Biology Reference
In-Depth Information
Box 5.3 Nanostring nCounter System for Mulplex Gene Expression Analysis
The nCounter assay can be used to detect several types of
nucleic acid molecule, including mRNA, DNA and micro-
RNAs. The nCounter assay is based on direct imaging of mRNA
molecules of interest that are detected using target-specific,
color-coded probe pairs
[273]
. It does not require the
conversion of mRNA to cDNA via reverse transcription or the
amplification of the resulting cDNA via PCR. A pair of
sequence-specific probes
contiguous positions allows for a large diversity of color-based
barcodes, each designating a different gene transcript, that can
be mixed together in a single reaction for hybridization and still
be individually resolved and identified. The methodology offers
the flexibility of multiplexing up to 800 reporter
capture probe
e
pairs within a single reaction.
The target mRNA is mixed in solution with a large excess of
the reporter and capture probe pairs, so that each targeted
transcript finds its corresponding probe pair. After hybridiza-
tion, excess unbound probes are washed away and the
complexes, comprising target mRNA bound to specific repor-
ter
the capture and reporter probes
e
detects each target gene of interest. The capture probe contains,
from 5
0
to 3
0
,a35
e
50-base sequence complementary to the
target mRNA, a short sequence common to all capture probes,
and a biotin affinity tag that provides a molecular handle for the
attachment of target genes to facilitate detection. The reporter
probe contains, from 3
0
to 5
0
, a second 35
e
capture probe pairs, are isolated. The biotin label at the 3
0
end of the capture probes is used to attach the complexes to
streptavidin-coated slides. An electric field is applied to orient
and extend the tripartite complexes on the surface of the slide to
facilitate imaging and detection of the color-coded molecules.
A microscope objective and a CCD camera are used to image
the immobilized complexes. The number of molecules for
a particular mRNA species is counted by decoding the unique
pattern of the fluorescent colors encoded in each reporter
probe. The protocol is performed from start to finish on the
nCounter System, which is designed to provide hybridization,
post-hybridization processing, and digital data acquisition
capabilities in one simple workflow. The integrated system is
composed of two instruments: the fully automated nCounter
Prep Station for post-hybridization processing and the Digital
Analyzer for imaging, data collection, and data processing.
e
50-base sequence
(complementary to the same target mRNA, near or contiguous
with the target-specific sequence in the capture probe partner),
a short sequence common to all reporter probes, and a color-
coded molecular barcode. The common sequences included in
all capture and reporter probes facilitate the removal of
unbound excess probes during post-hybridization steps. The
barcode contained in each reporter probe is composed of
a linearized single-stranded M13 DNA molecule annealed to
a series of six complementary RNA segments, each labeled with
one of four spectrally non-overlapping fluorescent dyes. The
arrangement of the differently colored RNA segments creates
a unique color code for each target gene of
e
interest. The
different combinations of
the four distinct colors at six
information from cell surface receptors to effector mole-
cules to regulate the response and functions of cells, tissues
and organisms. Aberrant signaling due to misregulation of
protein phosphorylation and dephosphorylation cascades is
associated with many disease states, including most types
of cancer.
A multiplex approach using phospho-specific anti-
bodies and intracellular phospho-specific flow cytometry
[254,255]
to monitor changes in the level of phosphoryla-
tion of multiple key protein nodes in primary leukemic cells
has been shown to have great potential in understanding
how signaling through a network is co-opted in cancer cells
to produce the aberrant phenotypes
[256]
. Phospho-specific
antibodies to Stat1, Stat3, Stat5, Stat6, p38, and Erk1/2
were used to profile primary cells from patients with acute
myeloid leukemia at basal state and following cytokine
stimulation. Phosphorylation profiles of the six signaling
proteins in acute myeloid leukemia cells were compared to
those in normal blood cells to distinguish the leukemic
signal transduction network from the healthy network.
Using the same methodology, it is also possible to measure
the effects of perturbations (genetic or small molecule) on
these signaling events in cancer cells compared to normal
cells with the aim of identifying potential drug targets.
Furthermore, one could determine the effect of such
perturbations in either attenuating or enhancing the
response of the cancer cells to other environmental cues.
Multicolor flow cytometry
[257]
has been used to
measure 11 phosphoproteins and phospholipids simulta-
neously in response to stimulatory or inhibitory perturba-
tions (small molecule inhibitors of key signaling
components) to determine the effects of each condition on
the cellular signaling networks in naive CD4
รพ
T primary
cells. Bayesian network analysis was applied in order to
infer causal connections between components of the
network. Key to the success of this application was the use
of the phosphorylation signatures of the 11 phosphoproteins
and phospholipids in response to stimuli and perturbations.
Both studies discussed above use phospho-specific
antibodies to key signaling or response proteins
[256,257]
.
Such studies are limited by the availability of specific
antibodies that recognize phosphorylated residues on
proteins of interest. Proteome-scale MS-based studies
provide one of the most comprehensive analyses of phos-
phorylation and do not depend on antibodies
[258
260]
.
MS-based technologies provide highly quantitative and
direct measurements that can detect the activities of many
phosphorylation pathways simultaneously. The KAYAK
e
