188 results about "Paraffin embedded" patented technology

The present invention provides novel methods for analyzing gene expression levels from fresh or aged (more than one year old) formalin-fixed, paraffin-embedded tissue (“FFPET”) samples that comprise pre-hybridizing a labeled nucleic acid sample prepared from the formalin-fixed, paraffin-embedded tissue sample with a first microarray, hybridizing the unbound labeled nucleic acid sample with a second microarray, and detecting the labeled nucleic acid sample bound to the second microarray. The pre-hybridization step results in an increase in the specific gene signals in subsequent hybridizations with high density gene expression arrays. The first microarray used for the pre-hybridization step can be either a new or used microarray. Importantly, from a cost-savings perspective, the inventors determined that when the first microarray used for the pre-hybridization step is a previously used microarray, the results of the subsequent hybridization on a second microarray are nearly identical to the results obtained when the pre-hybridization was carried out using a new or previously unused microarray.

The present invention concerns prognostic markers associated with cancer. In particular, the invention concerns prognostic methods based on the molecular characterization of gene expression in paraffin-embedded, fixed samples of cancer tissue, which allow a physician to predict whether a patient is likely to respond well to treatment with an EGFR inhibitor.

The present invention concerns prognostic markers associated with EGFR positive cancer. In particular, the invention concerns prognostic methods based on the molecular characterization of gene expression in paraffin-embedded, fixed tissue samples of EGFR-expressing cancer, which allow a physician to predict whether a patient is likely to respond well to treatment with an EGFR inhibitor.

This invention provides methods of quantitating nucleic acids from problematic samples, such as aged samples, formalin fixed samples, paraffin embedded samples, samples with aneuploid cells, and cells with fragmented nucleic acids. Methods include techniques to efficiently solublize the nucleic acids under non-denaturing conditions from preserved clinical samples without resort to organic extractions, to normalize cell counts regardless of aneuploidy, to access the fragmentation state of the nucleic acids, and to provide standard curves for degraded nucleic acid samples.

Method and apparatus for processing paraffin embedded samples, e.g., to disassociate paraffin from tissue components and / or other biomolecules from the paraffin. The sample may be exposed to focused acoustic energy while held in a vessel containing a non-solvent, aqueous solution. Disassociated paraffin may be emulsified into the liquid or otherwise separated from the sample.

The present invention concerns prognostic markers associated with cancer. In particular, the invention concerns prognostic methods based on the molecular characterization of gene expression in paraffin-embedded, fixed samples of cancer tissue, which allow a physician to predict whether a patient is likely to respond well to treatment with an EGFR inhibitor.

The present invention relates to prognostic methods which are useful in medicine, particularly cancer chemotherapy. The object of the invention to provide a method for assessing TS and / or ERCC1 expression levels in fixed or fixed and paraffin embedded tissues and prognosticate the probable resistance of a patient's tumor to treatment with 5-FU and oxaliplatin-based therapies by examination of the amount of TS and / or ERCC1 mRNA in a patient's tumor cells and comparing it to a predetermined threshold expression level for those genes. More specifically, the invention provides to oligonucleotide primer pairs ERCC1 and TS and methods comprising their use for detecting levels of ERCC1 and TS mRNA, respectively.

Aspects of the present invention relate to compositions and methods for providing DNA fragments from an archived sample (e.g., paraffin-embedded and / or fixed-tissue biopsies, etc). Particular aspects provide methods whereby high yields of DNA are isolated as well as a substantial portion of the DNA consists of long DNA fragments, and where the isolated genomic DNA is free of associated or cross-linked contaminants like proteins, peptides, amino acids or RNA. The methods are facile, cost-effective, an are characterized by high reproducibility and reliability. Particular aspects provide methods for providing DNA fragments derived from an archived sample, wherein the yield of DNA before, for example, an amplification step is at least 20%, and amplicons up to a length of about 1,000 base pairs are amplifiable.

