Nanoliter high throughput quantitative PCR
Tom Morrison, James Hurley, Javier Garcia, Karl Yoder, Arrin Katz, Douglas Roberts,
Jamie Cho, Tanya Kanigan, Sergey E. Ilyin1, Daniel Horowitz1, James M. Dixon1and
Colin J.H. Brenan*
BioTrove Inc., 12 Gill Street, Suite 4000, Woburn, MA 01810, USA and1Johnson & Johnson Pharmaceutical
Research & Development, LLC, Spring House, PA 19477, USA
Received June 15, 2006; Revised and Accepted August 16, 2006
Understanding biological complexity arising from
patterns of gene expression requires accurate and
precise measurement of RNA levels across large
numbers of genes simultaneously. Real time PCR
(RT-PCR) in a microtiter plate is the preferred
method for quantitative transcriptional analysis but
scaling RT-PCR to higher throughputs in this fluidic
format is intrinsically limited by cost and logistic
considerations. Hybridization microarrays measure
the transcription of many thousands of genes
simultaneously yet are limited by low sensitivity,
dynamic range, accuracy and sample throughput.
The hybrid approach described here combines the
superior accuracy, precision and dynamic range of
RT-PCR with the parallelism of a microarray in an
array of 3072 real time, 33 nl polymerase chain
reactions (RT-PCRs) the size of a microscope slide.
RT-PCR is demonstrated with an accuracy and
precision equivalent to the same assay in a 384-
well microplate but in a 64-fold smaller reaction
volume, a 24-fold higher analytical throughput and a
Central to research into cell survival, growth and differentia-
tion in normal and diseased states is the ability to quantify
altered patterns of gene expression. Oligonucleotide (1,2)
and cDNA (3) hybridization microarrays have emerged as
the leading quantitative tool for analyzing transcription of
many thousands of genes in a sample simultaneously (4)
yet have known limitations in analytical performance and
sample throughput (5–7). Real time or quantitative poly-
merase chain reaction (qPCR) (8) is the superior alternative
because of its high accuracy, precision and dynamic range
and, as a consequence, is the reference assay for calibration
and validation of microarray data (9). However, scaling
qPCR to analyze larger numbers of genes and samples
simultaneously is intrinsically prohibited by the logistics
and cost of the assay in its current microliter format in
96- or 384-well microplates.
High throughput PCR strategies have focused on smaller
reaction volumes and follow one of two fluidics methods.
Fast sequential analysis is exemplified by monolithic,
functionally integrated lab-on-a-chip devices that flow a sam-
ple bolus through fixed temperature zones of a microma-
chined channel for target sequence PCR amplification,
followed by sequence specific capture by hybridization and
electrochemical detection (10), fluorescence detection (11)
or electrophoretic separation with fluorescent detection (12).
With quantitative performance similar to a microarray, detec-
tion sensitivity is further constrained by sample throughput
and the increased potential for cross-contamination from
processing samples in a common microchannel.
Many of these problems are mitigated in a parallel fluidics
approach. Miniaturized versions of microplates based on
high-density arrays of wells etched in a planar substrate is
the basis for nanoliter- (13–16) or picoliter-scale PCR (17)
in an array format. Other embodiments include PCR in
microdroplets on a patterned hydrophobic–hydrophilic sur-
face (18) or in a 2D array of communicating microchannels
(19–21). Reports of quantitative nucleic acid measurement
in these devices have focused on limiting dilution schemes
(22–24), which is clearly not high-throughput. Achieving
high areal densities of physically independent reaction con-
tainers (>4/mm2) requires stringent fluidic isolation between
adjacent containers and a high degree of environmental con-
trol to prevent cross-contamination and evaporative loss
during temperature cycling. Despite these challenges, parallel
micro- or nanofluidics offers throughput advantages by
thermal cycling and imaging many reactions at once to
quantify target copy number in multiple genes and samples,
simultaneously. Imaging reactions in parallel allows for
longer integration times, improves detected signal-to-noise
ratios and benefits PCR specificity and sensitivity by requir-
ing fewer temperature cycles to detect a given target copy
number. Shorter cycle times are facilitated by rapid heat
transfer across proportionally larger surface areas as the
reaction volume is reduced.
