U.S. patent application number 14/688372 was filed with the patent office on 2015-08-06 for method for cross-instrument comparison of gene expression data.
The applicant listed for this patent is APPLIED BIOSYSTEMS, LLC. Invention is credited to Mark F. Oldham, David W. Ruff, Mark E. Shannon.
Application Number | 20150220685 14/688372 |
Document ID | / |
Family ID | 37024561 |
Filed Date | 2015-08-06 |
United States Patent
Application |
20150220685 |
Kind Code |
A1 |
Shannon; Mark E. ; et
al. |
August 6, 2015 |
Method for Cross-Instrument Comparison of Gene Expression Data
Abstract
A method for determining bias across two domains comprising gene
expression data. The method can comprise (a) providing a first
domain and a second domain; (b) obtaining information indicative of
a bias within the first domain; (c) obtaining information
indicative of a bias within the second domain; and (d) using the
information indicative of the bias within the first domain and the
information indicative of the bias within the second domain to
produce an indication of bias across the two domains.
Inventors: |
Shannon; Mark E.; (San
Francisco, CA) ; Oldham; Mark F.; (Emerald Hills,
CA) ; Ruff; David W.; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APPLIED BIOSYSTEMS, LLC |
Carlsbad |
CA |
US |
|
|
Family ID: |
37024561 |
Appl. No.: |
14/688372 |
Filed: |
April 16, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12558428 |
Sep 11, 2009 |
9026371 |
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14688372 |
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11086253 |
Mar 22, 2005 |
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12558428 |
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10944673 |
Sep 17, 2004 |
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11086253 |
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10944668 |
Sep 17, 2004 |
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10944673 |
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10913601 |
Aug 5, 2004 |
7233393 |
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10944668 |
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60504500 |
Sep 19, 2003 |
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60504052 |
Sep 19, 2003 |
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60589224 |
Jul 19, 2004 |
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60589225 |
Jul 19, 2004 |
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60601716 |
Aug 13, 2004 |
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60504052 |
Sep 19, 2003 |
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60589224 |
Jul 19, 2004 |
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60601716 |
Aug 13, 2004 |
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Current U.S.
Class: |
702/19 |
Current CPC
Class: |
C40B 30/04 20130101;
G16B 25/00 20190201; C12Q 1/686 20130101 |
International
Class: |
G06F 19/20 20060101
G06F019/20 |
Claims
1. A method for cross-instrument platform comparison of gene
expression data, the method comprising: generating first cycle
threshold data sets on a first instrument platform, wherein the
first cycle threshold data sets are for at least one of a first
domain sample, a first domain reference, and a first domain
calibrator data set; generating second cycle threshold data sets on
a second instrument platform, wherein the second cycle threshold
data sets are for at least one of a second domain sample, a second
domain reference, and a second domain calibrator data set;
calculating a first domain .DELTA.C.sub.T given for a first domain
sample data set from the first instrument platform given by:
.DELTA.C.sub.Tfirst=C.sub.Tq first-C.sub.Tendo first, wherein
C.sub.Tq first is a threshold cycle for a first domain sample data
set, and C.sub.Tendo first is a threshold cycle for a first domain
reference data set; calculating a first domain .DELTA.C.sub.T given
for a first domain calibrator data set from the first instrument
platform given by: .DELTA.C.sub.Tcb first=C.sub.Tq cb
first-C.sub.Tendo first, wherein C.sub.Tq cb first is a threshold
cycle for a first domain calibrator data set, and C.sub.Tendo first
is a threshold cycle for a first domain reference data set;
calculating a second domain .DELTA.C.sub.T for a second domain
sample data set from the second instrument platform given by:
.DELTA.C.sub.Tsecond=C.sub.Tq second-C.sub.Tendo second; wherein
C.sub.Tq second is a threshold cycle for a second domain sample
data set, and C.sub.Tendo second is a threshold cycle for a second
domain reference data set; calculating a second domain
.DELTA.C.sub.T for a second domain calibrator set from the second
instrument platform given by: .DELTA.C.sub.Tcb second=C.sub.Tq cb
second-C.sub.Tendo second; wherein C.sub.Tq cb second is a
threshold cycle for a second domain calibrator data set, and
C.sub.Tendo second is a threshold cycle for a second domain
reference data set; calculating a first domain
.DELTA..DELTA.C.sub.T for the first instrument platform given by;
.DELTA..DELTA.C.sub.Tfirst=.DELTA.C.sub.Tfirst-.DELTA.C.sub.Tcb
first, calculating a second domain .DELTA..DELTA.C.sub.T for the
second instrument platform given by
.DELTA..DELTA.C.sub.Tsecond=.DELTA.C.sub.Tq second-.DELTA.C.sub.Tcb
second, determining a measure of preamplifaction or amplification
bias within the first domain and within the second domain to
produce an indication of bias across the two domains, wherein the
measure of measure of preamplifaction or amplification bias is
given by, .DELTA..DELTA..DELTA.CT=.DELTA..DELTA.C.sub.T
first-.DELTA..DELTA.C.sub.T second; and outputting the measure of
preamplifaction or amplification bias across the two domains to a
view or report of an analysis session.
