Biology Reference
In-Depth Information
Box 5.2 Luminex xMAP technology for multiplex gene expression analysis
The Luminex xMAP Technology (http: //
www.luminexcorp.
com
) can be used to perform a wide variety of multiplex
assays on the surface of 5.6 mm polystyrene microspheres. Each
microsphere or bead is uniquely color-coded internally using
precise concentrations of red and infrared fluorescent dyes,
resulting in 500 spectrally distinct beads. This feature allows
multiplexing of 1
excites the fluorescent dye associated with the reporter mole-
cule that is used to detect the analyte of interest.
Peck and colleagues
[253]
developed the Luminex xMAP
technology for gene expression signature analysis. Messenger
RNA (mRNA) transcripts from each sample are captured on
immobilized poly-dT in 384-well plates and are reverse tran-
scribed to complementary DNA (cDNA). For each gene/tran-
script of interest two oligonucleotide probes are designed. The
5
0
probe contains a 20 nt sequence complementary to the T7
primer site, a unique 24 nt sequence that serves as a barcode,
and a 20 nt sequence complementary to the transcript of
interest. Each 3
0
probe is phosphorylated at its 5
0
end and
contains a 20 nt sequence contiguous with the gene-specific
fragment of the 5
0
probe followed by a 20 nt T3 primer site.
Probe pairs for the transcripts of interest are mixed with cDNA
from each sample, unbound probes are removed, and probe
pairs annealed to contiguous regions of target mRNAs are
ligated together to yield synthetic 104 nt templates for ampli-
fication. Universal T3 and 5
0
-biotinylated T7 primers are used
to amplify the templates by PCR. The resulting biotinylated and
bar-coded amplicons are hybridized to a pool of spectrally
distinct microspheres. Each microsphere presents on its surface
a distinct capture probe complementary to one of the barcodes.
The hybridization reactions are finally reacted with streptavi-
din-phycoerythrin to fluorescently label biotin labels. Captured
labeled transcripts of interest are quantified and beads decoded
in the Luminex analyzer as described above. Luminex xMAP
assays are carried out in a 96-well plate format, with up to 500
genes being measured in each well/sample.
500 analytes in a single sample. The surface
chemistry of the microspheres allows capture reagents to be
efficiently coupled to the beads to facilitate the measurement of
different kinds of analyte in the sample. For example, capture
reagents may include oligonucleotides, antibodies, peptides,
enzyme substrates, or receptors, thus offering a wide range of
applications, including gene expression analysis, detection of
single nucleotide polymorphisms, protein expression analysis,
detection of protein
e
protein interactions, quantification of
antibody affinity and epitope mapping, serum analyte profiling
and detection of enzyme/substrate or receptor
e
ligand reac-
tions. After the analyte of interest (transcript, antibody, antigen,
ligand, or substrate) is captured from the sample on the surface
of the beads, the reactions are quantified in the Luminex
analyzer, an instrument that combines high-tech fluidics based
on the principles of flow cytometry and laser optics for signal
detection and processing. In the analyzer, the microspheres
pass through the detection chamber in a single file such that the
reaction between the surface coated capture reagent and the
analyte of interest can be quantitatively measured for each
bead. In the detection chamber, a red laser or light-emitting
diode (LED) is first used to classify each microsphere to one out
of the 500 spectrally different sets. A second laser or LED
e
within a single reaction in thousands of samples in a cost-
effective manner
[253]
. (http: //
www.luminexcorp.com
)
GE-HTS has also been recently adopted to dissect the
regulatory network controlling the transcriptional response
of mouse primary dendritic cells to pathogens
[215]
. A 118-
gene signature that defines the response of mouse primary
dendritic cells to infection by pathogens was established
using expression profiling of dendritic cells exposed to
different pathogen-derived components (virus, Gram-
positive and Gram-negative bacteria). The gene expression
signature was then used to screen 125 transcription factors,
including proteins that modify chromatin and proteins that
bind RNA, for their role(s) in coordinating cellular
response to pathogen infection. The reconstructed network
model composed of the transcription factors and their
cognate upstream signaling pathways helps to explain how
pathogen-sensing pathways achieve specificity in their
response to different microbial populations. The study used
a screening platform called NanoString nCounter Gene
Expression Assay (see
Box 5.3
for details on the Nano-
string technology). The nCounter Gene Expression Assay
is a robust and highly reproducible method for detecting the
expression of up to 800 genes in a single reaction. The
biggest advantages of this platform are its high sensitivity,
requirement for very small amounts of total RNA as start-
ing material, and the lack of any enzymatic reactions to
convert total RNA to cDNA and amplification of resulting
cDNA by polymerase chain reaction (PCR).
Direction of Information Flow from
Phosphorylation Signatures
Similar to gene expression signatures, the biological state
of a cell can also be inferred from the phosphorylation
profile of proteins that are themselves components of the
cellular signaling network, as well as of proteins that form
a part of the cellular response to signals impinging on the
cell. Protein phosphorylation is a widespread post-trans-
lational modification and plays important roles in most
biological processes in eukaryotic cells. The addition of
phosphate groups on substrate proteins by kinases modu-
lates the overall function of the substrates by directing their
activity, localization and stability. Extensive protein-phos-
phorylation-mediated signaling networks direct the flow of
