Biology Reference
In-Depth Information
INTRODUCTION
of gene expression microarray technology in
1995
89
provided researchers with technology
needed to fabricate high-throughput highlymulti-
plexed miniaturized arrays or
cient way to identify cancer
patients who will respond to a given therapy is
to determine, prior to treatment initiation, which
potential drug target molecules are responsible
for the pathogenesis of that patient
The most ef
”
Prior to development of RPMA, immunoassays
and protein microarrays were generally
sandwich-style assays in which one antibody
was used to capture the analyte of interest and
a second antibody, directed against a different
epitope on the same protein, was used as a detec-
tion molecule.
13,36,56,71
An early iteration of future
RPMAwas a
“
microarrays.
s individual
tumor.
1,29,37,40,46,55,58,79
Molecular pro
'
ling using
gene expression arrays has shown considerable
potential for the classi
cation of patient responses
to chemotherapy.
10,11
Nevertheless,
transcript
“
”
whichwas constructedby
manually depositing protein samples on
a membrane. Dot blotting was labor intensive
and could only accommodate a few samples per
blot. Advances
dot blot,
pro
ling, and genomic sequencing, by itself,
provides an incomplete picture of the ongoing
activated protein signaling network that is
driving the cancer at the functional
level.
42,44,46,51,62,63,80,89,90,97
Gene transcript levels
have not been found to correlate signi
in
technology
related to
molecular pro
ling, such as laser capture
microdissection,
23,92
pin-and-ring and quill-pin-
style robotic arrayers, and commercially available
phosphospeci
cantly
with protein expression or the functional (often
phosphorylated) forms of the encoded proteins.
RNA transcripts also provide little information
about protein
e
protein interactions and the state
of the cellular signaling pathways.
4,43,57,67,74,89,90
Importantly, most current therapeutics are
directed at protein targets, and these targets
are
c antibodies, were the basis of
microarrays.
55,56,64
“
antigen-down
”
The term
“
reverse phase protein microarray
”
was coined by Paweletz et al. in the
rst paper
describing the technology.
76
The term
“
reverse
phase
refers to the fact that the analyte (antigen)
is immobilized as a capture molecule, rather than
immobilizing an antibody as the capture mole-
cule.
56
Each RPMA microarray consists of
a self-contained assay composed of duplicate/
triplicate samples, controls, and calibrators that
are analyzed with one class of antibody and
ampli
”
often
protein
kinases
and/or
their
substrates.
12,43,55
The activation state of
these
proteins and these networks
fluctuate constantly
depending on the cellular microenvironment and
the disease state. Consequently, pro
ling kinase
driven signal pathways represents a rich source
for new molecular targeted therapeutics.
99
Tech-
nologies, such as reverse-phase protein microar-
rays
cation chemistry. Paweletz et al. used
RPMA to construct the
(RPMAs),
56,72,74,76,79
first quantitative multi-
plexed cell signaling protein analysis of tissue
cells. In this
which can broadly
pro
le and assess the activity of protein signaling
pathways containing the targets of molecular
inhibitors, are becoming an essential technology
that complements genomic analysis
13,39
in the
quest for realizing the goal of patient-tailored
therapy.
first experimental application of
RPMA, the technology was used to discover
the importance of the pro-survival pathway
during the transition from in situ to invasive
prostate cancer. RPMA provided the sensitivity
and precision to measure phosphorylated signal
proteins in small samples of laser capture micro-
dissected captured directly from the invading
edge of the tumor.
76
Since 2001, terms used in
the literature include lysate array,
83
ORI
GIN OF RPMA TECHNOLO
GY
RPMAis adirect descendent
56,71,74,76,79
ofmini-
aturized immunoassays.
20,21,22
The introduction
reverse
lysate microarrays,
87
phase
and
protein
Search WWH ::
Custom Search