2. The method of claim 1, wherein the first domain sample data set
and the second domain sample data set are based on the same target
DNA.
3. The method of claim 1, wherein the at least two instrument
platforms are polynucleotide amplification instrumentation and
microarray instrumentation.
4. The method of claim 3, wherein PCR polynucleotide amplification
instrumentation is any of quantitative PCR, real time quantitative
PCR, qualitative PCR, and multiplexed PCR.
5. The method of claim 3, wherein the microarray instrumentation is
any of a hybridization array, a DNA microchip, or a high-density
sequence oligonucleotide array.
6. The method of claim 1, wherein the first domain reference and
the second domain reference are each independently an endogenous
reference or a passive internal reference.
7. The method of claim 1, wherein the first domain reference and
the second domain reference are the same.
8. The method of claim 1, wherein the data obtained from a
microarray is converted into a .DELTA..DELTA.CT format using the
equation: R=(1/2) .DELTA..DELTA.CT; wherein R is the resulting
measurement from the microarray.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of patent application
Ser. No. 12/558,428 filed Sep. 11, 2009, which is a continuation of
patent application Ser. No. 11/086,253 filed Mar. 22, 2005, now
abandoned, which is a continuation-in-part of patent application
Ser. No. 10/944,673 filed on Sep. 17, 2004, now abandoned, and
patent application Ser. No. 10/944,668 filed on Sep. 17, 2004, now
abandoned. patent application Ser. No. 10/944,673 claims a benefit
to U.S. Provisional Application No. 60/504,500 filed on Sep. 19,
2003; U.S. Provisional Application No. 60/504,052 filed on Sep. 19,
2003; U.S. Provisional Application No. 60/589,224 filed Jul. 19,
2004; U.S. Provisional Application No. 60/589,225 filed on Jul. 19,
2004; and U.S. Provisional Application No. 60/601,716 filed on Aug.
13, 2004. Patent application Ser. No. 10/944,668 is a
continuation-in-part of patent application Ser. No. 10/913,601
filed on Aug. 5, 2004, now U.S. Pat. No. 7,233,393, and further
claims the benefit of U.S. Provisional Application No. 60/504,052
filed on Sep. 19, 2003; U.S. Provisional Application No. 60/589,224
filed Jul. 19, 2004; and U.S. Provisional Application No.
60/601,716 filed on Aug. 13, 2004.
[0002] All literature and similar materials cited in this
application, including but not limited to, patents, patent
applications, articles, books, treatises, and internet web pages,
regardless of the format of such literature and similar materials,
are expressly incorporated by reference in their entirety for any
purpose. In the event that one or more of the incorporated
literature and similar materials differs from or contradicts this
application, including but not limited to defined terms, term
usage, described techniques, or the like, this application
controls.
INTRODUCTION
[0003] Currently, genomic analysis, including that of the estimated
30,000 human genes is a major focus of basic and applied
biochemical and pharmaceutical research. Such analysis may aid in
developing diagnostics, medicines, and therapies for a wide variety
of disorders. However, the complexity of the human genome and the
interrelated functions of genes often make this task difficult.
There is a continuing need for methods and apparatus to aid in such
analysis.
DRAWINGS
[0004] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0005] The skilled artisan will understand that the drawings,
described herein, are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way.
[0006] FIG. 1 is a flowchart illustrating the use of a database
system according to some embodiments;
[0007] FIG. 2 is a flowchart illustrating a process for determining
bias.
[0008] FIG. 3 is a graph exemplifying a comparison of amplification
with IVT and multiplex preamplification.
[0009] FIG. 4 is a graph exemplifying a .DELTA..DELTA.C.sub.T
comparison of a brain and a liver sample.
[0010] FIG. 5 is a graph exemplifying a .DELTA..DELTA.C.sub.T
comparison between four different sample inputs.
[0011] FIG. 6 is a graph exemplifying a
.DELTA..DELTA..DELTA.C.sub.T of a liver and a brain sample with IVT
preamplification.
[0012] FIG. 7 is a graph exemplifying a
.DELTA..DELTA..DELTA.C.sub.T of a liver and a brain sample with
multiplex preamplification.
[0013] FIG. 8 is a flowchart illustrating a process for determining
bias between two gene expression platforms.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0014] The following description of various embodiments is merely
exemplary in nature and is in no way intended to limit the present
teachings, applications, or uses. Although the present teachings
will be discussed in various embodiments as relating to
polynucleotide amplification, such as PCR, such discussion should
not be regarded as limiting the present teaching to only such
applications.
