U.S. patent application number 13/242911 was filed with the patent office on 2012-01-19 for methods, panels of identification markers, and kits for identifying forensic samples.
This patent application is currently assigned to APPLIED BIOSYSTEMS, LLC. Invention is credited to John W. Burns, Rixun N. FANG, Manohar R. Furtado.
Application Number | 20120015832 13/242911 |
Document ID | / |
Family ID | 44773314 |
Filed Date | 2012-01-19 |
United States Patent
Application |
20120015832 |
Kind Code |
A1 |
FANG; Rixun N. ; et
al. |
January 19, 2012 |
METHODS, PANELS OF IDENTIFICATION MARKERS, AND KITS FOR IDENTIFYING
FORENSIC SAMPLES
Abstract
Methods for identifying forensic samples using panels of markers
and gene expression profiling, including without limitation, mRNA
profiling, miRNA profiling, or both, are disclosed. Panels of
markers for identifying certain tissue samples and certain body
fluid samples are also disclosed. Kits for expediting performance
of certain of the disclosed methods are provided.
Inventors: |
FANG; Rixun N.; (Menlo Park,
CA) ; Burns; John W.; (Austin, TX) ; Furtado;
Manohar R.; (San Ramon, CA) |
Assignee: |
APPLIED BIOSYSTEMS, LLC
Carlsbad
CA
|
Family ID: |
44773314 |
Appl. No.: |
13/242911 |
Filed: |
September 23, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11751845 |
May 22, 2007 |
8039234 |
|
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13242911 |
|
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60747927 |
May 22, 2006 |
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Current U.S.
Class: |
506/7 ;
506/16 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/178 20130101; C12Q 2600/16 20130101 |
Class at
Publication: |
506/7 ;
506/16 |
International
Class: |
C40B 30/00 20060101
C40B030/00; C40B 40/06 20060101 C40B040/06 |
Claims
1-6. (canceled)
7. A set of primers specific for a panel of markers comprising, at
least one brain marker comprising at least one of miR-125b,
miR-128a, miR-128b, miR-129, miR-135, and miR-153; at least one
muscle marker comprising at least one of miR-1d, miR-133a,
miR-133b, miR-296, miR-208; at least one kidney marker comprising
at least one of miR-192, miR-204, miR-215, and miR-216; at least
one thymus marker comprising at least one of miR-96 and miR-182; at
least one testes marker comprising at least one of miR-10b and
let-7e; and at least one placenta marker comprising at least one of
miR-141 and miR-23a.
8. A set of primers specific for a panel of markers comprising at
least one mRNA marker and at least one miRNA marker.
9. (canceled)
10. (canceled)
11. A method for identifying a forensic sample containing nucleic
acid comprising: combining a set of primer pairs to specifically
amplify each gene in a panel of markers, wherein the panel of
markers comprise at least one brain marker comprising at least one
of miR-125b, miR-128a, miR-128b, miR-129, miR-135, and miR-153; at
least one muscle marker comprising at least one of miR-1d,
miR-133a, miR-133b, miR-296, miR-208; at least one kidney marker
comprising at least one of miR-192, miR-204, miR-215, and miR-216;
at least one thymus marker comprising at least one of miR-96 and
miR-182; at least one testes marker comprising at least one of
miR-10b and let-7e; and at least one placenta marker comprising at
least one of miR-141 and miR-23a with at least some of the nucleic
acid from the sample and a polymerase, wherein the at least one
first primer in the set of marker-specific primers is designed to
amplify at least a portion of a first member of a panel of markers
and at least one second primer in the set of marker-specific
primers is designed to amplify at least a portion of a second
member of the panel of markers; generating an expression profile
for the panel of markers; and identifying the forensic sample.
12. The method of claim 11, wherein at least one marker-specific
primer comprises a marker-specific primer pair.
13. (canceled)
14. The method of claim 11, wherein the generating comprises a
reporter probe, a substrate, a nucleic acid dye, a microfluidics
device, or combinations thereof.
15. The method of claim 11, wherein the polymerase comprises a
DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase and
a DNA-dependent DNA polymerase.
16. The method of claim 11, wherein the generating comprises a
first amplification reaction comprising a multiplicity of different
gene-specific primer pairs and a second amplification reaction
comprising a single-plex amplification reaction.
17. The method of claim 11, wherein the second amplification
reaction comprises a multiplicity of different single-plex
amplification reactions performed in parallel.
18. The method of claim 17, wherein the multiplicity of parallel
single-plex amplification reactions comprise a microfluidics
device.
19. The method of claim 11, wherein at least one gene-specific
primer comprises a miRNA-specific primer.
20. The method of claim 19, wherein the at least one miRNA-specific
primer comprises at least one linker probe; and further comprising
a reporter probe.
21. The method of claim 20, wherein the generating comprises a
first amplification reaction and a second amplification
reaction.
22. A kit comprising of a set of primers specific for a panel of
markers, wherein the set of primers consists of primer pairs to
specifically amplify each of the markers in the panel of markers,
wherein the panel of markers comprise at least one brain marker
comprising at least one of miR-125b, miR-128a, miR-128b, miR-129,
miR-135, and miR-153; at least one muscle marker comprising at
least one of miR-1d, miR-133a, miR-133b, miR-296, miR-208; at least
one kidney marker comprising at least one of miR-192, miR-204,
miR-215, and miR-216; at least one thymus marker comprising at
least one of miR-96 and miR-182; at least one testes marker
comprising at least one of miR-10b and let-7e; and at least one
placenta marker comprising at least one of miR-141 and miR-23a,
wherein the set of primers consists of 21 or fewer primer
pairs.
23. The kit of claim 22, further comprising one or more of a
polymerase; nucleotide triphosphates (NTPs), wherein the NTPs can
be ribonucleotide triphosphates (rNTPs) and/or deoxyribonucleotide
triphosphates (dNTPs); a reporter probe; a nucleic acid dye; a
miRNA linker probe; an internal reference dye; a control sequence
or internal standard; a reporter group, wherein the reporter group
includes an NTP comprising a reporter group; or combinations
thereof.
24. The kit of claim 22, further including a microarray and/or a
microfluidics device.
25. The kit of claim 22, wherein the polymerase comprises a
DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase and
a DNA-dependent DNA polymerase.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
11/751,845, filed May 22, 2007, now allowed, which claims a
priority benefit under 35 U.S.C. .sctn.119(e) from U.S. Application
No. 60/747,927, filed May 22, 2006, the contents of which are
incorporated herein by reference.
FIELD
[0002] Methods, panels of markers for generating gene expression
profiles, and kits for identifying a forensic sample are
provided.
INTRODUCTION
[0003] Body fluids and stains are frequently encountered at crime
scenes, including but not limited to, blood, semen, saliva, and
vaginal secretions. Traditional methods for identifying body fluids
are often labor-intensive, time-consuming, and expensive. There is
a need for more efficient, less expensive body fluid identification
techniques. The identification of tissue samples is also important
to forensic scientists.
[0004] Advances in gene expression analysis have allowed scientists
to gain a better understanding of, among other things,
developmental biology and carcinogenesis. Expression profiles have
identified highly expressed genes based on cell and/or tissue type.
However, these techniques typically require relatively large
amounts of messenger RNA (mRNA) or microRNA (miRNA) to obtain
reliable results. Forensic samples are often small and the quantity
of nucleic acid obtained from such samples is correspondingly low.
If appropriate quantities of nucleic acid could be obtained from
typical crime scene samples, gene expression analysis could be
employed by forensic scientists for determining the source of such
samples, particularly if panels of suitable body fluid-specific
markers and tissue-specific markers were available to facilitate
identification.
SUMMARY
[0005] The present teachings are directed to methods, marker
panels, and kits for identifying a forensic sample by determining
the relative concentration of the RNA species that correspond to a
panel of markers comprising genes that are differentially expressed
in various samples, including mRNA markers, miRNA markers, or both.
The methods permit the identification of forensic samples,
including without limitation, tissue or organ samples (collectively
referred to in this specification as a "tissue sample") or body
fluid samples using certain tissue marker panels and body fluid
marker panels of the current teachings. According to certain
disclosed methods, nucleic acid is obtained from a sample and a
gene expression profile for the markers in the panel is generated,
typically using a marker-specific primer pair for each marker. By
comparing the gene expression profile generated from the sample
with the known expression profile of those markers in certain
tissue samples and body fluids, the sample can generally be
identified. According to certain methods, a reverse transcriptase
or a DNA-dependent DNA polymerase with reverse transcriptase
activity, for example but not limited to, Tth DNA polymerase is
bined with a gene-specific first primer for each target to be
analyzed and a DNA complement of the RNA target is generated. This
is followed by PCR amplification using a gene-specific primer pair
or a gene-specific primer and a universal primer for each marker.
Certain of the disclosed methods employ two PCR amplification
reactions, including without limitation, a first PCR reaction that
is performed for a limited cycle multiplex reaction followed by a
second amplification reaction, often performed as a single-plex
reaction (see, e.g., U.S. Pat. No. 6,605,451 (Marmaro and Gerdes);
U.S. Patent Application Publication No. 2004/0175733 A1 (Andersen
and Ruff); and Dolganov et al., Genome Research 11:1473-83, 2001).
