BIOMARKERS FOR DIFFERENTIATING MELANOMA FROM BENIGN NEVUS IN THE SKIN

Disclosed is a method for diagnosing melanoma in a human subject, as well as a method for providing a prognosis to a human subject who is at risk of developing melanoma recurrence, and a method for determining the stage of melanoma in a human subject, comprising the step of determining the level of expression of phosphatase and actin regulator 1 (PHACTR1) gene, or fragments thereof, either alone or in combination with the level of expression of secreted integrin-binding phosphoprotein (SPP1), preferentially expressed antigen in melanoma (PRAME), growth differentiation factor 15 (GDF 15), and chemokine C-X-C motif ligand 10 (CXCL10) genes. Further, the invention relates to a diagnostic kit, comprising at least one substance for detection of the expression of PHACTR1, or fragments thereof, either alone or in combination with the detection of SPP1, PRAME, GDF15, and CXCL10, for the diagnosis or prognosis of melanoma.

In situ detection of nucleotide variants in high noise samples, and compositions and methods related thereto

The invention relates to methods of in situ detection of a nucleic acid variation of a target nucleic acid in a sample, including single nucleotide variations, multi-nucleotide variations or splice sites. The method can comprise the steps of contacting the sample with a probe that detects the nucleic acid variation or splice site and a neighbor probe; contacting the sample with pre-amplifiers that bind to the nucleic acid variation probe or splice site probe and neighbor probe, respectively; contacting the sample with a collaboration amplifier that binds to the pre-amplifiers; and contacting the sample with a label probe system, wherein hybridization of the components forms a signal generating complex (SGC) comprising a target nucleic acid with the nucleic acid variation or splice site, the probes and amplifiers; and detecting in situ signal from the SGC on the sample. The invention also provides samples, tissue slides, and kits relating to detection of nucleic acid variations, including ...

Ultra sensitive method for in situ detection of nucleic acids

Disclosed is a method for in situ detection of one or more target nucleic acids based on a combination of an in situ hybridization (ISH) assay method and a general ISH signal amplification method. This new method produces high signal intensity and while keeps low background noise of signal amplification. The result can be consistently reproduced and the method can be easily adopted for routine clinic diagnostic use. Further, the invention relates to a kit, comprising the components of the ISH assay and a general ISH signal amplification assay, for sensitive detection of one or more target nucleic acids.

Methods and reagents for identifying rare fetal cells in the maternal circulation

This invention provides methods and compositions useful for identifying and diagnosing rare fetal cells in a mixed cell population such as a maternal blood sample. The methods entail the use of specific nucleic acid probes that hybridize to fetal cell associated RNAs to identify the rare fetal cells or antibodies that bind to polypeptides encoded by the fetal cell associated RNAs for fetal cell detection. The cells detected by the methods of the present invention are useful for diagnosing the fetal cells for a genetic trait of interest, such as trisomy 21. Novel methods for simultaneous screening for fetal cells and diagnosing the fetal cells are also provided. Compositions comprising the fetal cell associated nucleic acids of the invention and their encoded proteins are also provided. The present invention further provides kits useful for practicing the present methods.

IN SITU DETECTION OF NUCLEOTIDE VARIANTS IN HIGH NOISE SAMPLES, AND COMPOSITIONS AND METHODS RELATED THERETO

The invention relates to methods of in situ detection of a nucleic acid variation of a target nucleic acid in a sample, including single nucleotide variations, multi-nucleotide variations or splice sites. The method can comprise the steps of contacting the sample with a probe that detects the nucleic acid variation or splice site and a neighbor probe; contacting the sample with pre-amplifiers that bind to the nucleic acid variation probe or splice site probe and neighbor probe, respectively; contacting the sample with a collaboration amplifier that binds to the pre-amplifiers; and contacting the sample with a label probe system, wherein hybridization of the components forms a signal generating complex (SGC) comprising a target nucleic acid with the nucleic acid variation or splice site, the probes and amplifiers; and detecting in situ signal from the SGC on the sample. The invention also provides samples, tissue slides, and kits relating to detection of nucleic acid variations, including single nucleotide variations, multi-nucleotide variations or splice sites, of a target nucleic acid. 1. A method of in situ detection of a single nucleotide variation of a target nucleic acid in a sample of fixed and permeabilized cells , comprising:(A) contacting the sample with a single nucleotide variation probe (SP) and a neighbor probe (NP), wherein the SP comprises a target anchor segment (SPAT) that can specifically hybridize to a region of the target nucleic acid comprising the single nucleotide variation and a pre-amplifier anchor segment (SPAP), and wherein the NP comprises a target anchor segment (NPAT) that can hybridize to a region of the target nucleic acid adjacent to the binding site of the SP and a pre-amplifier anchor segment (NPAP);(B) contacting the sample with an SP pre-amplifier (SPM) and an NP pre-amplifier (NPM), wherein the SPM comprises a segment that can bind to the SP and comprises two or more SP collaboration anchors (SPCAs), and wherein the NPM ...