[0015] In general, gene expression is a process by which a gene's
coded information is converted into the structures present and
operating in the cell. Gene expression is a multi-step process that
begins with transcription and translation and is followed by
folding, post-translational modification and targeting. The amount
of protein that a cell expresses depends on the tissue, the
developmental stage of the organism and the metabolic or
physiologic state of the cell. Expressed genes can include those
that are transcribed into mRNA and then translated into protein and
those that are transcribed into RNA but not translated into
protein. In various embodiments, gene expression can be studied
using analytical techniques such as polymerase chain reaction
(PCR), Northern blots, serial analysis of gene expression (SAGE)
microarrays, hybridization arrays, and high density oligonucleotide
arrays.
[0016] Briefly, by way of background, PCR can be used to amplify a
sample of target Deoxyribose Nucleic Acid (DNA) for analysis.
Typically, the PCR reaction involves copying the strands of the
target DNA and then using the copies to generate additional copies
in subsequent cycles. Each cycle doubles the amount of the target
DNA present, thereby resulting in a geometric progression in the
number of copies of the target DNA. The temperature of a
double-stranded target DNA is elevated to denature the DNA, and the
temperature is then reduced to anneal at least one primer to each
strand of the denatured target DNA. In some embodiments, the target
DNA can be a cDNA. In some embodiments, primers are used as a
pair--a forward primer and a reverse primer--and can be referred to
as a primer pair or primer set. In some embodiments, the primer set
comprises a 5' upstream primer that can bind with the 5' end of one
strand of the denatured target DNA and a 3' downstream primer that
can bind with the 3' end of the other strand of the denatured
target DNA. Once a given primer binds to the strand of the
denatured target DNA, the primer can be extended by the action of a
polymerase. In some embodiments, the polymerase can be a
thermostable DNA polymerase, for example, a Taq polymerase. The
product of this extension, which sometimes may be referred to as an
amplicon, can then be denatured from the resultant strands and the
process can be repeated. Temperatures suitable for carrying out the
reactions are well known in the art. Certain basic principles of
PCR are set forth in U.S. Pat. Nos. 4,683,195, 4,683,202,
4,800,159, and 4,965,188, each issued to Mullis et al.
[0017] In some embodiments, PCR can be conducted under conditions
allowing for quantitative and/or qualitative analysis of one or
more target DNA. Accordingly, detection probes can be used for
detecting the presence of the target DNA in an assay. In some
embodiments, the detection probes can comprise physical (e.g.,
fluorescent) or chemical properties that change upon binding of the
detection probe to the target DNA. Some embodiments of the present
teaching can provide real time fluorescence-based detection and
analysis of amplicons as described, for example, in PCT Publication
No. WO 95/30139 and U.S. patent application Ser. No.
08/235,411.
[0018] In some embodiments, assay can be a homogenous
polynucleotide amplification assay, for coupled amplification and
detection, wherein the process of amplification generates a
detectable signal and the need for subsequent sample handling and
manipulation to detect the amplified product is minimized or
eliminated. Homogeneous assays can provide for amplification that
is detectable without opening a sealed well or further processing
steps once amplification is initiated. Such homogeneous assays can
be suitable for use in conjunction with detection probes. For
example, in some embodiments, the use of an oligonucleotide
detection probe, specific for detecting a particular target DNA can
be included in an amplification reaction in addition to a DNA
binding agent of the present teachings. Homogenous assays among
those useful herein are described, for example, in commonly
assigned U.S. Pat. No. 6,814,934.
[0019] In some embodiments, methods are provided for detecting a
plurality of targets. Such methods include those comprising forming
an initial mixture comprising an analyte sample suspected of
comprising the plurality of targets, a polymerase, and a plurality
of primer sets. In some embodiments, each primer set comprises a
forward primer and a reverse primer and at least one detection
probe unique for one of the plurality of primer sets. In some
embodiments, the initial mixture can be formed under conditions in
which one primer elongates if hybridized to a target.
[0020] In some embodiments for amplification of a polynucleotide,
assay can comprise a preamplification product, wherein one or more
polynucleotides in an analyte has been amplified prior to being
deposited in at least one of the plurality of wells. In some
embodiments, these methods can further comprise forming a plurality
of preamplification products by subjecting an initial analyte
comprising a plurality of polynucleotides to at least one cycle of
PCR to form a detection mixture comprising a plurality of
preamplification products. The detection mixture of
preamplification products can be then used for further
amplification using a PCR. In some embodiments, preamplification
comprises the use of isothermal methods.
[0021] In some embodiments, a two-step multiplex amplification
reaction can be performed wherein the first step truncates a
standard multiplex amplification round to boost a copy number of
the DNA target by about 100-1000 or more fold. Following the first
step, the resulting product can be divided into optimized secondary
single amplification reactions, each containing one or more of the
primer sets that were used previously in the first or multiplexed
booster step. The booster step can occur, for example, using an
aqueous target or using a solid phase archived nucleic acid. See,
for example, U.S. Pat. No. 6,605,452, Marmaro.