Certain embodiments of the disclosed methods include multiplex
assays for quantitating a multiplicity of different small RNA
species; other embodiments are directed to single-plex assays for
detecting or quantitating a single small RNA species, including
without limitation a series of two or more single-plex assays
performed in parallel. Typically the multiplicity of different RNA
species that are amplified and quantitated are members of a panel
of markers, each of which is indicative of a particular tissue or
body fluid. In certain embodiments, an internal standard, for
example but not limited to a mRNA transcript of housekeeping gene,
is also amplified and quantitated as a control.
[0006] In other embodiments, mRNA and/or miRNA profiling comprises
a microarray, including but not limited to a planar array and a
bead-based array, wherein the relative concentration of two
different species of miRNA and/or mRNA are determined using
well-known hybridization-based techniques and the sample is
identified. In certain embodiments, a bead-based array comprises
flow cytometry.
[0007] Kits for performing certain of the instant methods are also
disclosed. Certain kit embodiments include at least one pair of
primers for each of the markers in an identification panel, wherein
each primer pair is designed to amplify one of the markers in the
panel; appropriate nucleotide triphosphates (NTPs, including
without limitation ribonucleotide triphosphates (rNTPs) and/or
deoxyribonucleotide triphosphates (dNTPs), as appropriate); and a
suitable polymerase. Certain kits comprise a RNA-dependent DNA
polymerase and a DNA-dependent DNA polymerase. Some kits further
comprise a reporter probe (sometimes referred to as a "non-priming
oligonucleotide probe") for each marker amplicon; a nucleic acid
dye; a miRNA linker probe (see U.S. Patent Application Pub. No. US
2005/0266418); an internal reference dye; a control sequence or
internal standard; a reporter group, including without limitation
an NTP comprising a reporter group; or combinations thereof. In
certain embodiments, kits comprise a promoter-primer for each RNA
sequence to be amplified, a polymerase, appropriate NTPs, a
reporter group, including without limitation a NTP comprising a
reporter group, or combinations thereof. In certain embodiments,
kits comprise a microarray, a microfluidics device, a reaction
vessel, or combinations thereof.
[0008] These and other features of the present teachings are set
forth herein.
DRAWINGS
[0009] The skilled artisan will understand that the drawings,
described below, are for illustration purposes only. These figures
are not intended to limit the scope of the present teachings in any
way.
[0010] FIG. 1: schematically depicts an illustrative workflow
comprising certain aspects of various embodiments of exemplary
methods of the current teachings.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0011] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not intended to limit the scope of the
current teachings. In this application, the use of the singular
includes the plural unless specifically stated otherwise. For
example, "a forward primer" means that more than one forward primer
can be present, such as, one or more copies of a particular forward
primer species, as well as one or more copies of different forward
primer species. Also, the use of "comprise", "contain", and
"include", or modifications of those root words, for example but
not limited to, "comprises", "contained", and "including", are not
intended to be limiting.
[0012] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the described
subject matter in any way. All literature and similar materials
cited in this application, including patents, patent applications,
articles, books, and treatises 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
defines or uses a term in such a way that it contradicts that
term's definition in this application, this application controls.
While the present teachings are described in conjunction with
various embodiments, it is not intended that the present teachings
be limited to such embodiments. On the contrary, the present
teachings encompass various alternatives, modifications, and
equivalents, as will be appreciated by those of skill in the
art.
[0013] The term "or combinations thereof" as used herein refers to
all permutations and combinations of the listed items preceding the
term. For example, "A, B, C, or combinations thereof" is intended
to include at least one of: A, B, C, AB, AC, BC, or ABC, and if
order is important in a particular context, also BA, CA, CB, ACB,
CBA, BCA, BAC, or CAB. Continuing with this example, expressly
included are combinations that contain repeats of one or more item
or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and
so forth. The skilled artisan will understand that typically there
is no limit on the number of items or terms in any combination,
unless otherwise apparent from the context.
[0014] The term "corresponding" as used herein refers to at least
one specific relationship between the elements to which the term
relates. For example, but without limitation, a particular
marker-specific primer pair corresponds to the amplicon of that
marker that the pair amplifies; and a forward primer of a given
primer pair corresponds to the reverse primer of that primer
pair.
[0015] The terms "DNA polymerase" and "polymerase" are used in a
broad sense herein and refer to any polypeptide that catalyzes the
addition of deoxyribonucleotides or analogs of deoxyribonucleotides
to a nucleic acid polymer in a template dependent manner. For
example but not limited to, the sequential addition of
deoxyribonucleotides to the 3'-end of a primer that is annealed to
a nucleic acid template during a primer extension reaction.
Typically DNA polymerases include DNA-dependent DNA polymerases and
RNA-dependent DNA polymerases, including without limitation,
reverse transcriptases. Certain reverse transcriptases possess
DNA-dependent DNA polymerase activity under certain reaction
conditions, including AMV reverse transcriptase and MMLV reverse
transcriptase. Such reverse transcriptases with DNA-dependent DNA
polymerase activity may be suitable for use with the disclosed
methods and are expressly within the contemplation of the current
teachings. Descriptions of DNA polymerases can be found in, among
other places, Lehninger Principles of Biochemistry, 3d ed., Nelson
and Cox, Worth Publishing, New York, N.Y., 2000, particularly
Chapters 26 and 29; Twyman, Advanced Molecular Biology: A Concise
Reference, Bios Scientific Publishers, New York, N.Y., 1999;
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
& Sons, Inc., including supplements through May 2006
(hereinafter "Ausubel et al."); Lin and Jaysena, J. Mol. Biol.
271:100-11, 1997; Pavlov et al., Trends in Biotechnol. 22:253-60,
2004; and Enzymatic Resource Guide: Polymerases, 1998, Promega,
Madison, Wis. Expressly within the intended scope of the term DNA
polymerase are enzymatically active mutants or variants thereof,
including without limitation enzymes modified to confer different
temperature-sensitive properties (see, e.g., U.S. Pat. Nos.
5,773,258; 5,677,152; and 6,183,998; and DNA Amplification: Current
Techniques and Applications, Demidov and Broude, eds., Horizon
Bioscience, 2004, particularly in Chapter 1.1) and enzymatically
active fragments of certain DNA polymerases such as Klenow fragment
and Stoffel fragment.
[0016] The term "nucleic acid dye" as used herein refers to a
fluorescent molecule that is specific for a double-stranded
polynucleotide or that at least emits a substantially greater
fluorescent signal when associated with a double-stranded
polynucleotide than with a single-stranded polynucleotide.
Typically nucleic acid dye molecules associate with double-stranded
segments of polynucleotides by intercalating between the base pairs
of the double-stranded segment, by binding in the major or minor
grooves of the double-stranded segment, or both. Non-limiting
examples of nucleic acid dyes include ethidium bromide, DAPI,
Hoechst derivatives including without limitation Hoechst 33258 and
Hoechst 33342, intercalators comprising a lanthanide chelate (for
example but not limited to a napthalene diimide derivative carrying
two fluorescent tetradentate .beta.-diketone-Eu3+ chelates
(NDI-(BHHCT-Eu.sup.3+).sub.2), see, e.g., Nojima et al., Nucl.
Acids Res. Supplement No. 1, 105-06 (2001)), ethidium bromide, and
certain unsymmetrical cyanine dyes such as SYBR Green.RTM.,
PicoGreen.RTM. (both available from Molecular Probes-Invitrogen),
and BOXTO (TATAA Biocenter AB). An "unsymmetrical cyanine dye",
sometimes described in the art as an asymmetric cyanine dye or an
asymmetrical cyanine dye, refers to a dye molecule with the general
formula R.sub.2N[CH.dbd.CH].sub.nCH.dbd.NR.sub.2, where n is a
small number and the R groups typically comprise at least one
benzazole group and at least one quinoline group or at least one
pyridine group. Non-limiting examples of unsymmetrical cyanine dyes
include
[2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(ben-
zo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium] (SYBR.RTM.
Green),
[2-[N-bis-(3-dimethylaminopropyl)-amino)-amino]-4-[2,3-dihydro-3-methyl-(-
benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]
(PicoGreen.RTM.),
4-[(3-methyl-6-(benzothiazol-2-yl)-2,3-dihydro-(benzo-1,3-thiazole)-2-met-
hylidene)]-1-methyl-pyridinium iodide (BEBO), BOXTO, and BETO.
Descriptions of unsymmetrical cyanine dyes can be found in, among
other places, Karlsson et al., Nucl. Acids Res. 31:6227-34 (2003);
Zipper et al., Nucl. Acids Res. 32:e103 (2004); Bengtsson et al.,
Nucl. Acids Res. 31:e45 (2003); and Goransson et al., Asymmetric
cyanine dyes, DNA-Technology 2005, Chalmers University Technology
(2005).
[0017] The term "reporter group" is used in a broad sense herein
and refers to any identifiable tag, label, or moiety. The skilled
artisan will appreciate that many different species of reporter
groups can be used in the present teachings, either individually or
in combination with one or more different reporter group. In
certain embodiments, a reporter group emits a fluorescent, a
chemiluminescent, a bioluminescent, a phosphorescent, or an
electrochemiluminescent signal. Some non-limiting examples of
reporter groups include fluorophores, radioisotopes, chromogens,
enzymes, antigens including but not limited to epitope tags,
semiconductor nanocrystals such as quantum dots, heavy metals,
dyes, phosphorescence groups, chemiluminescent groups,
electrochemical detection moieties, binding proteins, phosphors,
rare earth chelates, transition metal chelates, near-infrared dyes,
electrochemiluminescence labels, and mass spectrometer-compatible
reporter groups, such as mass tags, charge tags, and isotopes (see,
e.g., Haff and Smirnov, Nucl. Acids Res. 25:3749-50, 1997; Xu et
al., Anal. Chem. 69:3595-3602, 1997; Sauer et al., Nucl. Acids Res.