*To whom correspondence should be addressed. Tel: +1 781 721 3615; Fax: +1 781 721 3601; Email: email@example.com
? 2006 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Published online 25 September 2006Nucleic Acids Research, 2006, Vol. 34, No. 18 e123
grounded in a high-density array of nanoliter reactions is
attractive because it combines the high precision, accuracy
and dynamic range of qPCR with the parallelism of a
microarray for simultaneous quantification of gene expression
across multiple genes and samples. For this to occur, two
challenges need to be overcome. The first is creation of a
simple interface for precise and accurate transfer of liquids
between the wells of a microplate to those of a nanoplate.
The second is achieving the accuracy, precision and sensitiv-
ity demanded by qPCR in a 96- or 384-well microplate but in
a substantially reduced reaction volume. Discovery of a facile
interface for speedy transfer of liquids between micro- and
nanoplates and identifying a robust approach to ensure
qPCR assay performance at the nanoliter-scale has been at
the leading edge of our development efforts.
We have solved these problems with an approach based on
through-hole arrays (25–27). Effectively thought of as a high-
density version of a microplate, our nanoplates combine the
high-throughput and reagent savings of a nanofluidic system
with the macroscale performance of qPCR in microplates. A
stainless steel (317 stainless steel) platen the size of a micro-
scope slide (25 mm · 75 mm · 0.3 mm) is photolithographi-
cally patterned and etched to form a rectilinear array of 3072,
320 mm diameter through-holes. The through-holes are
grouped in 48 subarrays of 64 holes each and spaced on a
4.5 mm pitch equal to that of wells in a 384-well microplate
(Figure 1). A series of vapor and liquid deposition steps
covalently attaches a PCR compatible polyethylene glycol
(PEG) hydrophilic layer amine-coupled to the interior surface
of each through-hole, and a hydrophobic fluoroalkyl layer
to the exterior surface of the platen. The differential
hydrophilic–hydrophobic coating facilitates precise loading
and isolated retention of fluid in each channel. Primer pairs
stored in 384-well microplates are transferred into individual
through-holes by an array of 48 slotted pins manipulated by a
4-axis robot (XYZ?) in an environmentally controlled cham-
ber to prevent evaporative loss during loading. Once a platen
is fully populated with primer pairs, the solvent is evaporated
in a controlled manner leaving the primers immobilized in a
PEG matrix on the inside surface of each through-hole. The
array loaded with primer is stored in an evacuated Mylar?
bag at ?20?C, ready for sample addition.
Up to 48 different, previously prepared cDNA samples at a
concentration of 32 ng/ml are mixed with off-the-shelf qPCR
reagents for SYBR Green PCR (see Materials and Methods;
PCR Mix) and dispensed into each sub-array (one sample per
sub-array) with an automated 48 pipette tip dispensing
device. A slotted cassette for holding the platen is assembled
by sandwiching a U-shaped glass-reinforced epoxy polymer
spacer between two microscope slides patterned with an opa-
que ink to optically mask background autofluorescence from
the spacer. A degassed, immiscible perfluorinated liquid
(Fluorinert?) is dispensed into the cassette, the platen
inserted and the assembly hermetically sealed with a plug
of ultraviolet (UV) curable epoxy.
Real time PCR (RT-PCR) occurs in a computer-controlled
imaging thermal cycler whose essential components are
two pairs of off-axis, high energy light emitting diode
(LED) excitation sources, a thermoelectric flat block
holding up to three encased arrays, two emission filters in a
computer-controlled filter wheel and a thermoelectrically-
cooled CCD camera. Under software control, the real time
method for 9216 PCR amplifications and dissociation curves
is implemented in <4 h. Post-acquisition data processing
generates fluorescence amplification and melt curves for
each through-hole in the array, from which cycle threshold
(CT) and melt temperature (Tm) are computed. All data are
stored in a flat file (*.csv) format for ready export to a
database or third party software for further analysis.