A method for isolating biomolecules from a fixed biological specimen embedded in paraffin, comprises the steps of bringing the specimen into contacta) with a solvent non-miscible with water and dissolving the paraffin in this solvent such as to form a liquid organic phase, andb) with an aqueous solution of at least one lysis substance and / or with water and at least one lysis substance such as to form an aqueous phase;isolation of the biomolecules from the aqueous phase. The invention further relates to a corresponding kit as well as to the use of such a kit for isolating biomolecules.

The present invention relates to prognostic methods which are useful in medicine, particularly cancer chemotherapy. The object of the invention to provide a method for assessing ERCC1 expression levels in fixed or fixed and paraffin embedded tissues and determine a platinum-based chemotherapy by examination of the amount of ERCC1 mRNA in a patient's tumor cells and comparing it to a predetermined threshold expression level. More specifically, the invention provides to oligonucleotide primer pair ERCC1 and methods comprising their use for detecting levels of ERCC1 mRNA.

The present invention relates to the field of cancer. More specifically, the present invention provides methods and compositions useful for diagnosing or predicting cancer in a patient. In one embodiment, a method for identifying a patient as having cancer comprises the steps of (a) providing a formalin-fixed, paraffin-embedded or fresh frozen sample of patient tissue; (b) steaming the sample in antigen retrieval buffer; (c) incubating the sample in hydrochloric acid (HCl); (d) incubating the sample with an affinity reagent specific for 5hmC under conditions to form a complex between the affinity reagent and 5-hydroxymethylcytosine (5hmC) present in the sample; (e) detecting the complexes formed between 5hmC and the affinity reagent with secondary detection reagents; (f) quantifying 5hmC levels; and (g) identifying the patient as having cancer if the 5hmC levels in the sample are reduced as compared to a control.

The invention discloses a kit for detecting EGFR gene mutation and an application of the kit. The kit is characterized by containing 20 specific amplification primers for an EGFR gene mutation site, 5 efficient blocking probes for wild sequences, and 4 EGFR gene specific TaqMan fluorescent probes. The kit can be used for detecting samples of which the mutation copy number is as low as 5-10 copies and the mutation content is as low as 0.1%. The kit disclosed by the invention can be used for simultaneously detecting 5 types of gene mutations of an EGFR gene, is high in sensitivity, simple to operate, low-cost in detection and wide in clinical application range, the samples can be fresh pathological tissues, paraffin embedded tissues, pleural fluid, serum or plasma, the detection speed is high, and the detection process can be finished in only 90 minutes.

The present application relates to methods for increasing stability of formalin fixed, optionally paraffin embedded biological material. In one example, DNAse inhibitors and / or RNAse inhibitors are combined with the biological material or formalin at the time of fixation or shortly prior.

Molecular characterization of disease has become the dominant trend in modern medicine, and it has recently become increasingly important to obtain qPCR, microarray, and next-generation sequencing (NGS) data for both research and clinical applications. Formalin-fixed, paraffin-embedded (“FFPE”) samples have become the standard way of storing clinical biopsies throughout the world. Unfortunately, the poor quality and quantity of nucleic acids obtained from such specimens has posed severe limitations on the types of studies that can be accomplished. Existing methods for retrieval of the biomolecules from their crosslinked matrices are poorly effective and rely on harsh conditions which further damage the biomolecules being extracted. The invention provides compositions and methods for retrieval, analysis and use of biomolecules, including nucleic acids, from such samples.

Autotaxin (ATX) is a prometastatic enzyme initially isolated from the conditioned media of human melanoma cells that stimulates a myriad of biological activities including angiogenesis and the promotion of cell growth, survival, and differentiation through the production of lysophosphatidic acid (LPA). ATX increases the aggressiveness and invasiveness of transformed cells, and ATX levels directly correlate with tumor stage and grade in several human malignancies. To study the role of ATX in the pathogenesis of malignant melanoma, we developed antibodies and small molecule inhibitors against recombinant human protein. Immunohistochemistry of paraffin embedded human tissue demonstrates that ATX levels are markedly increased in human primary and metastatic melanoma relative to benign nevi. Chemical screens identified several small molecule inhibitors with binding constants ranging from nanomolar to low micromolar. Cell migration and invasion assays with melanoma cell lines demonstrate that ATX markedly stimulates melanoma cell migration and invasion, an effect suppressed by ATX inhibitors. The migratory phenotype can be rescued by the addition of ATX's enzymatic product, LPA, confirming that the observed inhibition is linked to suppression of LPA production by ATX. Chemical analogues of the inhibitors demonstrate structure activity relationships important for ATX inhibition and indicate pathways for their optimization. These studies suggest that ATX is an approachable molecular target for the rational design of chemotherapeutic agents directed against human malignancies driven by the ATX / LPA axis, especially including malignant melanoma, among numerous others including breast and ovarian cancers.