31:e63, 2003).
[0018] The term reporter group also encompasses an element of
multi-element reporter systems, including without limitation,
affinity tags such as biotin:avidin, antibody:antigen, and the
like, in which one element interacts with one or more other
elements of the system in order to effect the potential for a
detectable signal. Some non-limiting examples of multi-element
reporter systems include an oligonucleotide comprising a biotin
reporter group and a streptavidin-conjugated fluorophore, or vice
versa; an oligonucleotide comprising a DNP reporter group and a
fluorophore-labeled anti-DNP antibody; and the like. Detailed
protocols for attaching reporter groups to nucleic acids can be
found in, among other places, Hermanson, Bioconjugate Techniques,
Academic Press, San Diego, 1996; Current Protocols in Nucleic Acid
Chemistry, Beaucage et al., eds., John Wiley & Sons, New York,
N.Y. (2000), including supplements through April 2005; and
Haugland, Handbook of Fluorescent Probes and Research Products,
9.sup.th ed., Molecular Probes, 2002.
[0019] Multi-element interacting reporter groups are also within
the intended scope of the term reporter group, such as
fluorophore-quencher pairs, including without limitation
fluorescent quenchers and dark quenchers (also known as
non-fluorescent quenchers). A fluorescent quencher can absorb the
fluorescent signal emitted from a fluorophore and after absorbing
enough fluorescent energy, the fluorescent quencher can emit
fluorescence at a characteristic wavelength, e.g., fluorescent
resonance energy transfer (FRET). For example without limitation,
the FAM-TAMRA pair can be illuminated at 492 nm, the excitation
peak for FAM, and emit fluorescence at 580 nm, the emission peak
for TAMRA. A dark quencher, appropriately paired with a fluorescent
reporter group, absorbs the fluorescent energy from the
fluorophore, but does not itself fluoresce. Rather, the dark
quencher dissipates the absorbed energy, typically as heat. Some
non-limiting examples of dark or nonfluorescent quenchers include
Dabcyl, Black Hole Quenchers, Iowa Black, QSY-7, AbsoluteQuencher,
Eclipse non-fluorescent quencher, certain metallic particles such
as gold nanoparticles, and the like. Certain dual-labeled probes
comprising fluorophore-quencher pairs can emit fluorescence when
the members of the pair are physically separated, for example but
without limitation, nuclease probes such as TaqMan.RTM. probes.
Other dual-labeled probes comprising fluorophore-quencher pairs can
emit fluorescence when the members of the pair are spatially
separated, for example but not limited to hybridization probes such
as molecular beacons or extension probes such as Scorpion primers.
Fluorophore-quencher pairs are well known in the art and used
extensively for a variety of reporter probes (see, e.g., Yeung et
al., BioTechniques 36:266-75, 2004; Dubertret et al., Nat. Biotech.
19:365-70, 2001; and Tyagi et al., Nat. Biotech. 18:1191-96,
2000).
[0020] In this application, a statement that one sequence is the
same as, substantially the same as, complementary to, or
substantially complementary to another sequence encompasses
situations where both of the sequences are completely the same as,
substantially the same as, or complementary or substantially
complementary to one another, and situations where only a portion
of one of the sequences is the same as, substantially the same as,
complementary to, or substantially complementary to a portion or
the entire other sequence.
[0021] U.S. Pat. No. 6,605,451 (Marmaro and Gerdes); U.S. Patent
Application Publication Nos. 2004/0175733 A1 (Andersen and Ruff)
and 2005/0266418 A1 (Chen and Ridzon); U.S. application Ser. No.
10/944,153 (Lao and Straus); and Dolganov et al., Genome Research
11:1473-83, 2001 are incorporated by reference in their entirety
for any purpose.
[0022] The term "marker" as used herein refers to a nucleic acid
sequence, such as a gene, that is included in a panel because it is
differentially expressed in one or more sample types. The term
"marker amplicon" or "amplicon" refers to the nucleic acid sequence
that is being amplified and analyzed to aid in the identification
of the sample from which expressed RNA was obtained. In certain
embodiments, one can identify or tentatively identify a sample
based on the expression profile of the markers in the panel.
Typically, a mRNA and/or miRNA is amplified according to the
current teachings and its expression level is determined. By
comparing the expression level of at least some of the markers in a
panel of the current teachings, the sample can be identified. An
amplicon can be double-stranded, single-stranded, or partially
double-stranded and partially single-stranded, including without
limitation, the separated component strands obtained from a
double-stranded amplification product.
[0023] As used herein, the terms "polynucleotide",
"oligonucleotide", and "nucleic acid" are used interchangeably and
refer to single-stranded and double-stranded polymers of nucleotide
monomers, including 2'-deoxyribonucleotides (DNA) and
ribonucleotides (RNA) linked by internucleotide phosphodiester bond
linkages, or internucleotide analogs, and associated counter ions,
e.g., H.sup.+, NH.sub.4.sup.+, trialkylammonium, Mg.sup.2+,
Na.sup.+, and the like. A polynucleotide may be composed entirely
of deoxyribonucleotides, entirely of ribonucleotides, or chimeric
mixtures thereof. Polynucleotides typically range in size from a
few monomeric units, e.g. 5-40 when they are sometimes referred to
in the art as oligonucleotides, to several thousands of monomeric
nucleotide units. Unless denoted otherwise, whenever a
polynucleotide sequence is represented, it will be understood that
the nucleotides are in 5' to 3' order from left to right and that
"A" denotes deoxyadenosine, "C" denotes deoxycytosine or possibly
5-methyldeoxycytosine (5mC), "G" denotes deoxyguanosine, "T"
denotes thymidine, and "U" denotes deoxyuridine, unless otherwise
noted.
[0024] Certain oligonucleotides of the current teachings can be
synthesized using well-known chemical synthesis methods. Detailed
descriptions of such techniques can be found in, among other
places, Current Protocols in Nucleic Acid Chemistry, Beaucage et
al., eds., John Wiley & Sons, New York, N.Y., including updates
through May 2006; and Nucleic Acids in Chemistry And Biology, 2d
ed., Blackburn and Gait, eds., Oxford University Press, 1996.
Automated DNA synthesizers useful for synthesizing target regions
and primers are commercially available from numerous sources,
including for example, the Applied Biosystems DNA Synthesizer
Models 381A, 391, 392, and 394 (Applied Biosystems, Foster City,
Calif.). Oligonucleotides can also be generated biosynthetically,
using in vivo methodologies and/or in vitro methodologies that are
well known in the art. Descriptions of such technologies can be
found in, among other places, Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Press (1989) (hereinafter
"Sambrook et al."); and Current Protocols in Nucleic Acid
Chemistry, Beaucage et al., eds., John Wiley & Sons, including
updates through May 2006.
[0025] The term "primer" refers to a polynucleotide that
selectively hybridizes to a marker or to a corresponding
primer-binding site of an amplification product; and allows the
synthesis of a sequence complementary to the corresponding
polynucleotide template from its 3' end. In certain embodiments, a
looped linker primer, sometimes referred to as a linker probe, is
used as a reverse marker-specific primer, particularly when miRNA
markers are to be amplified.
[0026] A "marker-specific primer pair" of the current teachings
comprises a forward marker-specific primer and a corresponding
reverse marker-specific primer. In some embodiments and under
suitable conditions, a marker-specific primer pair allows for
exponential amplification of the marker and/or marker amplicon. In
certain embodiments, the reverse marker-specific primer is employed
in a reverse transcription to generate a cDNA copy of the
corresponding mRNA or corresponding miRNA in the sample. In certain
embodiments, at least one such primer of a primer pair comprises a
universal primer-binding site to allow for a universal primer to be
employed in subsequent amplifications. In some embodiments, the
forward target-specific primer and/or the reverse marker-specific
primer further comprises an upstream tail portion that serves as a
primer-binding site for an additional primer, for example but not
limited to, a universal primer. In certain embodiments, at least
one forward marker-specific primer, at least one reverse
marker-specific primer, or at least one forward marker-specific
primer and at least one reverse marker-specific primer further
comprises at least one of: a reporter probe-binding site, an
additional primer-binding site, and a reporter group, for example
but not limited to a fluorescent reporter group. In certain
embodiments, a forward primer and the corresponding reverse primer
of a marker-specific primer pair and/or a universal primer pair
have different melting temperatures (Tm) to permit
temperature-based asymmetric PCR.
[0027] A variety of methods are available for obtaining nucleic
acid from a sample. In certain embodiments, total nucleic acid,
total, RNA, mRNA, or small RNA (including without limitation miRNA)
are obtained. The method by which the desired nucleic acid
population or subpopulation is obtained from the sample is not a
limitation of the current teachings, provided that reasonably high
quality nucleic acid is obtained. Commercially available nucleic
acid extraction systems include, among others, the flashPAGE.TM.
Fractionation System (Ambion) and the ABI PRISM.RTM. 6100 Nucleic
Acid PrepStation and the ABI PRISM.RTM. 6700 Nucleic Acid Automated
Work Station (Applied Biosystems); and nucleic acid sample
preparation reagents and kits are also commercially available,
including, RiboPure.TM., RNAqueous.RTM., mirVana.TM. PARIS kit, and
mirVana.TM. miRNA Isolation kit (Ambion) and Total RNA Isolation
Chemistry Kit (P/N 4328773), ABI PRISM.RTM. TransPrep Chemistry
Reagents, and NucPrep.RTM. Chemistry Reagents (Applied
Biosystems).