[0022] In some embodiments, preamplification methods can employ in
vitro transcription (IVT) comprising amplifying at least one
sequence in a collection of nucleic acids sequences. The processes
can comprise synthesizing a nucleic acid by hybridizing a primer
complex to the sequence and extending the primer to form a first
strand complementary to the sequence and a second strand
complementary to the first strand. The primer complex can comprise
a primer complementary to the sequence and a promoter region in
anti-sense orientation with respect to the sequence. Copies of
anti-sense RNA can be transcribed off the second strand. The
promoter region, which can be single or double stranded, can be
capable of inducing transcription from an operably linked DNA
sequence in the presence of ribonucleotides and a RNA polymerase
under suitable conditions. Suitable promoter regions may be
prokaryote viruses, such as from T3 or T7 bacteriophage. In some
embodiments, the primer can be a single stranded nucleotide of
sufficient length to act as a template for synthesis of extension
products under suitable conditions and can be poly (T) or a
collection of degenerate sequences. In some embodiments, the
methods involve the incorporation of an RNA polymerase promoter
into selected cDNA molecule by priming cDNA synthesis with a primer
complex comprising a synthetic oligonucleotide containing the
promoter. Following synthesis of double-stranded cDNA, a polymerase
generally specific for the promoter can be added, and anti-sense
RNA can be transcribed from the cDNA template. The progressive
synthesis of multiple RNA molecules from a single cDNA template
results in amplified, anti-sense RNA (aRNA) that serves as starting
material for cloning procedures by using random primers. The
amplification, which will typically be at least about 20-40,
typically to 50 to 100 or 250-fold, but can be 500 to 1000-fold or
more, can be achieved from nanogram quantities or less of cDNA.
[0023] In some embodiments, a two stage preamplification method can
be used to preamplify assay in one vessel by IVT and, for example,
this preamplification stage can be 100 X sample. In the second
stage, the preamplified product can be divided into aliquots and
preamplified by PCR and, for example, this preamplification stage
can be 16,000 X sample or more.
[0024] In some embodiments, the preamplification can be a multiplex
preamplification, wherein the analyte sample can be divided into a
plurality of aliquots. Each aliquot can then be subjected to
preamplification using a plurality of primer sets for DNA targets.
In some embodiments, the primer sets in at least some of the
plurality of aliquots differ from the primer sets in the remaining
aliquots. Each resulting preamplification product detection mixture
can then be dispersed into at least some of a plurality of wells of
a microplate comprising an assay having corresponding primer sets
and detection probes for further amplification and detection
according to the methods described herein. In some embodiments, the
primer sets of assay in each of the plurality of wells can
correspond to the primer sets used in making the preamplification
product detection mixture. The resulting assay in each of the
plurality of wells thus can comprise a preamplification product and
primer sets and detection probes for amplification for DNA targets,
which, if present in the analyte sample, have been
preamplified.
[0025] Since a plurality of different sequences can be amplified
simultaneously in a single reaction, the multiplex preamplification
can be used in a variety of contexts to effectively increase the
concentration or quantity of a sample available for downstream
analysis and/or assays. In some embodiments, because of the
increased concentration or quantity of target DNA, significantly
more analyses can be performed with multiplex amplified samples
than can be performed with the original sample. In many
embodiments, multiplex amplification further permits the ability to
perform analyses that require more sample or a higher concentration
of sample than was originally available. In such embodiments,
multiplex amplification enables downstream analysis for assays that
could not have been possible with the original sample due to its
limited quantity. In some embodiments, the plurality of aliquots
can comprise 16 aliquots with each of the 16 aliquots comprising
about 1536 primer sets. In such embodiments, a sample comprising a
whole genome for a species, for example a human genome, can be
preamplified. In some embodiments, the plurality of aliquots can be
greater than 16 aliquots. In some embodiments, the number of primer
sets can be greater than 1536 primer sets. In some embodiments, the
plurality of aliquots can be less than 16 aliquots and the number
of primer sets can be greater than 1536 primer sets. For examples
of such embodiments, see PCT Publication No. WO 2004/051218 to
Andersen and Ruff.
[0026] In some embodiments, multiplex methods are provided wherein
assay comprises a first universal primer that binds to a complement
of a first target, a second universal primer that binds to a
complement of a second target, a first detection probe comprising a
sequence that binds to the sequence comprised by the first target,
and a second detection probe comprising a sequence that binds to a
sequence comprised by the second target. In some embodiments, at
least some of a plurality of wells of a microplate comprise a
solution operable to perform multiplex PCR. The first and second
detection probes can comprise different labels, for example,
different fluorophores such as, in non-limiting example, VIC and
FAM. Sequences of the first and second detection probes can differ
by as little as one nucleotide, two nucleotides, three nucleotides,
four nucleotides, or greater, provided that hybridization occurs
under conditions that allow each detection probe to hybridize
specifically to its corresponding detection probe.