MATERIALS AND METHODS
Through-hole array fabrication
Sheets comprised of 12 arrays attached by thin tabs to a sup-
port frame were purchased from Tech-Etch Inc. (Plymouth,
MA). The arrays are fabricated by double-sided wet-etching
of a photolithographically-patterned 300 mm sheet of
317 stainless steel resulting in the hole pattern shown in
Figure 1. The sheets are cleaned for 2 h in 10% RBS
35 (Pierce) at 50?C, rinsed in reverse osmosis de-ionized
(RODI) salt water and dried with a stream of dry
(7-octenyltrimethoxysilane, Gelest) is vapor deposited [5 h,
100?C in a vacuum oven (VWR)] followed by a 30 min
Next, the vinyl groups inside the through-holes are selec-
tively oxidized by first immersing the sheet in a 1l bath of
ethanol to overcome the surface tension of the hydrophobic
coating followed by immersion in 1L of RODI water. The
sheet is next slowly passed through a layer of 30 ml of an
oxidation solution (5 mM KMnO4and 19.5 mM NaIO4) float-
ing on 1l of Fluorinert? (FC3283), incubated for 2 h, rinsed
in RODI water and dried in a stream of dry nitrogen gas.
33 nL reaction
Figure 1. A stainless steel platen (317 stainless steel) the size of a microscope
slide (25 mm · 75 mm · 0.3 mm) is photolithographically patterned and wet
etched to form a rectilinear array of 3072 micro-machined, 320 mm diameter
holes of 33 nl each. The 48 groups of 64 holes are spaced at 4.5 mm to match
the pitch of the wells in a 384-well microplate. A PCR compatible PEG
hydrophilic layer is amine-coupled to the interior surface of each hole and a
hydrophobic fluoroalkyl layer is vinyl-coupled to the exterior surface of the
platen, resulting in the retention in individual, isolated containers of PCR
reagents and sample introduced onto the array.
e123 Nucleic Acids Research, 2006, Vol. 34, No. 18
PAGE 2 OF 9
After oxidation, a hydrophilic PEG layer is deposited inside
the through-holes by repeating the previous steps except
replacing the oxidation solution with 30 ml of 15 mg/ml
EDC (Pierce) and 5 mg/ml PEG 5000 (Nektar-Synasia) in
HEPES buffer (pH 7.5). After incubation for 2 h, the sheet
is removed and dried overnight at 100?C under vacuum. A
second hydrophobic layer is added by vapor deposition of
heptadecafluorotriethoxysilane (Gelest) for 2 h, 150?C in a
vacuum oven followed by a 30 min cure with NH3(g).
Finally, the array sheets are rinsed in RODI water to remove
the physisorbed PEG layer, thus exposing the underlying
covalently linked hydrophilic PEG.
RNA samples were converted to randomly primed first strand
cDNA using High Capacity cDNA Archive Kit (Applied
BioSystems, Foster City, CA). To reduce non-specific prod-
uct formation during qPCR, the cDNA sample was heated
to 75?C for 10 min to inactivate the reverse transcriptase;
snap chilled on ice for 5 min, then treated 1 h with
1.3 U/ml Exonuclease I (Amersham Biosciences, Piscataway,
NJ). The Exonuclease I is heat inactivated at 85?C for 10 min
and the resulting cDNA solution is stored at ?20C.
The PCR master mix consists of 1· LightCycler? FastStart
DNA Master SYBR Green I (Roche Applied Science, Indi-
anapolis, IN), 0.2% (w/v) Pluronic F-68 (Gibco, Carlsbad,
CA), 1 mg/ml BSA (Sigma–Aldrich, St. Louis, MO),
1:4000 SYBR Green I (Sigma–Aldrich), 0.5% (v/v) Glycerol
(Sigma–Aldrich), 8% (v/v) Formamide (Sigma–Aldrich) and
sample. For each kinase test of 507 assays, 66 ml of reaction
mix was required.
Kinase genes were selected based on their classification in
Gene Ontology (www.geneontology.org) and their presence
in the RefSeq database (www.ncbi.nlm.nih.gov/RefSeq).