Ultra sensitive method for in situ detection of nucleic acids

Disclosed is a method for in situ detection of one or more target nucleic acids based on a combination of an in situ hybridization (ISH) assay method and a general ISH signal amplification method. This new method produces high signal intensity and while keeps low background noise of signal amplification. The result can be consistently reproduced and the method can be easily adopted for routine clinic diagnostic use. Further, the invention relates to a kit, comprising the components of the ISH assay and a general ISH signal amplification assay, for sensitive detection of one or more target nucleic acids.

Methods to further enhance signal amplification for the in situ detection of nucleic acids

The present invention relates to detection of nucleic acids and provides a composition comprising a Signal Generating Complex, wherein the composition comprises: (A) a pair of target probes (TPs), wherein a first TP of the pair of TPs comprises a nucleic acid sequence comprising two segments; (B) a pair of base PPAs comprising the first and second base PPAs, wherein the first base PPA comprises a nucleic acid sequence comprising three segments; (C) a set of extension PPAs comprising the first and second extension PPAs, wherein the first extension PPA comprises a nucleic acid sequence comprising two segments; (D) a plurality of pre-amplifiers (PAs), wherein the PAs comprise a nucleic acid sequence comprising three segments; (E) a plurality of amplifiers (AMPs), wherein the AMPs comprise a nucleic acid sequence comprising two segments; and (F) a plurality of label probes (LPs), wherein the LPs comprise a nucleic acid sequence comprising two segments.

Methods and reagents for identifying rare fetal cells in the maternal circulation

This invention provides methods and compositions useful for identifying and diagnosing rare fetal cells in a mixed cell population such as a maternal blood sample. The methods entail the use of specific nucleic acid probes that hybridize to fetal cell associated RNAs to identify the rare fetal cells or antibodies that bind to polypeptides encoded by the fetal cell associated RNAs for fetal cell detection. The cells detected by the methods of the present invention are useful for diagnosing the fetal cells for a genetic trait of interest, such as trisomy 21. Novel methods for simultaneous screening for fetal cells and diagnosing the fetal cells are also provided. Compositions comprising the fetal cell associated nucleic acids of the invention and their encoded proteins are also provided. The present invention further provides kits useful for practicing the present methods.

IN SITU DETECTION OF NUCLEOTIDE VARIANTS IN HIGH NOISE SAMPLES, AND COMPOSITIONS AND METHODS RELATED THERETO

The invention relates to methods of in situ detection of a nucleic acid variation of a target nucleic acid in a sample, including single nucleotide variations, multi-nucleotide variations or splice sites. The method can comprise the steps of contacting the sample with a probe that detects the nucleic acid variation or splice site and a neighbor probe; contacting the sample with pre-amplifiers that bind to the nucleic acid variation probe or splice site probe and neighbor probe, respectively; contacting the sample with a collaboration amplifier that binds to the pre-amplifiers; and contacting the sample with a label probe system, wherein hybridization of the components forms a signal generating complex (SGC) comprising a target nucleic acid with the nucleic acid variation or splice site, the probes and amplifiers; and detecting in situ signal from the SGC on the sample. The invention also provides samples, tissue slides, and kits relating to detection of nucleic acid variations, including ...