[0027] In some embodiments, multiplex PCR can be used for relative
quantification, where one primer set and detection probe amplifies
the target DNA and another primer set and detection probe amplifies
an endogenous reference. In some embodiments, the present teaching
provide for analysis of at least four DNA targets in each of a
plurality of wells and/or analysis of a plurality of DNA targets
and a reference in each of a plurality of wells.
[0028] In some embodiments, as seen in FIG. 1, a plurality of
microplates having assay filled thereon can be analyzed as
described herein with sequence detection system, such as a PCR
system to generate data. In some embodiments, this data can be
stored in a gene expression analysis system database 736. Software
can then be used to generate gene expression analysis information
738.
[0029] In some embodiments, a gene expression analysis system can
utilize computer software that organizes analysis sessions into
studies and stores them in database 738. An analysis session can
comprise the results of running microplate in sequence detection
system. To analyze session data, one can load an existing study
that contains analysis session data or create a new study and
attach analysis session data to it. Studies can be opened and
reexamined an unlimited number of times to reanalyze the analysis
session data or to add other analysis sessions to the analysis.
[0030] In some embodiments, gene expression analysis system
database 736 stores the analyzed data for each microplate run on
sequence detection system as an analysis session in database 736.
The software can identify each analysis session by marking indicia
of the associated microplate and the date on which it was created.
Once analysis sessions have been assigned to a study, various
functions can be performed. These functions comprise, but are not
limited to, designating replicates, removing outliers, filtering
data out of a particular view or report, correction of
preamplification values via stored values, and computation of gene
expression values.
[0031] In various embodiments, real time PCR is adapted to perform
quantitative real time PCR (qRT-PCR). In various embodiments, two
different methods of analyzing data from qRT-PCR experiments can be
used: absolute quantification and relative quantification. In some
embodiments, absolute quantification can determine an input copy
number of the target DNA of interest This can be accomplished by
relating a signal from a detection probe to a standard curve. In
various embodiments, relative quantification can describe the
change in expression of the target DNA relative to a reference or a
group of references such as, for an example, an untreated control,
an endogenous control, a passive internal reference, an universal
reference RNA, or a sample at time zero in a time course study.
When determining absolute quantification, the expression of the
target DNA can be compared across many samples, for example, from
different individuals, from different tissues, from multiple
replicates, and/or serial dilution of standards in one or more
matrices. In various embodiments of the present teachings, qRT-PCR
can be performed using relative quantification and the use of
standard curve is not required. Relative quantification can compare
the changes in steady state target DNA levels of two or more genes
to each other with one of the genes acting as an endogenous
reference which may be used to normalize a signal from a sample
gene. In various embodiments, in order to compare between
experiments, resulting fold differences from the normalization of
sample to the reference can be expressed relative to a calibrator
sample. In some embodiments, the calibrator sample is included in
each assay 1000. The gene expression analysis system can determine
the amount of target DNA, normalized to a reference, by
determining
.DELTA.C.sub.T=C.sub.Tq-C.sub.Tendo
where C.sub.T is the threshold cycle for detection of a fluorophore
in real time PCR; C.sub.Tq is the threshold cycle for detection of
a fluorophore for a target DNA in assay 1000; and C.sub.Tendo is
the threshold cycle for detection of a fluorophore for an
endogenous reference or a passive internal reference in assay.
[0032] In some embodiments, a gene expression analysis system can
determine the amount of target DNA, normalized to a reference and
relative to a calibrator, by determining:
.DELTA..DELTA.C.sub.T=.DELTA.C.sub.Tq-.DELTA.C.sub.Tcb
[0033] where C.sub.Tq is the threshold cycle for detection of a
fluorophore for the target DNA in assay 1000; C.sub.Tcb is the
threshold cycle for detection of a fluorophore for a calibrator
sample; .DELTA.C.sub.Tq is a difference in threshold cycles for the
target DNA and an endogenous reference; and .DELTA.C.sub.Tcb is a
difference in threshold cycles for the calibrator sample and the
endogenous reference If .DELTA..DELTA.C.sub.T is determined, the
relative quantity of the target DNA can be determined using a
relationship of relative quantity of the target DNA can be equal to
2.sup.-.DELTA..DELTA.C.sub.T. In various embodiments,
.DELTA..DELTA.C.sub.T can be about zero. In some embodiments,
.DELTA..DELTA.C.sub.T can be less than .+-.1. In various
embodiments, the above calculations can be adapted for use in
multiplex PCR (See, for example, Livak et al. Applied Biosystems
User Bulletin #2, updated October 2001 and Livak and Schmittgen,
Methods (25) 402-408 (2001).