[0028] The terms "amplifying" and "amplification" are used in a
broad sense and refer to any technique by which a target region, an
amplicon, or at least part of an amplicon, is reproduced or copied
(including the synthesis of a complementary strand), typically in a
template-dependent manner, including a broad range of techniques
for amplifying nucleic acid sequences, either linearly or
exponentially. Some non-limiting examples of amplification
techniques include primer extension, including the polymerase chain
reaction (PCR), RT-PCR, asynchronous PCR (A-PCR), and asymmetric
PCR, strand displacement amplification (SDA), multiple displacement
amplification (MDA), nucleic acid strand-based amplification
(NASBA), rolling circle amplification (RCA), transcription-mediated
amplification (TMA), and the like, including multiplex versions
and/or combinations thereof. Descriptions of certain amplification
techniques can be found in, among other places, Molecular Cloning,
A Laboratory Manual, Cold Spring Harbor Press, 3d ed., 2001
(hereinafter "Sambrook and Russell"); Sambrook et al.; Ausubel et
al.; PCR Primer: A Laboratory Manual, Diffenbach, Ed., Cold Spring
Harbor Press (1995); Msuih et al., J. Clin. Micro. 34:501-07
(1996); PCR The Basics, 2d ed., McPherson & Moller, Taylor
& Francis (2006; hereinafter "McPherson"); Nucleic Acid
Protocols Handbook, Rapley, Humana Press (2000), Totowa, N.J.
(hereinafter "Rapley"); U.S. Pat. Nos. 6,027,998 and 6,511,810; PCT
Publication Nos. WO 97/31256 and WO 01/92579; Ehrlich et al.,
Science 252:1643-50 (1991); Favis et al., Nature Biotechnology
18:561-64 (2000); Protocols & Applications Guide, rev. 9/04,
Promega, Madison, Wis.; and Rabenau et al., Infection 28:97-102
(2000).
[0029] In certain embodiments of the instant teachings, an
amplification reaction comprises at least one cycle of
amplification, for example, but not limited to, the steps of:
selectively hybridizing a primer to a marker sequence or an
amplicon (or complements of either, as appropriate); synthesizing a
strand of nucleotides in a template-dependent manner using a
polymerase; and denaturing the resulting nucleic acid duplex to
separate the strands. The cycle may or may not be repeated.
[0030] Amplification can comprise thermocycling or can be performed
isothermally. In some embodiments, amplifying comprises a
thermocycler, for example but not limited to a GeneAmp.RTM. PCR
System 9700, 9600, 2700, or 2400 thermocycler (all from Applied
Biosystems). In certain embodiments, single-stranded amplicons are
generated in an amplification reaction, for example but not limited
to asymmetric PCR or A-PCR.
[0031] Primer extension according to the present teachings is an
amplification process comprising elongating a primer that is
annealed to a template in the 5' to 3' direction using a suitable
polymerase. According to certain embodiments, with appropriate
buffers, salts, pH, temperature, and appropriate NTPs, a suitable
polymerase can incorporate nucleotides complementary to the
template strand starting at the 3'-end of an annealed primer, to
generate a complementary strand. In certain embodiments, a DNA
polymerase is used for primer extension lacks or substantially
lacks 5'-exonuclease activity, 3'-exonuclease activity, or both. In
some embodiments, primer extension comprises reverse transcription
and the DNA polymerase comprises a RNA-dependent DNA polymerase,
such as a reverse transcriptase, or a DNA-dependent DNA polymerase
that under certain conditions comprises reverse transcriptase
activity, for example but not limited to, Thermus thermophilus
(Tth) DNA polymerase, recombinant Tth DNA polymerase (rTth), or
Thermus species Z05 (TZ05) DNA polymerase (see, e.g., Smith et al.,
in PCR Primer, at pages 211-219). In certain embodiments, a reverse
transcriptase that exhibits DNA-dependent DNA polymerase activity
under suitable conditions, including but not limited to AMV reverse
transcriptase is employed. In certain embodiments, primer extension
comprises a RNA-dependent DNA polymerase and a DNA-dependent DNA
polymerase. Descriptions of certain primer extension reactions can
be found in, among other places, Sambrook et al., Sambrook and
Russell, and Ausubel et al.
[0032] In certain embodiments, an amplification reaction comprises
multiplex amplification, in which a multiplicity of different
markers, a multiplicity of different marker amplicons, or both, are
simultaneously amplified using a multiplicity of different primer
pairs (see, e.g., Henegariu et al., BioTechniques 23:504-11, 1997;
and Rapley, particularly in Chapter 79). Certain embodiments of the
disclosed methods comprise a multiplex amplification reaction and a
single-plex amplification reaction, including a multiplicity of
single-plex reactions performed in parallel (see, e.g., U.S. Pat.
No. 6,605,451 (Marmaro and Gerdes); U.S. Patent Application
Publication No. 2004/0175733 A1 (Andersen and Ruff); and Dolganov
et al., Genome Research 11:1473-83, 2001).
[0033] In certain embodiments, an amplifying reaction comprises
asymmetric PCR. According to certain embodiments, asymmetric PCR
comprises an amplification composition comprising (i) at least one
primer pair in which there is an excess of one primer, relative to
the corresponding primer of the primer pair, for example but not
limited to a five-fold, a ten-fold, or a twenty-fold excess; (ii)
at least one primer pair that comprises only a forward primer or
only a reverse primer; (iii) at least one primer pair that, during
given amplification conditions, comprises a primer that results in
amplification of one strand and a corresponding primer that is
disabled; or (iv) at least one primer pair that meets the
description of both (i) and (iii) above. Consequently, when the
marker or an amplification product is amplified, an excess of one
strand of the subsequent amplification product (relative to its
complement) is generated. Descriptions of asymmetric PCR, can be
found in, among other places, McPherson, particularly in Chapter 5;
and Rapley, particularly in Chapter 64.
[0034] In certain embodiments, one may use at least one primer pair
wherein the melting temperature (Tm.sub.50) of one of the primers
is higher than the Tm.sub.50 of the other primer, sometimes
referred to as A-PCR (see, e.g., Published U.S. Patent Application
No. US 2003-0207266 A1). In certain embodiments, the Tm.sub.50 of
the forward primer is at least 4-15.degree. C. different from the
Tm.sub.50 of the corresponding reverse primer. In certain
embodiments, the Tm.sub.50 of the forward primer is at least
8-15.degree. C. different from the Tm.sub.50 of the corresponding
reverse primer. In certain embodiments, the Tm.sub.50 of the
forward primer is at least 10-15.degree. C. different from the
Tm.sub.50 of the corresponding reverse primer. In certain
embodiments, the Tm.sub.50 of the forward primer is at least
10-12.degree. C. different from the Tm.sub.50 of the corresponding
reverse primer. In certain embodiments, in at least one primer
pair, the Tm.sub.50 of a forward primer differs from the Tm.sub.50
of the corresponding reverse primer by at least about 4.degree. C.,
by at least about 8.degree. C., by at least about 10.degree. C., or
by at least about 12.degree. C.
[0035] In certain embodiments of A-PCR, in addition to the
difference in Tm.sub.50 of the primers in a primer pair, there is
also an excess of one primer relative to the other primer in the
primer pair. In certain embodiments, there is a five- to
twenty-fold excess of one primer relative to the other primer in
the primer pair. In certain embodiments of A-PCR, the primer
concentration is at least 50 nM.
[0036] In A-PCR according to certain embodiments, one may use
conventional PCR in the first cycles of amplification such that
both primers anneal and both strands of a double-stranded amplicon
are amplified. By raising the temperature in subsequent cycles of
the same amplification reaction, however, one may disable the
primer with the lower T.sub.m such that only one strand is
amplified. Thus, the subsequent cycles of A-PCR in which the primer
with the lower T.sub.m is disabled result in asymmetric
amplification. Consequently, when the target region or an
amplification product is amplified, an excess of one strand of the
subsequent amplification product (relative to its complement) is
generated.
[0037] According to certain embodiments of A-PCR, the level of
amplification can be controlled by changing the number of cycles
during the first phase of conventional PCR cycling. In such
embodiments, by changing the number of initial conventional cycles,
one may vary the amount of the double-stranded amplification
products that are subjected to the subsequent cycles of PCR at the
higher temperature in which the primer with the lower T.sub.m is
disabled.
[0038] Certain methods of optimizing amplification reactions are
known to those skilled in the art. For example, it is known that
PCR may be optimized by altering times and temperatures for
annealing, polymerization, and denaturing, as well as changing the
buffers, salts, and other reagents in the reaction composition.
Optimization may also be affected by the design of the primers
used. For example, the length of the primers, as well as the
G-C:A-T ratio may alter the efficiency of primer annealing, thus
altering the amplification reaction. Descriptions of amplification
optimization can be found in, among other places, James G. Wetmur,
"Nucleic Acid Hybrids, Formation and Structure," in Molecular
Biology and Biotechnology, pp. 605-8, (Robert A. Meyers ed., 1995);
McPherson, particularly in Chapter 4; Rapley; and Protocols &
Applications Guide, rev. 9/04, Promega.
