Fluidigm has developed a novel nanofluidic chip that provides
reliable single-copy sensitivity and absolute quantification of target
nucleic acid sequences. The Digital Isolation and Detection (DID) chip
works by partitioning a sample/assay (TaqMan® assays) mixture into
hundreds to tens of thousands of reaction chambers, where real-time QPCR
reactions are continuously monitored by our Dynamic Array Reader. Only
those chambers containing copies of target sequence produce signal, making
it possible to calculate copy number in the entire sample extremely
accurately. This note describes experiments using a DID chip to quantify
TIMP-3 transcript and relates results to those from the dynamic array
described in the preceding application note.
Introduction
When first developed, PCR was semi-quantitative at best. Advancements,
such as exogenous competitive sequences, improved quantification accuracy
and precision, and now real-time PCR (RT PCR) has become the technique of
choice for sequence detection and quantification. Now, RT PCR can
routinely achieve 2-fold discrimination when sufficient replicate
reactions are run to provide statistical confidence. Despite these
advancements, reliable single-copy detection and absolute quantification
remain unmet needs. DID chips enable scientists to easily and quickly
determine absolute copy number of nucleic acid sequences down to a single
copy.
Experimental Conditions
Figure 1. DID chip. The DID chip
consists of a carrier and an integrated fluidic circuit (IFC)
center. The carrier contains 12 sample/reagent inputs, each of
which holds a volume of 7.5 µL. The IFC partitions each mixture
into 1,200 isolated reaction chambers.
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Fluidigm has developed DID architectures
with differing numbers of sample/assay inputs and densities of reactions
per input. The DID chip described here (Figure 1) contains inputs for 12
sample/assay mixtures, and its architecture partitions 7.5 µL of fluid
for each input into 1,200 reaction chambers.
Our instrumentation was used to drive
the sample/assay mixtures from the wells in the carrier into the
appropriate reaction chambers. Sample/assay mixtures consisted of a serial
5-fold dilution of Random-primed BD™ qPCR Human Reference cDNA (BD
Biosciences) already mixed with TaqMan® Universal PCR Master Mix (Applied
Biosystems), TIMP-3 primers and Black Hole Quencher™ DNA probe (IDT).
The mixtures consisted of the following cDNA amounts: 5 pg, 1 pg, 1 pg,
200 fg, 200 fg, 40 fg, 40 fg, 8 fg, 8 fg, 1.6 fg, 1.6 fg, and no cDNA.
Reaction- and thermal-cycling conditions were as described for TIMP-3 in
the preceding note. Our dynamic array reader was used to perform thermal
cycling, imaging, and data collection and our proprietary software was
used for quantitative analysis.
Results
Figure 2. Absolute quantification of
TIMP-3 transcript on a DID chip. The image shown is for FAM
(excitation 485 nm; emission 525 nm) after 37 cycles of PCR. RT
PCR data for each partition is available but not shown. The white
spots indicate the presence of TIMP-3 transcript.
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As shown in Figure 2, white spots
indicate the location of reaction chambers positive for TIMP-3. The sum of
TIMP-3 positive wells in each section is consistent with the TIMP-3 cDNA
copies that were measured in the 5 pg, 1 pg, and 200 fg cDNA loads, at
approximately 1 copy per 16 fg cDNA. This consistency demonstrates that
single-copy detection in the DID chip is extremely reproducible and
reliable. The cDNA loads of 40 fg and below showed less consistency due to
sampling error.