[0034] In some embodiments, assay can be preamplified, as discussed
herein, in order to increase the amount of target DNA prior to
distribution into a plurality of wells of a microplate. In some
embodiments, assay can be collected, for example, via a needle
biopsy that typically yields a small amount of sample. Distributing
this sample across a large number of wells can result in variances
in sample distribution that can affect the veracity of subsequent
gene expression computations. In such situations, assay can be
preamplified using, for example, a pooled primer set to increase
the number of copies of all target DNA simultaneously.
[0035] In various embodiments, preamplification processes can be
non-biased, such that all target DNA are amplified similarly and to
about the same power. In such embodiments, each target DNA can be
amplified reproducibly from one input sample to the next input
sample. For example, if target DNA X is initially present in sample
A at 100 target molecules, then after 10 cycles of PCR
amplification (1000-fold), 100,000 target molecules should be
present. Continuing with the example, if target DNA X is initially
present in sample B at 500 target molecules, then after 10 cycles
of PCR amplification (1000-fold), 500,000 target molecules should
be present. In this example, the ratio of target DNA X in samples
A/B remains constant before and after the amplification
procedure.
[0036] In various embodiments, a minor proportion of all target DNA
can have an observed preamplification efficiency of less than 100%.
In such embodiments, if the amplification bias is reproducible and
consistent from one input sample to another, then the ability to
accurately compute comparative relative quantitation between any
two samples containing different relative amounts of target can be
maintained. Continuing the example from above and assuming 50%
reproducible amplification efficiency, if target DNA X is initially
present in sample A at 100 target molecules, then after 10 cycles
of PCR amplification (50% of 1000-fold), 50,000 target molecules
should be present. Further continuing the example, if target X is
initially present in sample B at 500 target molecules, then after
10 cycles of PCR amplification (50% of 1000-fold), 250,000 target
molecules should be present. In this example, the ratio of template
X in samples A/B remains constant before and after the
amplification procedure and is the same ratio as the 100%
efficiency scenario.
[0037] In various embodiments, an unbiased amplification of each
target DNA (x, y, z, etc.) can be determined by calculating the
difference in CT value of the target DNA (x,y,z, etc.) from the
C.sub.T value of a selected endogenous reference, and such
calculation is referred to as the .DELTA.C.sub.T value for each
given target DNA, as described above. In various embodiments, a
reference for a bias calculation can be non-preamplified, amplified
target DNA and an experimental sample can be a preamplified
amplified target DNA. In some embodiments, the standard sample and
experimental sample can originate from the same sample, for
example, same tissue, same individual and/or same species. In
various embodiments, comparison of .DELTA.C.sub.T values between
the non-preamplified amplified target DNA and preamplified
amplified target DNA can provide a measure for the bias of the
preamplification process between the endogenous reference and the
target DNA (x, y, z, etc.).
[0038] In various embodiments, the difference between the two
.DELTA.C.sub.T values (.DELTA..DELTA.C.sub.T) can be zero and as
such there is no bias from preamplification. This is explained in
greater detail below with reference to FIG. 2. In some embodiments,
the gene expression analysis system can be calibrated for potential
differences in preamplification efficiency that can arise from a
variety of sources, such as the effects of multiple primer sets in
the same reaction. In some embodiments, calibration can be
performed by computing a reference number that reflects
preamplification bias. Reference number similarity for a given
target DNA across different samples is indicative that the
preamplification reaction .DELTA.C.sub.Ts can be used to achieve
reliable gene expression computations.
[0039] In various embodiments of the present teaching, a gene
expression analysis system can compute these reference numbers by
collecting a sample (designated as Sample A (S.sub.A)) and
processing it with one or more protocols. A first protocol
comprises running individual PCR gene expression reactions for each
target DNA (T.sub.x) relative to an endogenous reference (endo),
such as, for example, 18s or GAPDH. These reactions can yield cycle
threshold values for each target DNA relative to the endogenous
control; as computed by:
.DELTA.C.sub.T not preamplifiedT.sub.xS.sub.A=C.sub.T not
preamplifiedT.sub.xS.sub.A-C.sub.T notpreamplifiedendo
[0040] A second protocol can comprise running a single PCR
preamplification step on assay with, for example, a pooled primer
set. In various embodiments, the pooled primer set can contain
primers for each target DNA. Subsequently, the preamplified product
can be distributed among a plurality of wells of a microplate. PCR
gene-expression reactions can be run for each preamplified target
DNA (T.sub.x) relative to an endogenous reference (endo). These
reactions can yield cycle threshold values for each preamplified
target DNA relative to the endogenous control, as computed by:
.DELTA.C.sub.T preamplifiedT.sub.xS.sub.A=C.sub.T
preamplifiedT.sub.xS.sub.A-C.sub.T preamplified
endoT.sub.xS.sub.A
A difference between these .DELTA.C.sub.T not preamplified
T.sub.xS.sub.A and .DELTA.C.sub.T preamplified T.sub.xS.sub.A can
be computed by:
.DELTA.C.sub.TT.sub.xS.sub.A=.DELTA.C.sub.T not
preamplifiedT.sub.xS.sub.A-.DELTA.C.sub.T preamplified
TxS.sub.A
[0041] In various embodiments, a value for
.DELTA..DELTA.C.sub.TT.sub.xS.sub.A can be zero or close to zero,
which can indicate that there is no bias in the preamplification of
target DNA T.sub.x. In various embodiments, a negative
.DELTA..DELTA.C.sub.T T.sub.xS.sub.A value can indicate the
preamplification process was less than 100% efficient for a given
target DNA (T.sub.x). For example, when using an IVT
preamplification process, a percentage of target DNA with a
.DELTA..DELTA.C.sub.T of +/-1 C.sub.T of zero can be .about.50%, as
shown in FIG. 3. In another example, when using a multiplex
preamplification process, a percentage of target DNA with a
.DELTA..DELTA.CT of +/-1 C.sub.T of zero can be .about.90%, as
shown in FIG. 4.