The disclosure provides methods for the use of gene expression measurements to classify or identify neuroendocrine cancer in samples obtained from a subject in a clinical setting, such as in cases of formalin fixed, paraffin embedded (FFPE) samples.

The invention discloses a preparation method of a transmission electron microscope sample of a paraffin-embedded section tissue. The preparation method comprises the steps of (1) regulating the temperature of a laboratory to be 22-30 DEG C, and regulating the humidity of the laboratory to be smaller than or equal to 45%; (2) providing a glass slide with the paraffin-embedded section tissue; (3) preparing various pure embedding mediums; (4) preparing isolating rings of EP pipes; (5) preparing EP pipes for top buckling and embedding or prefabricated embedding blocks; (6) dewaxing and hydrating; (7) arranging the isolating rings of the EP pipes; (8) fixing and rinsing; (9) dehydrating and soaking; (10) carrying out top buckling and embedding; (11) polymerizing; (12) stripping; and (13) repairing, slicing, dyeing as well as observing and collecting images by an electron microscope. The preparation method has the beneficial effects that the amount of reagents can be saved, the errors caused by the volatilization of the reagents can be reduced, and meanwhile, the obtained sample can be completely stripped from the glass slide, so that the ultra microstructure of the paraffin-embedded section tissue can be further observed and researched conveniently.

The application relates to the examining field of gene mutation and particularly relates to a PCR micro droplet kit for examining human EGFR gene mutation. The kit includes a nucleic acid amplification reagent; a reference substance and micro droplet PCR generated oil. The nucleic acid amplification reagent includes a primer and a probe for examining the No. 18 extron mutant, a primer and a probe for examining the No. 19 extron mutant, a primer and a probe for examining the No. 20 extron mutant, and a primer and a probe for examining the No. 21 extron mutant. The present application can be used for examining the seven species of the No. 18, 19, 20, and 21 extron mutants for the EGFR genes in the DNAs extracted from the paraffin-embedded pathology tissue slices and the peripheral blood dissociate DNAs. The present application provides a reference for the patients to choose the tumor molecularly targeted drug remedy and monitor. The present invention has absolute ration, high specificity, accuracy and high sensitivity.

The invention provides a quantitative realtime RT-PCR assay for detection of metastatic breast, gastric, pancreas or colon cancer cells or metastatic melanoma. The assay allows to predict disease recurrence and survival in patients with AJCC stage I and II, and III disease using multimarker panels. The method for detecting metastatic melanoma cells utilizes panels of markers selected from a group consisting of MAGE-A3, GalNAcT, MART-1, PAX3, Mitf, TRP-2, and Tyrosinase. The method for detecting metastatic breast, gastric, pancreas or colon cancer cells in paraffin-embedded samples utilizes panels of markers selected from a group consisting of C-Met, MAGE-A3, Stanniocalcin-1, mammoglobin, HSP27, GalNAcT, CK20, and β-HCG.

Compositions and methods useful in determining the major morphological types of lung cancer are provided. The methods include detecting expression of at least one gene or biomarker in a sample. The expression of the gene or biomarker is indicative of the lung tumor subtype. The compositions include subsets of genes that are monitored for gene expression. The gene expression is capable of distinguishing between normal lung parenchyma and the major morphological types of lung cancer. The gene expression and somatic mutation data are useful in developing a complete classification of lung cancer that is prognostic and predictive for therapeutic response. The methods are suited for analysis of paraffin-embedded tissues. Methods of the invention include means for monitoring gene or biomarker expression including PCR and antibody-based detection. The biomarkers of the invention are genes and / or proteins that are selectively expressed at a high or low level in certain tumor subtypes. Biomarker expression can be assessed at the protein or nucleic acid level.