Primer pairs biased towards the 30end of these genes were
obtained from either PrimerBank (28) and http://pga.mgh.
harvard.edu/primerbank or designed using Primer3 [http://
frodo.wi.mit.edu/cgi-bin/primer3_www.cgi]. Primers were
ordered from a commercial supplier (Sigma-Genosys, www.
sigma-genosys.com) and their performance was validated
with the following process. An ABI 7900 (Applied BioSys-
tems Inc., Foster City, CA) was used to test if each primer
set run against either five tissue cDNA library (qPCR
Human Reference cDNA, BD BioSciences, Franklin Lakes,
NJ) or 37 tissue cDNA library (BD Quick-Clone II Human
Universal cDNA, BD BioSciences, Franklin Lakes, NJ)
could generate a product rising above 0.2 DRn. Amplicon
mobility in 4% agarose E-gels (Invitrogen, Carlsbad, CA)
was measured from images captured with a gel imager
(AlphaImager, AlphaInnotech, San Leandro, CA) and pro-
cessed by software (Quantity One 1-D Analysis Software;
BioRad, Hercules, CA) to confirm the primer set made a
product within 10% of the predicted length. Amplicons
generated from primer pairs passing the above criteria were
pooled and gel purified to remove fragments <80 and >400
bp in size. This pool was used as a source of template in
subsequent PCR array validation experiments. All amplicons
were sequenced; ?70% matched all or part of the expected
target sequence while although the remainder could not be
confirmed by sequencing, these amplicons nonetheless
matched the expected size of the predicted amplified product.
The primer concentration where each primer set fails to
produce a product in the PCR array was determined. The
working primer concentration was adjusted to 8-fold above
this concentration to ensure that small quantities of primer
carryover between holes during sample loading will reduce
interference with PCR in adjacent holes. Assays were demon-
strated to have at least a 95% confidence of detecting 4-fold
change at >100 copies by first measuring a <0.5 CTSTD
across greater than five replicates and then demonstrating a
DCTshift of 2 +/?1 cycle for a sample diluted 4-fold. The
template for these experiments was the pooled amplicon
template in 1 ng total RNA equivalent randomly primed
liver cDNA, with an average of 200 +/?100 starting copy
number. A pass rate of 60% was observed with this validation
Real time thermal cycler protocol
The PCR array thermal cycling protocol consisted of 10 min,
92?C polymerase activation step followed by 35 cycles of 15 s
@ 92C, 1 min @ 55?C and 1 min @ 72?C (imaging step).
Following amplification, amplicon dissociation was measured
by cooling the PCR array to 65?C then slowly heated to
92?C @ 1?/min, with images collected every 0.25?C.
Human umbilical vein endothelial cells (HUVEC
(EGM(tm)-2) were purchased from CAMBREX BIO SCI-
ENCE (Walksersville, MD). Recombinant Human TNF-a
(10 ng/ml in EGM?-2), purchased from R & D Systems
Inc. (Minneapolis, MN) was added to HUVEC cultures that
were ?50% confluent in T150 tissue culture flasks. Cultures
were incubated for 4 h at 37C, 5% CO2. Medium was removed
and 2.5 ml of TRI Reagent?(Molecular Research Center) was
added to lyse cells. RNA was purified using an RNeasy Mini
Kit (Qiagen Inc.) and reverse transcription was done using a
High Capacity cDNA Archive Kit (Applied Biosystems).
Samples were treated with Exonuclease I (10 U/ml)
HUVECand Endothelial CellMedium-2
Baseline qPCR performance: uniformity, precision,
accuracy, sensitivity and dynamic range
To examine amplification uniformity, three arrays were
uniformly loaded with PCR mastermix, CycA primer pairs
and amplicon, resulting in an average of 500 starting copies
of amplicon per through-hole. OD260of purified amplicon
was used to independently confirm the number of starting
copies per hole. An image generated by subtracting pixel
values of the first and 19th cycling image (Figure 2A)
provides a visualization of amplicon replication in each
PAGE 3 OF 9
Nucleic Acids Research, 2006, Vol. 34, No. 18e123
through-hole of the array. Dark holes resulting from a sample
loading failure are detected by the absence of SYBR Green
fluorescence. Fluidics errors are typically <2% and are stoch-
astically distributed amongst the through-holes as failed
PCRs (Figure 2B). The dimmer holes along the first and
last column of the array image are not from reduced yield
of PCR product but rather from reduced SYBR fluorescence