METHODS TO FURTHER ENHANCE SIGNAL AMPLIFICATION FOR THE IN SITU DETECTION OF NUCLEIC ACIDS

The present invention relates to detection of nucleic acids and provides a composition comprising a Signal Generating Complex, wherein the composition comprises: (A) a pair of target probes (TPs), wherein a first TP of the pair of TPs comprises a nucleic acid sequence comprising two segments; (B) a pair of base PPAs comprising the first and second base PPAs, wherein the first base PPA comprises a nucleic acid sequence comprising three segments; (C) a set of extension PPAs comprising the first and second extension PPAs, wherein the first extension PPA comprises a nucleic acid sequence comprising two segments; (D) a plurality of pre-amplifiers (PAs), wherein the PAs comprise a nucleic acid sequence comprising three segments; (E) a plurality of amplifiers (AMPs), wherein the AMPs comprise a nucleic acid sequence comprising two segments; and (F) a plurality of label probes (LPs), wherein the LPs comprise a nucleic acid sequence comprising two segments. 1. A composition comprising a Signal Generating Complex (SGC) , wherein the composition comprises:(A) a pair of target probes (TPs), wherein a first TP of the pair of TPs comprises a nucleic acid sequence comprising two segments, (i) a segment comprising a binding site for a first segment of a target nucleic acid, and (ii) a segment comprising a binding site for a first base pre-pre-amplifier (base PPA); and wherein a second TP of the pair of TPs comprises a nucleic acid sequence comprising two segments, (i) a segment comprising a binding site for a second segment of the target nucleic acid, and (ii) a segment comprising a binding site for a second base PPA;(B) a pair of base PPAs comprising the first and second base PPAs, wherein the first base PPA comprises a nucleic acid sequence comprising three segments, (i) a segment that binds to the first base PPA binding site of the first TP, (ii) a segment comprising a plurality of first pre-amplifier binding segment repeats (first PA-BSRs), and (iii) a segment comprising a ...

BIOMARKERS FOR DIFFERENTIATING MELANOMA FROM BENIGN NEVUS IN THE SKIN

Disclosed is a method for diagnosing melanoma in a human subject, as well as a method for providing a prognosis to a human subject who is at risk of developing melanoma recurrence, and a method for determining the stage of melanoma in a human subject, comprising the step of determining the level of expression of phosphatase and actin regulator 1 (PHACTR1) gene, or fragments thereof, either alone or in combination with the level of expression of secreted integrin-binding phosphoprotein (SPP1), preferentially expressed antigen in melanoma (PRAME), growth differentiation factor 15 (GDF15), and chemokine C-X-C motif ligand 10 (CXCL10) genes. Further, the invention relates to a diagnostic kit, comprising at least one substance for detection of the expression of PHACTR1, or fragments thereof, either alone or in combination with the detection of SPP1, PRAME, GDF15, and CXCL10, for the diagnosis or prognosis of melanoma. 1. A method for diagnosing melanoma in a human subject suspected of melanoma , comprising the following steps:(a) obtaining a tissue sample from said human subject, wherein said tissue sample comprises a plurality of melanocytes;(b) determining the level of expression of at least phosphatase and actin regulator 1 (PHACTR1) gene, or fragments thereof; and(c) optionally determining the level of expression of one or more additional markers of melanoma, or fragments thereof;thereby diagnosing the presence of melanoma based on the expression levels in said tissue sample.2. The method of claim 1 , wherein said determining the level of expression comprises determining the level of expression of a set of genes comprising PHACTR1 and one or more of genes selected from the group consisting of secreted integrin-binding phosphoprotein (SPP1) claim 1 , preferentially expressed antigen in melanoma (PRAME) claim 1 , growth differentiation factor 15 (GDF15) claim 1 , and chemokine C-X-C motif ligand 10 (CXCL10) genes.3. The method of claim 2 , wherein said determining the ...