[0039] In some embodiments, unincorporated primers, unincorporated
dNTPs, amplification reagents, or combinations thereof, are
separated from an amplification product by, for example but not
limited to, gel or column purification, sedimentation, filtration,
beads, including streptavidin-coated beads, magnetic separation, or
hybridization-based pull out, including annealing amplification
products comprising hybridization tags to a solid support. A number
of kits and reagents for performing such separation techniques are
commercially available, including the Wizard.RTM. MagneSil.TM. PCR
Clean-Up System (Promega), the MinElute PCR Purification Kit, the
QIAquick Gel Extraction Kit, the QIAquick Nucleotide Removal Kit,
the QIAquick 96 PCR Purification Kit or BioRobot Kit (all from
Qiagen, Valencia, Calif.), Dynabeads.RTM. (Dynal Biotech), or the
ABI PRISM.RTM. Duplex.TM. 384 Well F/R Sequence Capture Kit
(Applied Biosystems P/N 4308082). In some embodiments, an
amplification product is not purified prior to a subsequent
amplifying reaction.
[0040] In certain embodiments, the disclosed methods and kits
comprise a solid support. Non-limiting examples of solid supports
include, agarose, sepharose, polystyrene, polyacrylamide, glass,
membranes, silica, semiconductor materials, silicon, organic
polymers; optically identifiable micro-cylinders; biosensors
comprising transducers; appropriately treated or coated reaction
vessels and surfaces, for example but not limited to, micro
centrifuge or reaction tubes, wells of a multiwell microplate, and
glass, quartz or plastic slides and/or cover slips; and beads, for
example but not limited to magnetic beads, paramagnetic beads,
polymer beads, metallic beads, dye-impregnated or labeled beads,
coated beads, glass beads, microspheres and nanospheres. In some
embodiments, a solid support is used in a separating and/or
detecting step, for example but not limited to, for purifying
and/or analyzing amplification products. Those in the art will
appreciate that any number of solid supports may be employed in the
disclosed methods and kits and that the shape and composition of
the solid support is generally not limiting. It is to be
appreciated that a solid support may be porous or non-porous, and
may have a smooth or even surface or an irregular or uneven
surface. In certain embodiments, a solid support comprises a
microarray, a bead array, or a bead for use in a flow cytometric
assay (see, e.g., Shingara et al., RNA 11:1461-70, 2005: Lu et al.,
Nature 435(9):834-38, 2005). A variety of exemplary arrays and
bead-based systems for use in the current teachings are
commercially available from, among other sources, Applied
Biosystems; Ambion, Austin, Tex.; Illumina, San Diego, Calif.;
Affymetrix, Sunnyvale, Calif.; and Luminex, Austin, Tex.
[0041] In some embodiments, the methods of the current teachings
comprise a Q-PCR reaction. The term "quantitative PCR", or "Q-PCR"
refers to a variety of methods used to quantify the results of the
polymerase chain reaction for specific nucleic acid sequences. Such
methods typically are categorized as kinetics-based systems, that
generally determine or compare the amplification factor, such as
determining the threshold cycle (C.sub.t), or as co-amplification
methods, that generally compare the amount of product generated
from simultaneous amplification of target and standard templates.
Many Q-PCR techniques comprise reporter probes, nucleic acid dyes,
or both (see, e.g., Kubista et al., Mol. Aspects. of Med. 27
(2-3):95-125 (2006)). For example but not limited to TaqMan.RTM.
probes (Applied Biosystems), i-probes, molecular beacons, Eclipse
probes, scorpion primers, Lux.TM. primers, FRET primers, ethidium
bromide, SYBR.RTM. Green I (Molecular Probes), ethidium bromide,
BOXTO (TATAA Biocenter AB), and PicoGreen.RTM. (Molecular
Probes).
[0042] In some embodiments, the disclosed methods and kits comprise
a microfluidics device, "lab on a chip", or micrototal analytical
system (.mu.TAS). In some embodiments, sample preparation is
performed using a microfluidics device. In some embodiments, an
amplification reaction is performed using a microfluidics device.
In some embodiments, a Q-PCR reaction is performed using a
microfluidic device, for example but not limited to a TaqMan.RTM.
Low Density Array card (Applied Biosystems). Descriptions of
exemplary microfluidic devices can be found in, among other places,
Published PCT Application Nos. WO/0185341 and WO 04/011666;
Kartalov and Quake, Nucl. Acids Res. 32:2873-79, 2004; and Fiorini
and Chiu, BioTechniques 38:429-46, 2005.
[0043] A "panel of markers" or "panel" is a select group of genes
that are differentially expressed in various tissues or that are
present in characteristic levels in specific body fluids, such that
by determining the mRNA and/or miRNA expression profile of a tissue
or body fluid sample for at least some of the markers in the panel,
one can determine the identity of the sample, for example but not
limited to, saliva, blood, brain, or muscle. Those in the art will
understand that different tissues, cell types, and body fluids have
defined sets of highly expressed or uniquely expressed genes and
instructed by the current teachings, will appreciate that these
distinctive expression patterns can be useful in identifying the
tissue or body fluid from which the nucleic acid was obtained. It
will also be appreciated by those in the art that additional
markers for inclusion in such panels can be identified by routine
expression analysis methods, wherein the expression profiles of
various tissues or body fluids are compared with each other.
Exemplary panels of markers useful for identifying human samples
are shown in Tables 1-3.
[0044] In certain embodiments, an amplification reaction comprises
multiplex amplification, in which a multiplicity of different
target nucleic acids and/or a multiplicity of different
amplification product species are simultaneously amplified using a
multiplicity of different primer pairs. Certain embodiments of the
disclosed methods comprise a two step amplification comprising a
multiplex amplification reaction and a single-plex amplification
reaction, including a multiplicity of single-plex or lower-plexy
reactions (for example but not limited to a two-plex, a three-plex,
a four-plex, a five-plex, or a six-plex reaction) performed in
parallel. In certain embodiments, the multiplex first amplification
reaction comprises a reverse transcription reaction or a reverse
transcription reaction is performed followed by a multiplex first
amplification reaction. In certain embodiments, a multiplex first
amplification reaction is performed for a limited number of cycles,
for example but not limited to 5 cycles, 6 cycles, 7 cycles, 8
cycles, 9 cycles, 10 cycles, 11 cycles, 12 cycles, 13 cycles, 14
cycles, 15 cycles, 16 cycles, 17 cycles, 18 cycles, 19 cycles, 20
cycles, 21 cycles, 22 cycles, 23 cycles, 24 cycles, or 25 cycles.
In certain embodiments, a parallel single-plex second amplification
reaction comprises a microfluidics device, for example but not
limited to a TaqMan.RTM. Low Density Array (Applied
Biosystems).
[0045] In certain embodiments, one or more markers are amplified,
for example by the reverse-transcription polymerase chain reaction
("RT-PCR"), using a plurality of amplification primer pairs, each
of which is suitable for amplifying a different marker and/or
marker amplicon. Because a plurality of different marker sequences
are amplified simultaneously in a single reaction, the multiplex
amplifications may be used in a variety of contexts to effectively
increase the concentration or quantity of a sample available for
downstream analyses and/or assays. Once the sample has been
multiplex amplified according to the methods described herein, it
may be divided into aliquots, with or without prior dilution, for
subsequent analyses. Owing to its increased concentration and
quantity, significantly more analyses or assays can be performed
with the multiplex amplified sample than could have been performed
with the original sample. In many embodiments, multiplex
amplification even permits the ability to perform assays or
analyses that require more sample, or a higher concentration of
sample, than was originally available. For example, after a
1000.times. multiplex amplification, subsequent assays could then
be performed at 1000.times. less sample volume.
[0046] Certain embodiments of the instant methods comprise an RT
reaction followed by a two-step amplification reaction. In a first
step, a plurality of different target sequences in a panel of
markers are multiplex amplified by PCR in the presence of a
plurality of different amplification primer pairs or sets,
generating a plurality of different first amplification products.
In certain embodiments, in a second step the diluted or undiluted
multiplex amplification product is divided into a plurality of
reaction vessels, one of the first amplification products in each
vessel is single-plex amplified in the presence of a set of
amplification primers for amplifying that first amplification
product (such as a marker amplicon) and the single-plex
amplifications monitored for the accumulation of second
amplification product. In certain such embodiments, the second
amplification reaction further comprises a reporter probe, a
nucleic acid dye, a reference dye, or combinations thereof. In
certain embodiments, a small number of first amplification products
in each vessel, e.g., 2, 3, 4, 5, or 6 different first
amplification products, are amplified in the presence of a set of
amplification primers suitable for amplifying each of the desired
first amplification products and the amplifications monitored for
the accumulation of second amplification products.
[0047] The accumulation of single-plex amplification product can be
monitored at the end of the reaction by conventional means, e.g.,
by chromatography, by electrophoresis, by binding a nucleic acid
dye, or by binding certain reporter probes. Alternatively, the
accumulation of single-plex amplification product can be monitored
as a function of time using well known methods, such as carrying
out the single-plex amplification in the presence of one or more
dyes or labels capable of producing a detectable signal upon
binding double-stranded polynucleotide (e.g., SYBR.RTM. Green I or
II, SYBR.RTM. Gold, ethidium bromide, or YO-PRO-1; Molecular
Probes, Eugene, Oreg.) or an oligonucleotide probe labeled with a
suitable labeling system (e.g. a TaqMan.RTM. probe, or other
suitable detector probe). The accumulation of a small number of
different second amplification products (e.g., 2, 3, 4, 5, or 6) in
the same reaction well or chamber can be monitored as a function of
time using a reporter probe for each of the second amplification
products, wherein each of the different reporter probes will, under
suitable conditions, emit a fluorescent wavelength that can be
distinguished from the other reporter probes.