intensity from non-uniformities in LED excitation, imaging
field of view and slight variations in optical path at the refrac-
tive index boundaries across the array. These fluorescent
signal differences (?6% CV, data not shown) are corrected
for in the instrument calibration. Residual optical differences
are taken into account by fluorescent baseline normalization
of the amplification curve for each through-hole prior to
crossing threshold (CT) calculation. The mean CT for
500 starting copies is 15.3 cycles, on average about 11 cycles
earlier than microplate-based qPCR systems. The CTshift
results from a higher concentration of amplified products
expected for PCR at reduced volumes (22) and an improved
CTcalling algorithm we developed by combining baseline
intercept with numerical modeling of the exponential ampli-
fication phase. The instrument precision at 500 starting copies
was +/?0.16 CT(or <12% CV on copy number), estimated
from the CT STD for >9100 amplification reactions
(Figure 2D). The CT distribution (Figure 2C) follows a
Gaussian distribution slightly skewed towards delayed
(higher) CTand with 0.6% of the through-holes having outlier
CTs > 3 STD from the mean. While the CTmap (Figure 2B)
indicates a small CTgradient spanning 0.32 cycles diagonally
from the middle of the array, outliers map stochastically (data
not shown). We speculate that the reduced PCR efficiency of
the outlier population may arise from random micro-scale
defects of the interior polymer surface coating and/or
contamination by random interfering particulates, but their
impact on data quality is substantially reduced with replicate
PCR array thermal uniformity was examined by melt curve
analysis of the amplicon products shown in Figure 3. Follow-
ing PCR, the array temperature was equilibrated to 65C,
slowly ramped to 92?C at 1?C per minute, and SYBR
Green fluorescence images were collected every 0.25?C to
record SYBR Green dye quenching on amplicon dissociation.
The product melt temperature, Tm, for each through-hole is
derived as the maximum of ?dF/dT, where T is the array
temperature and F is the fluorescence emission from a
through-hole at each temperature (29). The Tmdistribution
across the PCR array (Figure 3A) indicates the array center
was 1?cooler than at its edge. The Tm computed across
three arrays produced a similar Tmdistribution (Figure 3B)
and mean Tm, with an array-to-array Tm STD equal to
±0.28?C (Figure 3C).
sured by performing RT-PCR on a CycA amplicon titration.
Figure 4A depicts 12 replicate amplification profiles for each
log dilution, ranging from 107to 1 starting copies per hole
and a no template control. The high quality curves showed
>150-fold signal-to-noise, and >4 cycles exponential-phase
amplification. The CTcomputed from these curves were linear
14.515.0 15.516.0 16.5 17.0
Frequency from Arrays 1-3
% Failed PCR
% > 3 STD
CT STD No Outliers
Figure 2. Three through-hole arrays were loaded with PCR reagents containing 500 starting templates per hole and subjected to 32 cycles of PCR. The amplicon
product image (A) was generated by pixel-by-pixel subtraction of cycle image 1 from 19 for Array 1. The CTcolor map for Array 1 (B) indicates holes within CT
1 STD from average (white), CT< average ?1 STD (blue), CT> average +1 STD (red) and failed reactions (yellow). CTdistribution plot (C) for all three arrays,
and performance specifications table (D) for each array analyzed either independently or combined, summarize the instrumentation uniformity results.
e123Nucleic Acids Research, 2006, Vol. 34, No. 18
PAGE 4 OF 9
near perfect amplification efficiency based on slope of CT
versus log starting copy plot. The precision for measurements
>1000 starting copies was equivalent to the earlier PCR
uniformity experiment, averaging around 0.17 STD CT. The
no template control had no detectable product, indicating
carryover between subarrays is below the limit of detection.
Sensitivity can be estimated by measuring the frequency of
PCR positive reactions near the limit of assay detection. The
decreasing precision below 1000 copies appears to follow the
noise contribution based on the Poisson effect (Figure 4B).
Holes predicted to have an average of a single starting copy
produced 49 of 64 positive assays (77%); reasonably close
to the 63% predicted by Poisson statistics. Taken together,
the through-hole array demonstrated single copy sensitivity.