[0042] In various embodiments, an amplification efficiency can be
less than 100% for a particular target DNA, therefore
.DELTA..DELTA.C.sub.T is less than zero for the particular target
DNA. An example, as shown in FIG. 5, can be an evaluation of
.DELTA..DELTA.C.sub.T values for a group of target DNA from a
1536-plex for the multiplex preamplification process including four
different human sample input sources: liver, lung, brain and an
universal reference tissue composite. In this example, most
.DELTA..DELTA.C.sub.T values are near zero, however, some of the
target DNA have a negative .DELTA..DELTA.C.sub.T value but these
negative values are reproducible from one sample input source to
another. In various embodiments, a gene expression analysis system
can determine if a bias exists for target DNA analyzed for
different sample inputs.
[0043] In various embodiments of the present teachings, a gene
expression analysis system can use .DELTA..DELTA.C.sub.T values
computed for the same target DNA but in different samples (Sample A
(S.sub.A) and Sample B (S.sub.B)) in order to determine the
accuracy of subsequent relative expression computations. This
results in the equation,
.DELTA..DELTA..DELTA.C.sub.TT.sub.x=.DELTA..DELTA.C.sub.TT.sub.xS.sub.A--
.DELTA..DELTA.C.sub.TT.sub.xS.sub.B
[0044] In various embodiments a value for
.DELTA..DELTA..DELTA.L.sub.TT.sub.x can be zero or reasonably close
to zero which can indicate that the preamplified .DELTA.C.sub.T
values for T.sub.x (.DELTA.C.sub.T preamplified T.sub.xS.sub.A and
.DELTA.C.sub.T preamplified T.sub.xS.sub.B) can be used for
relative gene expression computation between different samples via
a standard relative gene expression calculation.
[0045] In some embodiments, a standard relative gene expression
calculation can determine the amount of the target DNA. In some
embodiments, a standard relative gene expression calculation
employs a comparative C.sub.T. In various embodiments, the above
methods can be practiced during experimental design and once the
conditions have been optimized so that the
.DELTA..DELTA..DELTA.C.sub.TT.sub.x is reasonably close to zero,
subsequent experiments only require the computation of the
.DELTA.C.sub.T value for the preamplified reactions. In various
embodiments, .DELTA..DELTA.C.sub.TT.sub.xS.sub.A values can be
stored in a database or other storage medium. In such embodiments,
these values can then be used to convert
.DELTA..DELTA.C.sub.TpreamplifiedT.sub.xS.sub.A values to
.DELTA..DELTA.C.sub.T not preamplifiedT.sub.xS.sub.A values. In
such embodiments, the .DELTA..DELTA.C.sub.T
preamplifiedT.sub.xS.sub.y values can be mapped back to a common
domain. In various embodiments, a not preamplified domain can be
calculated using other gene expression instrument platforms such
as, for example, a microarray. In various embodiments, the
.DELTA..DELTA.C.sub.TT.sub.xS.sub.A values need not be stored for
all different sample source inputs (S.sub.A) if it can be
illustrated that the .DELTA..DELTA.C.sub.T preamplifiedT.sub.x is
reasonably consistent over different sample source inputs. For
example, a distribution of .DELTA..DELTA..DELTA.C.sub.T for two
different sample inputs (liver and brain) are shown in FIG. 6 (IVT
preamplification) and FIG. 7 (multiplex preamplification).
[0046] In various embodiments, gene expression can be assessed with
microarray technology, which can provide a measure of the cellular
concentration of different mRNAs. In some embodiments, a microarray
can be a piece of glass or plastic on which single stranded pieces
of DNA are affixed in a microscopic array as probes. In some
embodiments, thousands of identical probes can be affixed at each
point in the array which can make effective detectors.
[0047] Typically arrays can be used to detect the presence of mRNAs
that may have been transcribed from different genes and which
encode different proteins. The RNA can be extracted from many
cells, ideally from a single cell type, then converted to cDNA. In
various embodiments, the cDNA may be amplified in quantity by PCR.