ULTRA SENSITIVE METHOD FOR IN SITU DETECTION OF NUCLEIC ACIDS

Disclosed is a method for in situ detection of one or more target nucleic acids based on a combination of RNAscope® method and a general ISH signal amplification method. This new method produces high signal intensity and while keeps low background noise of signal amplification. The result can be consistently reproduced and the method can be easily adopted for routine clinic diagnostic use. Further, the invention relates to a kit, comprising the components of RNAscope® assay and a general ISH signal amplification assay, for sensitive detection of one or more target nucleic acids. 1. A method of detecting at least one target nucleic acid , the method comprising:(a) providing a sample comprising or suspected of comprising said target nucleic acid;(b) providing at least one set of two or more capture probes capable of hybridizing to said target nucleic acid;(c) providing a signal generating multimer capable of hybridizing to said set of two or more capture probes, wherein said signal generating multimer comprises a label probe;(d) providing a signal amplification probe capable of binding to said label probe, wherein said signal amplification probe comprises a label;(e) hybridizing said target nucleic acid to said set of two or more capture probes;(f) capturing the signal generating multimer to said set of two or more capture probes and thereby capturing the signal generating multimer to said target nucleic acid;(g) capturing said signal amplification probe to said label probe and thereby capturing the signal amplification probe to said signal generating multimer; and(h) detecting the presence, absence, or amount of the label.2. The method of claim 1 , wherein said signal amplification probe comprises: a biotin molecule which is capable of conjugating to said label probe claim 1 , an avidin/streptavidin molecule which is capable of binding to said biotin molecule claim 1 , and additional biotin molecules being conjugated to Horse Radish Peroxidase (HRP) claim 1 , ...

ULTRA SENSITIVE METHOD FOR IN SITU DETECTION OF NUCLEIC ACIDS

Disclosed is a method for in situ detection of one or more target nucleic acids based on a combination of RNAscope® method and a general ISH signal amplification method. This new method produces high signal intensity and while keeps low background noise of signal amplification. The result can be consistently reproduced and the method can be easily adopted for routine clinic diagnostic use. Further, the invention relates to a kit, comprising the components of RNAscope® assay and a general ISH signal amplification assay, for sensitive detection of one or more target nucleic acids. 1. A method of detecting at least one target nucleic acid , the method comprising:(a) providing a sample comprising or suspected of comprising said target nucleic acid;(b) providing at least one set of two or more capture probes capable of hybridizing to said target nucleic acid;(c) providing a signal generating multimer capable of hybridizing to said set of two or more capture probes, wherein said signal generating multimer comprises a label probe;(d) providing a signal amplification probe capable of binding to said label probe, wherein said signal amplification probe comprises a label;(e) hybridizing said target nucleic acid to said set of two or more capture probes;(f) capturing the signal generating multimer to said set of two or more capture probes and thereby capturing the signal generating multimer to said target nucleic acid;(g) capturing said signal amplification probe to said label probe and thereby capturing the signal amplification probe to said signal generating multimer; and(h) detecting the presence, absence, or amount of the label.2. The method of claim 1 , wherein said signal amplification probe comprises: a biotin molecule which is capable of conjugating to said label probe claim 1 , an avidin/streptavidin molecule which is capable of binding to said biotin molecule claim 1 , and additional biotin molecules being conjugated to Horse Radish Peroxidase (HRP) claim 1 , ...

DIFFERENTIATION BETWEEN TRANSIENT AND PERSISTENT HIGH-RISK HPV INFECTION BY IN SITU HYBRIDIZATION

The invention relates to methods of categorizing a cervical tissue or cytology sample by performing an in situ hybridization assay using an antisense E6 or E7 probe on a cervical tissue sample, wherein the antisense E6 or E7 probe can simultaneously detect HPV DNA and HPV RNA; detecting the presence of HPV nucleic acid; and categorizing the cervical tissue sample based on HPV nucleic acid expression. 1. A method of categorizing a cervical tissue sample , comprising:(a) performing an in situ hybridization assay using an antisense E6 or E7 probe on a cervical tissue sample, wherein the antisense E6 or E7 probe can simultaneously detect high-risk HPV DNA and HPV RNA;(b) detecting the presence of HPV nucleic acid; and(c) categorizing the cervical tissue sample,wherein the presence of diffuse nuclear staining of HPV nucleic acid only in the superficial layer of the epithelium of the cervical sample indicates a transient state,wherein the presence of granular staining of HPV nucleic acid in the cytoplasm and/or the nucleus in the intermediate and basal layers of the epithelium of the cervical sample indicates a persistent state, orwherein the presence of diffuse nuclear staining of HPV nucleic acid in the superficial layer of the epithelium and granular staining of HPV nucleic acid in the cytoplasm and/or the nucleus of the intermediate layer of the epithelium of the cervical sample indicates a transition state.2. The method of claim 1 , wherein the transient state corresponds to a transient HPV infection state.3. The method of claim 1 , wherein the persistent state corresponds to a persistent HPV infection state.4. The method of claim 1 , wherein the transition state corresponds a transition HPV infection state that has an increased likelihood of transitioning to a persistent HPV infection state.5. The method of claim 1 , wherein detection of HPV DNA only in the superficial layer of the epithelium of the cervical tissue sample indicates a low-grade lesion.6. The method ...