Additional thermal cycling of these arrays show no increase
in assay positive holes, indicating that there is no intra
subarray carryover of primers at the level of single copy
qPCR measurement of differential expression of human
kinase genes between human heart and human liver
To assess the performance of the system with a biologically
relevant example, an array was constructed with primer pairs
targeting expression of 508 human kinases, 13 endogenous
(housekeeping) controls in quadruplicate and 208 negative
controls for analyzing four samples per array (see Materials
and Methods, Primer Validation). Normal human heart and
liver RNA from a commercial source was reverse transcribed
into cDNA equivalent and divided into two aliquots of 0.25
and 1 ng cDNA per through-hole. The pipette sample loader
transferred sample mixed with PCR mastermix into arrays
pre-loaded with the assay primer sets and the arrays were
processed according to the real time procedure described in
the Methods section.
Correlation of CT technical replicates for the heart
(Figure 5A) and liver (Figure 5B) samples at 1 and 0.25 ng
of cDNA per through-hole shows high assay precision and
accuracy across a large dynamic range of responses. With
1 ng cDNA per through-hole, 78% of the assays for the
heart sample have STD <0.5 while for liver, 72% of the
assays had this precision or better (Figure 5C). Assay accu-
racy was estimated from computing the mean CTdifference
between two different cDNA concentrations for the heart
and liver samples. A histogram of DCT for all assays in
both tissues shows a median CTof 1.8 ± 0.48 for heart and
1.7 ± 0.61 for the liver sample, indicative of a 4-fold change
in cDNA concentration. Negative controls in each subarray
Melting Point (C)
79.5 8080.5 81
Figure 3. Following 32 cycles of PCR, the arrays in Figure 2 were cooled
to 65?C then slowly heated to 92?C at 1 C/min. A dissociation curve
(F versus T) and Tm[(?dF/dT)max] was calculated for each sample from
fluorescent images collected every 0.25C. The heat map (A) for Array 1
indicates Tm spanning 79.5?C (yellow) to 81.3?C (light blue). Plot
(B) indicates the Tmdistribution for all three arrays and Table (C) breaks
down the Tmperformance either independently or combined.
Log Starting Copies
Log SYBR Fluorescence
y = -3.3x + 25.2
R2 = 0.99
Figure 4. qPCR Titration Curve. (A) depicts cycle-by-cycle log SYBR fluorescence of each through hole colored according to average starting amplicon copy
per hole [107to 1 plus no template control (gray), 12 of the 64 replicates depicted]. (B) indicates average CT(open circles) and STD CT(filled circles) plotted
against log starting copies (n ¼ 64); line formula and Pearsons Coefficient were calculated from dashed CTcurve. Predicted Poisson noise is indicated by the
PAGE 5 OF 9
Nucleic Acids Research, 2006, Vol. 34, No. 18e123
showed no detectable carry-over between through-holes
(0/1728 negative controls).
Differential gene expression between the heart and liver
samples at 1 ng cDNA per through-hole was determined by
comparing the difference in mean CTs for each gene in the
liver and heart assay populations (Figure 6A). Positive assays
for which an accurate CTcould be measured passed the fol-
lowing criteria: (i) the amplicon product was detected and
confirmed by melt curve analysis; (ii) target expression
greater than single copy in both tissues (CT < 25) and (iii)
each assay had a technical replicate >2. Eighty-three assays
were rejected based on these criteria and the remaining 442
positive assays were normalized relative to the geometric
mean of the same 13 housekeeping genes for each tissue.
Two subpopulations are observed when the difference in
expression for the same gene is compared between the two
expressed at less than 10 copies in one tissue but not the
other and (ii) the gene expressed in both tissues with an abun-
dance >10 copies but there is a significant difference in
expression between the tissues based on Student’s t-test
(two-tailed distribution and two-sample unequal variance)
with a P-value <0.005. Segmentation of the assay population
based on these criteria is not surprising given the physiolo-
gical differences between heart and liver tissue, even for
common functional pathways.
Consider the well-studied glycolysis pathway as a point
of comparison where multiple kinases play fundamental
roles in the multi-step oxidation of glucose to pyruvate and
ATP (30). The first step in this process is ATP-dependent
phosphorylation of glucose to the intermediate glucose-6-
phosphate by the liver-specific hexokinase isozyme gluco-
kinase (GCK; NM_000162). We detected insignificant
expression levels in the heart sample (<1 copy) compared
(>200000-fold higher). A similar pattern of tissue-specific
expression was recorded for the liver (PFKL; NM_002626)
and muscle (PFKM; NM_000289) isoforms of phospho-
fructose kinase; and the muscle (PKLR; NM_000298) and
liver (PKM2; NM_002654) isoforms of pyruvate kinase.