Fluorescent tags can be enzymatically incorporated into the or can
be chemically attached to strands of cDNA. In various embodiments,
a cDNA molecule that contains a sequence complementary to one of
the probes will hybridize via base pairing to the point at which
the complementary probes are affixed. In such embodiments, the
point on the array can then fluoresce when examined using a
microarray scanner. In some embodiments, the intensity of the
fluorescence can be proportional to the number of copies of a
particular mRNA that were present and thus roughly indicates the
activity or expression level of that gene.
[0048] In various embodiments, a microarray can be, for example, a
cDNA array, a hybridization array, a DNA microchip, a high density
sequence oligonucleotide array, or the like. In various
embodiments, a microarray can be available from a commercial source
such as, for example, Applied Biosystems, Affymetrix, Agilent,
Illumina, or Xeotron. In various embodiments, a microarray can be
made by any number of technologies including printing with
fine-pointed pins onto glass slides, photolithography using
pre-made masks, photolithography using dynamic micromirror devices,
or ink-jet printers. The lack of standardization in microarrays can
present an interoperability problem in bioinformatics, since it can
limit the exchange of array data.
[0049] In various embodiments, microarray output data can be in a
format of fluorescence intensity and in other embodiments,
microarray output data may be in a format of chemiluminescence
intensity. In various embodiments, an intensity value from a
microarray output data can be globally normalized. In some
embodiments, told difference values can be determined by
subtracting background noise and normalizing the array signal
intensity, then dividing experimental sample signal intensity by a
control sample signal intensity yielding net sample intensity. In
some embodiments, a control sample used to generate the control
sample signal intensity can be, for example, Stratagene.RTM., UHR
or the like. In some embodiments, a full difference can be
converted to a log.sub.2 by the following equation:
2.sup..DELTA..DELTA.C.sub.T=3.3 log.sub.10(net intensity sample
1/net intensity sample 2)
In such embodiments, microarray output data is in a
.DELTA..DELTA.C.sub.T format. In some embodiments, microarray
output data can be converted into a .DELTA..DELTA.C.sub.T format by
the following equation:
R=(1/2).sup..DELTA..DELTA.C.sub.T
where R is the resulting measurement from a microarray. Such
calculations are available commercially, such as GeneSpring from
Silicon Genetics. Other embodiments include converting microarray
output data into a .DELTA..DELTA.C.sub.T format using a Global
Pattern Recognition (GPR) algorithm which can convert intensity
values generated from microarrays from linear values to algorithmic
values and can use transformed intensity cutoffs to effect gene and
normalizer filters. In such embodiments, GPR, a software algorithm
for gene expression analysis is available from The Jackson
Laboratory. In various embodiments, microarray output data can be
in a standard language or format such as MAGE-ML (microarray and
gene expression markup language), MAML (microarray markup
language), or MIAME (minimum information about microarray
experiments). In various embodiments, such standardized formats and
language can be converted to a .DELTA..DELTA.C.sub.T format.
[0050] In various embodiments, after microarray output data is in a
.DELTA..DELTA.C.sub.T format, then real-time PCR data can be
directly compared to data from microarray platforms as shown in
FIG. 8. In various embodiments, a .DELTA..DELTA..DELTA.C.sub.T
calculation can be a validation tool to confirm that relative
quantitation data can be compared from one amplification/detection
process to another. In various embodiments,
.DELTA..DELTA..DELTA.C.sub.T calculation can be a validation tool
to confirm that relative quantitation data can be compared from one
sample input source to another sample input source, for example,
comparing a sample from liver to a sample from brain in the same
individual. In various embodiments, .DELTA..DELTA..DELTA.C.sub.T
calculation can be a validation tool to confirm that relative
quantitation data can be compared from one high-density sequence
detector system to another high-density sequence detection system.
In various embodiments, .DELTA..DELTA..DELTA.C.sub.T calculation
can be a validation tool to confirm that relative quantitation data
can be compared from one platform to another, for example, data
from real time PCR to data from a hybridization array is especially
valuable for cross-platform validation. In various embodiments,
real time PCR and hybridization array data can be directly
compared. In various embodiments, a TaqMan .DELTA..DELTA.C.sub.T
can be compared to a microarray output converted to the
.DELTA..DELTA.C.sub.T format. In such embodiments, the resultant
.DELTA..DELTA..DELTA.C.sub.T, if within +/-1 C.sub.T of zero, can
determine a high-degree of confidence that the actual fold
difference observed within each of the two platforms is
correlative.
[0051] In various embodiments, a correction, which can be a
quantity added to a calculated or observed value to obtain the true
value, may be used so that data generated on two different
platforms can be used together in further calculations and
analysis. Such embodiments allow for larger and sometimes more
complete data sets to be used in gene expression studies. In some
embodiments, the correction can be calculated from a resulting
.DELTA..DELTA..DELTA.C.sub.T. In various embodiments, a correction
can be a bias correction.
* * * * *