Certain Exemplary Kits
[0048] The instant teachings also provide kits designed to expedite
performing certain of the disclosed methods. Kits may serve to
expedite the performance of certain disclosed methods by assembling
two or more components required for carrying out the methods. In
certain embodiments, kits contain components in pre-measured unit
amounts to minimize the need for measurements by end-users. In some
embodiments, kits include instructions for performing one or more
of the disclosed methods. Preferably, the kit components are
optimized to operate in conjunction with one another.
[0049] In certain embodiments, kits comprise a first DNA
polymerase, a multiplicity of different primer pairs, wherein each
of the different primer pairs is designed to amplify a target
sequence of one marker of a panel of markers or a control sequence.
For example but not as a limitation, a kit designed to amplify a
panel of six different markers would comprise at least six
different primer pairs, at least one for each of the six markers.
In certain embodiments, kits further comprise a second DNA
polymerase. In some embodiments, kits comprise an amplification
primer pair. In some embodiments, a primer pair comprises a linker
probe (see Chen and Ridzon, U.S. Patent Application Publication No.
2005/0266418 A1) a forward primer, a reverse primer, any of which
in some embodiments comprise a universal priming sequence or the
complement of a universal priming sequence. In certain embodiments,
kits comprise at least one universal primer. In some embodiments,
kits comprise a forward primer, a reverse primer, or a forward
primer and a reverse primer that further comprises a reporter
group. In some such embodiments, the reporter group of a forward
primer of a primer pair is different from the reporter group of the
reverse primer of the primer pair. In some embodiments, kits
further comprise at least one of: a reverse transcriptase; a
reporter probe; a nucleic acid dye; a reporter group; a control
sequence, for example but not limited to an internal positive
control, such as a housekeeping gene; and a polynucleotide ladder
comprising molecular size or weight standards. In certain
embodiments, kits comprise at least one reporter probe for each
amplicon derived from a marker on the panel being evaluated.
III. EXEMPLARY EMBODIMENTS
Panels of Markers
[0050] The methods of the current teachings employ primer pairs
corresponding to markers from specific panels of markers to
generate appropriate gene expression profiles which are compared to
the expression profile for those markers from known tissues and
body fluids to identify the sample. The markers are typically
selected for inclusion in a particular panel because they represent
genes that are differentially expressed in a tissue or body fluid
of interest, for example but not limited to, a certain tissue or
body fluid that may be encountered at a crime scene. The panels of
the current teachings comprise mRNA markers or miRNA markers.
Certain panels of the current teachings comprise at least one mRNA
markers and at least one miRNA marker. In some embodiments, the
disclosed panels further comprise control markers such as
housekeeping genes or other internal control sequences.
[0051] An illustrative panel of the current teachings comprises:
(a) at least one marker for saliva, wherein the at least one saliva
marker comprises at least one of PRB4, PRB1, STATH, PRB3, and HTN3;
(b) at least one marker for semen, wherein the at least one semen
marker comprises at least one of SEMG1, SEMG2, TGM4, MCSP, PRM1,
and PRM2; (c) at least one marker for saliva containing mucus,
wherein the at least one saliva containing mucus marker comprises
at least one of PRB1, PRB3, and PRB4; and (d) at least one marker
for vaginal secretions, wherein the at least one vaginal secretion
marker comprises at least one of MUC4 and ESR1. In certain
embodiments, the panel further comprises: (e) at least one marker
for blood, wherein the at least one blood marker comprises at least
one of ANK1, SPTB, and PBGD; (f) at least one marker for menstrual
blood, wherein the at least one menstrual blood marker comprises
MMP11; or (g) at least one marker for blood, wherein the at least
one blood marker comprises at least one of ANK1, SPTB, and PBGD;
and at least one marker for menstrual blood, wherein the at least
one menstrual blood marker comprises MMP11.
[0052] In certain embodiments, a panel of markers comprises PRB4,
PRB1, STATH, SEMG1, SEMG2, TGM4, MCSP, ANK1, SPTB, PBGD, MUC4,
ESR1, and MMP11. In some embodiments, the panel further comprises
at least one of: PRB3, HTN3, PRM1, PRM2, PRB1, and PRB3.
[0053] Certain mRNA marker panels of the current teachings comprise
groups of markers comprise: (a) hCG1816257; (b) MMP11; (c) at least
one of UGT1A8, hCG2017793, APCS, and FGB; (d) at least one of
PNLIPRP1, REG1B, hCG2042161, and PRSS3; (e) TGM4; (f) FLJ46026; (g)
at least one of TH, FDX1, and QPCT; (h) at least one of hCG2040797
and KLK3; (i) at least one of PRM1, PRM2, MGC42718, SPA 17, and
MCSP; (j) at least one of PRB1, PRB3, and PRB4; (k) LGALS4; (l) at
least one of PGA5 and PGC; (m) at least one of SEMG1, SEMG2, and
CYSLTR2; (n) at least one of ANKRD1 and hCG1813636.1; (o) ANK1; (p)
TNNI1; and (q) PMP2. Certain panel embodiments comprise: at least
one marker from five of these groups (i.e., groups (a) through (q)
above); at least one marker from six of these groups; at least one
marker from seven of these groups; at least one marker from eight
of these groups; at least one marker from nine of these groups; at
least one marker from ten of these groups; at least one marker from
eleven of these groups; at least one marker from twelve of these
groups; at least one marker from thirteen of these groups; at least
one marker from fourteen of these groups; at least one marker from
fifteen of these groups; at least one marker from sixteen of these
groups; or at least one marker from each of these groups.
[0054] Certain miRNA marker panels comprise: at least brain marker
comprising at least one of miR-125b, miR-128a, miR-128b, miR-129,
miR-135, and miR-153; at least one muscle marker comprising at
least one of miR-1d, miR-133a, miR-133b, miR-296, miR-208; at least
one kidney marker comprising at least one of miR-192, miR-204,
miR-215, and miR-216; at least one thymus marker comprising at
least one of miR-96 and miR-182; at least one testes marker
comprising at least one of miR-10b and let-7e; at least one
placenta marker comprising at least one of miR-141 and miR-23a; or
combinations thereof.
[0055] Some panel embodiments of the current teachings comprise at
least one mRNA marker and at least one miRNA marker selected from
Tables 1 and 3; Tables 2 and 3; or Tables 1, 2, and 3. Certain
marker panel embodiments comprise a multiplicity of markers
selected form Table 1, a multiplicity of markers selected form
Table 2, or a multiplicity of markers selected form Table 3.
[0056] Exemplary Method Embodiments.
[0057] According to certain methods of the current teachings, the
nucleic acid from a forensic sample is obtained using any suitable
method known in the art. The nucleic acid is combined with a DNA
polymerase and a set of different marker-specific primers, where
each marker-specific primer is designed to amplify one of the
markers in the panel of interest, to form a reaction composition.
In certain embodiments, the reaction composition further comprises
a linker probe. For illustration purposes but not as a limitation,
the set of marker-specific primers for a panel comprising fifteen
different markers would include at least fifteen different primers.
Typically, the set of different marker-specific primers comprises a
set of different marker-specific primer pairs, each comprising a
forward and a reverse marker-specific primer. In some embodiments,
the DNA polymerase comprises a DNA-dependent DNA polymerase and/or
an RNA-dependent DNA polymerase, for example, a reverse
transcriptase. In some embodiments, the DNA polymerase comprises a
DNA-dependent DNA polymerase that, under certain conditions,
polymerizes reverse transcription, for example but not limited to
Tth DNA polymerase. In yet other embodiments, the DNA polymerase
comprises a RNA-dependent DNA polymerase with both reverse
transcription and DNA-dependent DNA polymerase activity, for
example but not limited to, certain retroviral reverse
transcriptases such as AMV RT.
[0058] The reaction composition is incubated under conditions
suitable for reverse transcription to occur and cDNA is generated.
The cDNA is amplified by PCR to generate an expression profile. In
some embodiments, the gene expression profile is generated using
conventional RT-PCR techniques, including without limitation,
qRT-PCR techniques known in the art. In certain embodiments, the
PCR comprises a two step reaction, for example but not limited to,
the method of Anderson and Ruff (U.S. Patent Application
Publication No. US 2004/0175733 A1). The expression profile is
compared to known expression profiles obtained from know (control)
body fluids and tissues. Based on this comparison, the sample can
be identified.
[0059] In one illustrative method, a body fluid stain from a crime
scene is rehydrated and suspended in an appropriate buffer. Total
RNA is extracted from the rehydrated body fluid and combined in a
reaction mixture comprising rTth DNA polymerase, a mix of dNTPs,
Mn(OAc).sub.2, and the following ten primer pairs: Hs00864002_m1,
Hs00818764_m1, Hs00162389, Hs00268141_m1, Hs00268143_m1,
Hs00165820_m1, Hs00609297_m1, Hs00366414_m1, Hs00174860_m1, and
Hs00171829_m1 (available from Applied Biosystems). These primer
pairs correspond to a panel of body fluid markers comprising
markers for saliva, semen, blood, vaginal secretion and menstrual
blood (see, e.g., Table 1). The reaction mixture is incubated under
conditions suitable for cDNA to be produced, then the reaction
mixture is thermocycled for twenty cycles to generate marker
amplicons. The thermocycled reaction mixture is diluted ten-fold,
then divided into ten wells of a 384 well micro-card, wherein each
such well comprises a reporter probe designed to anneal with one of
the ten marker amplicons and the corresponding primer pair for
amplifying that marker amplicon. The plate is loaded into an
Applied Biosystems 7900HT Fast Real-Time PCR System and
thermocycled. The single-plex amplifications are monitored in
real-time for the accumulation of amplification products to
generate an expression profile for this ten marker panel. The
resulting expression profile is compared with the expression
profiles obtained for saliva, semen, blood, vaginal secretion, and
menstrual blood to determine whether the sample can be
identified.