In situ detection of nucleotide variants in high noise samples, and compositions and methods related thereto

The invention relates to methods of in situ detection of a nucleic acid variation of a target nucleic acid in a sample, including single nucleotide variations, multi-nucleotide variations or splice sites. The method can comprise the steps of contacting the sample with a probe that detects the nucleic acid variation or splice site and a neighbor probe; contacting the sample with pre-amplifiers that bind to the nucleic acid variation probe or splice site probe and neighbor probe, respectively; contacting the sample with a collaboration amplifier that binds to the pre-amplifiers; and contacting the sample with a label probe system, wherein hybridization of the components forms a signal generating complex (SGC) comprising a target nucleic acid with the nucleic acid variation or splice site, the probes and amplifiers; and detecting in situ signal from the SGC on the sample. The invention also provides samples, tissue slides, and kits relating to detection of nucleic acid variations, including single nucleotide variations, multi-nucleotide variations or splice sites, of a target nucleic acid.

An ultra sensitive method for in situ detection of nucleic acids

The invention provides a method of detecting at least one target nucleic acid in a cell by in situ hybridization, a fixed and permeabilized cell, a tissue section comprising a cell, and a kit for detecting at least one target nucleic acid in a cell.

Biomarkers for differentiating melanoma from benign nevus in the skin

Disclosed is a method for diagnosing melanoma in a human subject, as well as a method for providing a prognosis to a human subject who is at risk of developing melanoma recurrence, and a method for determining the stage of melanoma in a human subject, comprising the step of determining the level of expression of phosphatase and actin regulator 1 (PHACTR1) gene, or fragments thereof, either alone or in combination with the level of expression of secreted integrin-binding phosphoprotein (SPP1), preferentially expressed antigen in melanoma (PRAME), growth differentiation factor 15 (GDF15), and chemokine C-X-C motif ligand 10 (CXCL10) genes. Further, the invention relates to a diagnostic kit, comprising at least one substance for detection of the expression of PHACTR1, or fragments thereof, either alone or in combination with the detection of SPP1, PRAME, GDF15, and CXCL10, for the diagnosis or prognosis of melanoma.

Methods to further enhance signal amplification for the in situ detection of nucleic acids

Differentiation between transient and persistent high risk hpv infection by in situ hybridization

The invention relates to methods of categorizing a cervical tissue or cytology sample by performing an in situ hybridization assay using an antisense E6 or E7 probe on a cervical tissue sample, wherein the antisense E6 or E7 probe can simultaneously detect HPV DNA and HPV RNA; detecting the presence of HPV nucleic acid; and categorizing the cervical tissue sample based on HPV nucleic acid expression.

Methods to further enhance signal amplification for the in situ detection of nucleic acids

The present invention relates to detection of nucleic acids and provides a composition comprising a Signal Generating Complex, wherein the composition comprises: (A) a pair of target probes (TPs), wherein a first TP of the pair of TPs comprises a nucleic acid sequence comprising two segments; (B) a pair of base PPAs comprising the first and second base PPAs, wherein the first base PPA comprises a nucleic acid sequence comprising three segments; (C) a set of extension PPAs comprising the first and second extension PPAs, wherein the first extension PPA comprises a nucleic acid sequence comprising two segments; (D) a plurality of pre-amplifiers (PAs), wherein the PAs comprise a nucleic acid sequence comprising three segments; (E) a plurality of amplifiers (AMPs), wherein the AMPs comprise a nucleic acid sequence comprising two segments; and (F) a plurality of label probes (LPs), wherein the LPs comprise a nucleic acid sequence comprising two segments.

Differentiation between transient and persistent high risk hpv infection by in situ hybridization