a dramatically lower
expression of several miRNAs (including miR-130a,
221 and 146a) in serum versus plasma, but higher
expression of other miRNAs (including miR-122). These
data were consistent for four other patients tested (data
not shown), all of which were obtained from the same
source.
A
B
mir-221-3p
mir-146a-5p
mir-122-5p
Group Signal
mir-130a-3p
Figure 5: Firefly Analysis Workbench expression analysis across sample collection methods including potassium EDTA (K2EDTA), lithium heparin
(LiHep), sodium citrate (NaCit), sodium heparin (NaHep), and sera. (a) Heat map showing miRNA expression profiles of serum and plasma samples,
(b) expression of miRNAs that were differentially expressed across sample types.
In conclusion, the Multiplex Circulating miRNA Assay
provides researchers with a robust method to profile
miRNAs in crude biofluid digests. The assay is sensitive,
specific, and enables high-throughput analysis required
for biomarker validation studies. The use of crude
biofluid digests eliminates a major source of
Related publications
Chapin SC, Pregibon DC, Doyle PS (2011). Rapid microRNA Profiling on Encoded
Gel Microparticles. Angew Chem Int Ed 50, 2289–2293.
Chapin S, Pregibon DC, Doyle PS (2009). High-throughput flow alignment of
barcoded hydrogel microparticles. Lab Chip 9, 3100–9.
Dendukuri D, Pregibon DC, Collins J, Hatton TA, Doyle PS (2006). Continuous flow
lithography for high-throughput microparticle synthesis. Nat Mater 5, 365–369.
Pregibon DC, Doyle PS (2009). Optimization of Encoded Hydrogel Particles for
Nucleic Acid Quantification. Anal Chem 81, 4873–81.
Pregibon DC, Toner M, Doyle PS (2007). Multifunctional Encoded Particles for
High-Throughput Biomolecule Analysis. Science, 315, 1393–1396.
Discover more at abcam.com
pre-analytical variability while minimizing workflow.
In addition to utilizing robust analytical methods, the
Firefly Analysis Workbench provides a means to rapidly
visualize and interpret experimental data. These tools will
provide researchers with a flexible and reliable method
for miRNA profiling across a broad range of applications.