In contrast, PGK1 (NM_000291), one of two isoforms of
phosphoglycerate kinase, shows a small yet significant
difference in expression between the heart and liver
sample (4.7-fold; P ¼ 6 · 10?7) yet is expressed at high
0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.v 3.6 4.0
CT (Heart Replicate 2)
CT (Liver Replicate 2)
CT (Heart Replicate 1)
CT (Liver Replicate 1)
Figure 5. Human heart and liver differential kinase gene expression. A Gene Ontology database was used to select 507 human kinase genes along with 13
housekeeping genes. Primers were designed, loaded into a through-hole array and SYBR Green PCR was used to measure transcript levels of normal human liver
(B) or heart (A) samples. Replicate 1 samples were at 1 ng/hole, whereas replicate 2 samples were at either 1 ng/hole (filled circles) or 0.25 ng/hole (open
circles). The solid diagonal lines represent 1:1 correlation for 1 ng cDNA/through-hole, and the dashed line the 1:1 correlation for 0.25 ng cDNA/through-hole.
The cumulative distribution of assay standard deviation at 1 ng/hole for both liver and heart samples (C) shows over 70% of the assays have a precision <0.5. DCT
for the difference in sample assay pair (Replicate 2–Replicate 1) is represented in histogram (D), liver indicated by unfilled bars and heart indicated by filled bars.
e123 Nucleic Acids Research, 2006, Vol. 34, No. 18
PAGE 6 OF 9
levels in both tissues, indicative of its known ubiquitous
expression. Expression of the second isoform, PGK2
(NM_138733), was not detected in either sample; this is
not surprising given that PGK2 is only expressed in
meiotic spermatocytes and postmeiotic spermatids during
To externally validate assay predictions, 21 assays showing
higher, equal and lower expression in liver than heart were
run in a microplate on a real time thermal cycler (ABI
7900). The resulting measurements showed a high degree
of concordance with the PCR array results (R2¼ 0.98) with
equivalent precision (Figure 6B).
qPCR measurement of differentially expressed genes in
TNF-a stimulated NK-b pathway
HUVEC TNF-a response was examined using both a kinase
array and a custom array. The custom array contained 26
TNF response pathway assays selected from a library of
2800 validated primer pairs (see Materials and Methods; Pri-
mer Validation). The layout of the custom array allows 48
samples per PCR array and the data analysis was similar to
the heart-liver comparison. Figure 7A depicts the DCT
between the TNF-a treated and vehicle treated HUVEC
Mean Liver ∆ ∆CT
Mean Heart ∆ ∆CT
ABI (∆∆CT Heart - Liver)
PCR Ar (∆∆CT Heart - Liver)
R2 = 0.98
Figure 6. Liver and heart kinase genes differential expression. CTdata from
both tissues at 1 ng/hole was normalized relative to the geometric mean of six
housekeeping genes; B2M (NM_004048), ACTB (NM_001101), EEF1A1
(NM_021130) and plotted as (DCT)Heart versus (DCT)Liver in (A). Open
circles are DCT measurements (n ¼ 3 replicates) and solid circles are
housekeeping genes normalizing dataset (n ¼ 4 replicates). Error lines
(dashed lines) are STD compared with linear fit to housekeeping genes (solid
line, R2¼ 0.99). Genes with tissue-specific expression (CT> 20 in one tissue,
CT< 20 in the other tissue) are indicated by crosses. Expression of individual
genes are indicated as follows: GCK (filled diamond), PGK1 (filled square),
PGK2 (open square), PKLR (filled triangle), PKM2 (open triangle),
PFKL (filled circle) and PFKM (open circle). Concordance data (B) for
21 transcript assays selected from 7 kinase genes spanning a 4000-fold
differential heart and liver expression, along with 12 housekeeping genes,
were used to perform microplate qPCR in an ABI 7900 at 3 replicates per
sample. Average DDCTwere calculated and compared with the same assays
in the PCR array; STD (error bars), and 1:1 correlation (dashed line).