[0060] In another exemplary method embodiment, the small RNA is
obtained from a tissue sample obtained from a crime scene using a
mirVana miRNA Isolation Kit (Ambion). The isolated RNA is combined
in a reaction mixture comprising MultiScribe.TM. reverse
transcriptase (Applied Biosystems), Ampli-Taq Gold DNA polymerase,
a mix of dNTPs, and twelve primer pairs comprising a linker probes
and corresponding forward primer for each marker of a human miRNA
panel selected from Table 3, consisting of hsa-mir-129,
hsa-mir-135, hsa-mir-1d, hsa-mir-133b, hsa-mir-296, hsa-mir-192,
hsa-mir215, hsa-mir-376, hsa-mir-148, hsa-mir-208, hsa-mir-182, and
hsa-mir-10b (see miRBase miRNA database at the http site:
microrna.sanger/ac/uk for miRNA sequences). Alternatively, primer
sets comprising unconventionally short target-binding portions
(see, Lao and Straus, U.S. patent application Ser. No. 10/944,153)
designed to amplify the markers of this exemplary miRNA panel can
be used in the reaction mixture in place of the primer pairs
comprising linker probes, described above. The reaction composition
is incubated under conditions suitable for generating complementary
DNA and then a first PCR amplification step is performed for
twenty-five cycles.
[0061] The reaction mixture is diluted and divided into twelve
aliquots that are transferred into wells of a 96-well plate that
each contain a primer pair suitable for amplifying one marker
amplicon and the nucleic acid dye Sybr.RTM. Green I. The plate is
thermocycled and the accumulation of single-plex amplification is
monitored after each cycle to generate an expression profile for
the exemplary twelve marker miRNA panel. The resulting expression
profile is compared with the expression profiles obtained with
brain, muscle, kidney, pancreas, liver, heart, thymus, and testes,
to determine whether the tissue sample can be identified.
[0062] Those in the art will appreciate that these two exemplary
methods of the current teachings illustrate the use of marker
panels to identify a forensic sample based on its gene expression
profile relative to corresponding expression profiles from known
tissues and body fluids.
[0063] Exemplary Kit Embodiments.
[0064] The instant teachings also provide kits designed to expedite
performing the subject methods. Kits serve to expedite the
performance of the methods of interest by assembling two or more
components required for carrying out the methods. Kits preferably
contain components in pre-measured unit amounts to minimize the
need for measurements by end-users. Kits preferably include
instructions for performing one or more of the disclosed methods.
Preferably, the kit components are optimized to operate in
conjunction with one another.
[0065] The instant kits comprise at least one primer pair for each
marker in a panel of markers, a DNA polymerase, a dNTP mixture, and
optionally a polymerase-compatible source of manganese ions.
Certain kits further comprise a reporter probe species for each
marker amplicon, a reaction vessel, and a nucleic acid dye.
[0066] The current teachings, having been described above, may be
better understood by reference to examples. The following examples
are intended for illustration purposes only, and should not be
construed as limiting the scope of the teachings herein in any
way.
Example 1
Exemplary Nucleic Isolation Technique
[0067] Nucleic acid is isolated from a sample using PrepMan
chemistry (Applied Biosystems) and a spin column comprising a
membrane as follows. Ten to thirty microliters (.mu.L) of liquid
sample (e.g., saliva or blood) is combined with reaction mix to a
final volume of 100 .mu.L, where the reaction mix includes 50 .mu.L
2.times.LYS buffer (20 mM Tris (pH 7.4), 2 mM EDTA (pH 8.0), 5%
Triton X-100, 150 mM NaCl), 4.0-24.0 .mu.L DEPC treated water
(Ambion, Austin Tex.; combined water and sample volume equals 34.0
.mu.L), 3 .mu.L PolyA RNA (200 ng/.mu.L, Sigma-Aldrich, St. Louis,
Mo.), and 8 .mu.L glycogen (5 .mu.g/.mu.l, Ambion) in a 1.5 mL
MicroAmp tube (Applied Biosystems). To this suspension is added 100
.mu.L 2.times.ncRNA buffer (Applied Biosystems) and the tube is
mixed by vortexing on a Baxter Scientific vortex for approximately
one minute. Optionally, the solution is passed through a 20-gauge
(or higher) needle (0.9 mm diameter) fitted to a sterile syringe.
The sample is incubated for ten minutes at room temperature, then
400 .mu.L of freshly prepared DNA precipitation solution (100 .mu.L
DNA precipitation solution 1 mixed with 300 .mu.L DNA precipitation
solution 2 (Applied Biosystems P/Ns 4325962 and 4325964,
respectively) is added to the tube.
[0068] A 1.times.ncRNA buffer solution is prepared by combining 300
.mu.L of 2.times.ncRNA buffer with an equal volume of DNA
precipitation solution 2. A Whatman QMA membrane (part no. 1851047)
is cut to size and placed into the inlet side of a spin column
(Micro Bio-Spin chromatography column, Bio-Rad, Hercules Calif.).
The spin column is placed in a 2 mL collection tube and the
membrane is pre-wet with 40 .mu.L of the 1.times.ncRNA lysis
buffer. The sample, including any precipitate that may have formed,
is placed in the top of the spin column assembly. The column is
centrifuged for 30 seconds at about 8000.times.g to remove most of
the liquid. The column containing nucleic acid from the sample is
sequentially washed with 500 .mu.L 1.times.ncRNA lysis buffer, 600
.mu.L DNA wash solution 1 (Applied Biosystems P/N 4325958), and
twice with 600 .mu.L DNA wash solution 2 (Applied Biosystems P/N
4325960), with a 30 second centrifugation at about 8000.times.g
between each wash step. The column is then centrifuged at about
10000.times.g for one minute.
[0069] The nucleic acid is eluted from the membrane by adding 30
.mu.L elution solution (Applied Biosystems P/N 4305893) to the spin
column, incubating for three minutes, centrifuging for thirty
seconds at about 10000.times.g, and collecting the first eluate.
The first eluate is placed back into the column and the procedure
is repeated. The second eluate is used for nucleic acid analysis,
including without limitation expression profiling. Those in the art
will appreciate that while the foregoing example provides a method
for isolating nucleic acid from a sample, a number of alternative
nucleic acid isolation methods can be effectively employed in the
disclosed methods, including but not limited to a variety of
commercially available kits and reagents from among other sources,
Ambion, Applied Biosystems, Qiagen, Stratagene, and Promega.
Example 2
Exemplary mRNA Profile Generation
[0070] A 2.times.RT master mix is prepared using the components of
the High-Capacity cDNA Archive Kit (Applied Biosystems). For each
sample to be evaluated, the master mix contains 3 .mu.L 10.times.
Reverse Transcription Buffer, 1.2 .mu.L 25.times.dNTPs, 3 .mu.L
random primers, 1.5 .mu.L MultiScribe.TM. Reverse Transcriptase(50
U/.mu.L), and 6.3 .mu.L nuclease-free water (15 .mu.L for each
sample). A 15 .mu.L volume of the 2.times.RT master mix and 15
.mu.L of the isolated nucleic acid from Example 1 are combined in a
well of 96 well reaction plate (or a MicroAmp tube, as
appropriate). The plate is sealed with adhesive film and briefly
centrifuged. The plate is loaded onto a GeneAmp.RTM. PCR System
9700 thermal cycler, the cycling conditions are set at 25.degree.
C. for ten minutes, 37.degree. C. for two hours, and then 4.degree.
C., and the reaction mixture in the plate is thermocycled to
generate cDNA.
[0071] A pool of primers and corresponding nuclease probes for each
marker in the panel to be evaluated is prepared by combining 5
.mu.L of at least one Assay on Demand TaqMan.RTM. Gene Expression
Assay (AOD, Applied Biosystems; each AOD assay comprising a
dye-labeled TaqMan.RTM. MGB probe and a corresponding primer pair
pre-designed for the marker) for each marker in the panel. The pool
is then diluted with nuclease-free water so that each of the AODs
is effectively diluted 1:50; for illustration purposes, an
exemplary marker panel includes twenty different AODs in a pooled
volume of 100 .mu.L (20 AODs.times.5 .mu.L/AOD), which is diluted
to a final volume of 250 .mu.L. For each cDNA to be pre-amplified,
a pre-amplification reaction mix is formed by combining 25 .mu.L
TaqMan.RTM. Universal PCR Master Mix (P/N 4304437), 2.5 .mu.L
AmpliTaq Gold DNA polymerase (5 U/.mu.L; P/N 4311816), 5 .mu.L of
the diluted AOD pool, and 15 .mu.L nuclease free water (final
volume 47.5 .mu.L per cDNA).
