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
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