This invention provides methods of quantitating nucleic acids from problematic samples, such as aged samples, formalin fixed samples, paraffin embedded samples, samples with aneuploid cells, and cells with fragmented nucleic acids. Methods include techniques to efficiently solublize the nucleic acids under non-denaturing conditions from preserved clinical samples without resort to organic extractions, to normalize cell counts regardless of aneuploidy, to access the fragmentation state of the nucleic acids, and to provide standard curves for degraded nucleic acid samples.

The invention is directed to methods and compositions for deparaffinizing paraffin-embedded biological samples for subsequent tissue staining. The compositions are microemulsions that may include water / oil / surfactant microemulsions, and optionally a cosurfactant. The microemulsions enable deparaffinization without the use of xylene or toluene, and also enable solvent exchange without the use of intermediary alcohol dehydration or alcohol rehydration compositions.

The invention relates to a method for extracting desoxyribonucleic acid from formalin fixed and paraffin embedded tissues, and belongs to the technical field of nucleic acid application in biology. The method comprises the following steps of: preparing special lysis solution, adding the lysis solution into paraffin section tissues, boiling at high temperature for 30 minutes, and centrifuging to take supernate; adding absolute ethanol for uniform mixing, and adding the mixed solution into a silicon membrane absorption column for centrifuging; rinsing by using protein-free liquid and salt-free rinsing liquid; and eluting by using elution buffer. In the method, a toxic reagent of dimethylbenzene is not used for dewaxing, and harm to bodies of experimenters is avoided; and precious protease Kis not needed to perform long-time incubation enzymolysis, the operation is simple and quick, the extracted desoxyribonucleic acid in genome is high in quality and stability, and the cost and time can be saved to the greatest degree. The extracted product is subjected to polymerase chain reaction (PCR) detection, long segments with about 750pb can be obtained through amplification, and the work in the aspects such as scientific research, biomedicine and the like is greatly facilitated.

Ligation-enhanced nucleic acid detection assay embodiments for detection of RNA or DNA are described. The assay embodiments rely on ligation of chimeric oligonucleotide probes to generate a template for amplification and detection. The assay embodiments are substantially independent of the fidelity of a polymerase for copying compromised nucleic acid. Very little background amplification is observed and as few as 1000 copies of target nucleic acid can be detected. Method embodiments are particularly adept for detection of RNA from compromised samples such as formalin-fixed and paraffin-embedded samples. Heavily degraded and cross-linked nucleic acids of compromised samples, in which classic quantitative real time PCR assays typically fail to adequately amplify signal, can be reliably detected and quantified.

Methods of reliably quantifying telomere length in cells or tissues that have been formalin fixed and paraffin embedded (FFPE) samples by quantitative polymerase chain reaction protocol and kits for use with such various methods are provided. The methods of the present invention may be used to predetermine an individual's response to treatment with a telomerase inhibitor, a telomere damaging agent or a telomerase activator.

The invention relates to a device for detecting copy number variation of FFPE (formalin-fixed and paraffin-embedded) samples. The device for detecting the copy number variation of the FFPE samples comprises a sequencing data acquisition module, a sequence comparison module, an earlier-stage data processing module, a normalization module, a background library screening module, a data fluctuation elimination module, a GC (guanine and cytosine) correction module and an output module. The device has the advantage of high detection sensitivity.

The invention provides a quantitative realtime RT-PCR assay for detection of metastatic breast, gastric, pancreas or colon cancer cells or metastatic melanoma. The assay allows to predict disease recurrence and survival in patients with AJCC stage I and II, and III disease using multimarker panels. The method for detecting metastatic melanoma cells utilizes panels of markers selected from a group consisting of MAGE-A3, GalNAcT, MART-1, PAX3, Mitf, TRP-2, and Tyrosinase. The method for detecting metastatic breast, gastric, pancreas or colon cancer cells in paraffin-embedded samples utilizes panels of markers selected from a group consisting of C-Met, MAGE-A3, Stanniocalcin-1, mammoglobin, HSP27, GalNAcT, CK20, and beta-HCG.