[0072] A 47.5 .mu.L volume of the pre-amplification mix and 2.5
.mu.L of the previously generated cDNA are combined in a well of 96
well reaction plate. The plate is sealed with adhesive film, then
the plate is vortexed for about ten seconds and briefly
centrifuged. The plate is loaded onto a GeneAmp.RTM. PCR System
9700 thermal cycler (Applied Biosystems), the cycling conditions
are set at 50.degree. C. for two minutes, 95.degree. C. for ten
minutes, 10-15 cycles of 95.degree. C. for fifteen seconds and
60.degree. C. for four minutes, and then 4.degree. C. The
pre-amplification mixture in the plate is thermocycled to generate
pre-amplified PCR product.
[0073] For each marker in the panel, a real-time PCR reaction is
performed in wells of a 96 well. For illustration purposes, for a
ten marker panel, each pre-amplified PCR product would be analyzed
in ten different wells or the plate, each containing a different
AOD corresponding to one of the markers of the panel. Controls, for
example certain housekeeping genes such as GAPDH or .beta.-actin
may be included in additional wells, as desired. To each well of
the reaction plate is added 25 .mu.L TaqMan.RTM. Universal PCR
Master Mix, No AmpErase.RTM. UNG (P/N 4324018), 2.5 .mu.L of the
appropriate AOD, 20 .mu.L nuclease free water, and 2.5 .mu.L of the
pre-amplification product. The plate is sealed with adhesive film,
then vortexed for about ten seconds and briefly centrifuged. Ten
microliters of each of these reaction mixtures is robotically
transferred (Beckman Multimek) to a corresponding well in each of
the four quadrants of a 384 well plate, the plate is sealed with an
adhesive film and briefly centrifuged. A real time PCR reaction is
performed using an Applied Biosystems 7900HT Real-Time PCR System
with a thermocycling profile of 50.degree. C. for two minutes,
95.degree. C. for ten minutes, forty cycles of 95.degree. C. for
fifteen seconds and 60.degree. C. for one minute, and the integral
data collection software determines the Ct values for each well.
After the run has completed, the Ct data is extracted from the
instrument, the .DELTA.Ct values are calculated, and the expression
profile of the marker panel is generated for each sample that was
evaluated. Based on the expression profile for a given sample, the
identity of that sample is determined.
[0074] Those in the art will appreciate that a pre-amplification
may not be necessary if the sample contains a sufficiently high
marker copy number and that the nucleic acid isolated from such
samples may be directly analyzed by real-time PCR to generate an
expression profile for the marker panel. Those in the art will also
appreciate that different mRNA and miRNA expression profiling
techniques can be employed, for example but not limited to array
analyses, including without limitation microarrays, bead arrays,
and filter arrays (see, e.g., Shingara et al., RNA 11:1461-70
(2005); Castoldi et al., RNA 12:1-8 (2006); Sioud et al.,
BioTechniques 37:574-80 (2004); and Lu et al., Nature 435:834-38
(2005).
[0075] The methods, panels of markers, and kits of the current
teachings have been described broadly and generically herein. Each
of the narrower species and sub-generic groupings falling within
the generic disclosure also form part of the current teachings.
This includes the generic description of the current teachings with
a proviso or negative limitation removing any subject matter from
the genus, regardless of whether or not the excised material is
specifically recited herein.
[0076] Although the disclosed teachings have been described with
reference to various applications, methods, and compositions, it
will be appreciated that various changes and modifications may be
made without departing from the teachings herein. The foregoing
examples are provided to better illustrate the present teachings
and are not intended to limit the scope of the teachings herein.
Certain aspects of the present teachings may be further understood
in light of the following claims.
TABLE-US-00001 TABLE 1 Human Body Fluid Identification Panel (mRNA)
Source Symbol Name AOD No..sup.1 Saliva PRB3 proline-rich protein
BstNI Hs00818925_m1 subfamily 3 PRB4 proline-rich protein BstNI
Hs00864002_m1 subfamily 4 HTN3 histatin3 HS00264790 PRB1
proline-rich protein BstNI Hs00818764_m1 subfamily 1 STATH
statherin Hs00162389 Semen SEMG1 semenogelin I Hs00268141_m1 SEMG2
semenogelin II Hs00268143_m1 TGM4 transglutaminase 4 (prostate)
Hs00162710_m1 PRM1 Protamine 1 Hs00358158_m1 PRM2 Protamine 2
Hs00172518_m1 MCSP mitochondrial capsule Hs00229076_m1
selenoprotein Blood ANK1 ankyrin 1 Hs00220867_m1 SPTB Beta-spectrin
Hs00165820_m1 PBGD Porphobilinogen deaminase Hs00609297_m1 Sputum
PRB1 proline-rich protein BstNI Hs00818764_m1 subfamily 1 PRB3
proline-rich protein BstNI Hs00818925_m1 subfamily 3 PRB4
proline-rich protein BstNI Hs00864002_m1 subfamily 4 Vaginal MUC4
mucin 4, tracheobronchial Hs00366414_m1 secretion ESR1 estrogen
receptor 1 Hs00174860_m1 MMP7 matrix metallopeptidase 7
Hs00159163_m1 (matrilysin, uterine) Menstral MMP11 matrix
metallopeptidase 11 Hs00171829_m1 blood (stromelysin 3)
.sup.1Assays-On-Demand Gene Expression Product Numbers (Applied
Biosystems, Foster City, CA)
TABLE-US-00002 TABLE 2 Human Tissue Identification Panel (mRNA)
Source Symbol Name AOD No..sup.2 Ovary hCG1816257 Hs01893192_s1
Brain (Whole) MMP11 Metalloproteinase domain 11 Hs00253742
Kidney/Liver UGT1A8 UDP glycosyltransferase 1 family, Hs01592482_m1
polypeptide A8 Liver/fetal liver hCG2017793 Hs00860044_m1
Hs00860044_m1 APCS amyloid P component, serum Hs00356632_g1 FGB
fibrinogen, B beta polypeptide Hs00170586_m1 Pancreas PNLIPRP1
pancreatic lipase-related protein 1 Hs00173824_m1 REG1B
regenerating islet-derived 1 beta Hs00359614_g1 (pancreatic stone
protein, pancreatic thread protein) hCG2042161 Hs00975170_m1 PRSS3
protease, serine, 3 (mesotrypsin) Hs00605637_m1 PNGPRP1 Prostate
TGM4 transglutaminase 4 (prostate) Hs00162710_m1 Spleen FLJ46026
FLJ46026 protein Hs01651932_m1 Adrenal Gland TH tyrosine
hydroxylase Hs00165941_m1 FDX1 ferredoxin 1 Hs00759864_s1 QPCT
glutaminyl-peptide Hs00202680_m1 cyclotransferase (glutaminyl
cyclase) Prostate hCG2040797 Hs01394126_m1 KLK3 kallikrein 3,
(prostate specific Hs00377590_s1 antigen) Testis PRM1 Protamine 1
Hs00358158_m1 PRM2 Protamine 2 Hs00172518_m1 MGC42718 hypothetical
protein MGC42718 Hs00542901_m1 SPA17 hypothetical protein MGC42718
Hs00255619_m1 Testis/Epididymus MCSP mitochondrial capsule
Hs00229076_m1 selenoprotein Throat/Trachea/Salivary Gland PRB1
proline-rich protein Hs00818764_m1 BstNI subfamily 1 PRB3
proline-rich protein Hs00818925_m1 BstNI subfamily 2 PRB4
proline-rich protein Hs00864002_m1 BstNI subfamily 4 Uterus/Trachea
MMP10 Metalloproteinase Hs00233987_m1 domain 10 Small intestine,
colon, LGALS4 lectin, galactoside- Hs00196223_m1 duodenum, cecum
binding, soluble, 4 (galectin 4) Stomach/small intestine PGA5
pepsinogen 5, group I Hs00380569_m1 (pepsinogen A) Stomach PGC
progastricsin Hs00160052_m1 (pepsinogen C) Ductus deferens/Seminal
Vesicle SEMG1 semenogelin I Hs00268141_m1 SEMG2 semenogelin II
Hs00268143_m1 CYSLTR2 cysteinyl leukotriene Hs00252658_s1 receptor
2 Fetal Heart ANKRD1 ankyrin repeat domain Hs00173317_m1 1 (cardiac
muscle) hCG1813636.1 Hs01395395_m1 Peripheral Blood/Throat ANK1
ankyrin 1 Hs00220867_m1 Skeletal Muscle TNNI1 troponin I, skeletal,
Hs00268531_m1 slow Spinal Cord PMP2 peripheral myelin Hs00160204_m1
protein 2 .sup.2Assays-On-Demand Gene Expression Product Numbers
(Applied Biosystems, Foster City, CA)
TABLE-US-00003 TABLE 3 Human Tissue Identification Panel (miRNA)
Source Name Brain hsa-miR-125b hsa-miR-128a, b hsa-miR-129
hsa-miR-135 hsa-miR-153 hsa-miR-219 Muscle (skeletal & heart)
hsa-miR-1d hsa-miR-133a, b hsa-miR-296 Kidney hsa-miR-192
hsa-miR-204 hsa-miR-215 hsa-miR-216 Pancreas hsa-miR-375
hsa-miR-376 Liver hsa-miR-122a hsa-miR-148 Heart hsa-miR-208 Thymus
hsa-miR-96 hsa-miR-182 Testes hsa-miR-10b hsa-let-7e Placenta
hsa-miR-141 hsa-miR-23a
* * * * *