Treated HUVEC (∆ ∆CT)
Control HUVEC (∆ ∆CT)
Log [cDNA] (ng/µL)
dCT (control - treated)
Figure 7. Transcript analysis of TNF-a treated HUVEC. PCR arrays were
used to make transcript measurements of cDNA generated from pooled
HUVEC cells treated with either TNF-a or vehicle. Samples were normalized
(DCT) by subtracting the geometric mean of 13 housekeeping genes from the
CTof each assay. (A) Correlates DCTbetween treated and untreated assays
(circles), triangles showing significant difference were, from left-to-right
genes CCL2 (NM_002982), SELE (NM_000450), TNFAIP2 (NM_006291),
TNFAIP3 (NM_006290), VCAM1 (P < 0.001, n > 2 technical replicates) and
targets detected in one sample but not the other (pluses). (B) Examines fold
difference in expression in SELE between treated and control HUVEC
measured in PCR array and microplate at differing cDNA input quantity
(error bars are STD, n > 3).
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Nucleic Acids Research, 2006, Vol. 34, No. 18 e123
(see Materials and Methods). Of the 533 targets screened, 406
were positive in both samples. Negative controls in each sub-
array showed no detectable carry-over between through-holes
(0/1224 negative controls). As predicted, the TNF-a treat-
ment increased selectin E (SELE; NM_000450) and vascular
cell adhesion molecule 1 (VCAM1; NM_001078) expression
level >100-fold (32,33). The dynamic range of SELE was
compared between microplate and PCR array (Figure 7B)
by diluting the sample and determining if the resulting
housekeeping-normalized DCTgave the same measurement
across the dilution series. The PCR array produced a similar
DCTuntil the SELE starting copy number reached single copy
in the PCR array hole, ?0.03 ng/hole.
A nanofluidic system for performing solution-phase RT-PCR
in an array of isolated through-holes overcomes limitations of
existing micro- or nanofluidic devices by implementing a par-
allel approach to fluidic handling that lends itself well to
interfacing with microtiter plates for simple and efficacious
transfer of liquid samples and reagents into the nanofluidic
structure using arrays of slotted pins or pipette tips. The reac-
tion containers are kept separate and distinct by selective and
controlled modification of the platen surface to make the
inside surfaces of the through-holes chemically distinct
from the outside surfaces. In the system described here, the
interior surface of each hole is modified to be hydrophilic
and the exterior surface hydrophobic.
We validated the system performance by carrying out thou-
sands of real time SYBR Green PCR assays for a number of
sample sets. With amplicon as the target, the baseline perfor-
mance of the system had the same dynamic range, sensitivity
and precision as PCR in a microplate but in 64-fold smaller
reaction volumes. The increase in CTvariability with decreas-
ing number of target amplicon was shown to follow a Poisson
distribution at low copy number, demonstrating the system is
capable of single copy detection. Further, the thermal unifor-
mity was demonstrated by the small spread in CTand Tmin a
uniformly loaded array. With volume miniaturization, we
identified the need to increase sample concentration to main-
tain a constant number of target molecules in the reaction
volume to obtain the high quality PCR observed.
When challenged with cDNA prepared from normal human
heart and liver RNA, the high replicate precision and accu-
racy enabled detection of tissue-specific expression of kinase
genes and discrimination in differential kinase expression
between the two tissue types. A large number of genes
showed small differences in expression (<2-fold) with a
high degree of significance (P < 0.001), suggesting the
system is potentially capable of revealing new patterns of
transcription across large numbers of genes. The data from
TNF-a stimulation of the NF-b pathway in HUVEC cells
demonstrates this point. The high sample throughput of
over 27 000 RT-PCR analyses per person per day, based on
a workflow of 3 h to prepare three arrays and three processing
runs per day per person, is a 24-fold increase in analytical
throughput based on current 384-well microplates and
opens new possibilities in genetic system analysis not
currently possible. Clearly, the nanofluidic PCR array has
the potential to span the space of genomic applications
requiring the parallelism of hybridization microarrays with
the specificity, accuracy, dynamic range and precision of
solution phase PCR.
The authors wish to thank John Linton, Leila Hasan, Elen
Mahima Santhanam and Amy Goldberger for their technical
assistance. Funding to pay the Open Access publication
charges for this article was provided by BioTrove Inc.
Conflict of interest statement. None declared.
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