129 results about "Paraffin embedded tissue" patented technology

The invention relates to methods of depleting RNA from a nucleic acid sample. The RNA may be any RNA, including, but not limited to, rRNA, tRNA, and mRNA. The method is useful for depleting RNA from a nucleic acid sample obtained from a fixed paraffin-embedded tissue (FPET) sample. The method may also be used to prepare cDNA, in particular, a cDNA library for further analysis or manipulation.

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 invention concerns an integrated, qRT-PCR-based system for analyzing and reporting RNA expression profiles of biological samples. In particular, the invention concerns a fully optimized and integrated multiplex, multi-analyte method for expression profiling of RNA in biological samples, including fixed, paraffin-embedded tissue samples. The gene expression profiles obtained can be used for the clinical diagnosis, classification and prognosis of various pathological conditions, including cancer.

The invention relates to methods of depleting RNA from a nucleic acid sample. The RNA may be any RNA, including, but not limited to, rRNA, tRNA, and mRNA. The method is useful for depleting RNA from a nucleic acid sample obtained from a fixed paraffin-embedded tissue (FPET) sample. The method may also be used to prepare cDNA, in particular, a cDNA library for further analysis or manipulation.

This invention relates to methods for the detection of one or more mRNA transcripts in paraffin-embedded tissue by “mRNA liberation in fixed-treated tissue or ‘MLIFTT’”. This method includes treating the tissue with ammonia-ethanol and sodium borohydride combined with pressure cooking of the tissue. The chemical treatments reduce the tissue autofluorescence and the physical treatments overcome the interference created by the fixation-induced chemical bonds. The methods of the present invention can be utilized to identify a plurality of mRNA transcripts in a microarray format.

The invention provides a method for manufacturing a paraffin-embedded tissue cell specimen. The method is characterized by comprising the following steps: precipitating and performing centrifugal treatment on a tissue cell specimen, subsequently extracting the tissue cell specimen, adding an environmental-friendly biologic synthesis agent, synchronously fixing, dehydrating, transparentizing and performing paraffin dipping treatment on the tissue cell specimen by an ultrasonic tissue treatment instrument or a medical microwave oven, slicing the paraffin-embedded tissue, and dyeing and sealing the paraffin-embedded tissue slice. The method has the advantages that the operation is rapid, no pollution is caused, the operation is simple and convenient, a dyeing process can be performed together with daily work, a molecular pathological examination can be also performed, and the result is stable. The positive detection rate and the accuracy in pathological diagnosis are improved.

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.

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 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 invention relates to methods of using fragmented RNA, such as RNA obtained from archived fixed paraffin-embedded tissue material (FPET RNA) or other clinically biopsied tissue specimens for universal gene expression profiling.

The invention discloses a primer, a probe and a detection kit for detection of an EML4-ALK fusion gene mutation. The primer and probe provided by the invention are SEQ ID NO 1-SEQ ID NO 24. The primer and probe of the invention can carry out specific detection on EML4-ALK fusion gene mutation. The present invention has the following advantages: (1) an established real-time fluorescent PCR system capable of simultaneously detecting 9 kinds of fusion mutation of EML4-ALK gene; (2) high sensitivity capable of detecting mutation of 5-10 copies; (3) simple operation, cheap detection and wide clinical application range; (4) a wide range of samples from fresh pathological tissue to paraffin embedded tissue or pleural fluid; and (5) high speed detection with a detection procedure taking only 90 minutes.

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.

A tissue chip for detecting autoantibody is composed of substrate and the matrix on the substrate. Said matrix may be paraffin wax coated tissue, frozen fresh tissue, or cell specimen. An indirect immunofluorescence technique is used to detect, screen and analyze a great deal of autoantibodies in the blood serum of autoimmunopathy patient.

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.

The present invention provides a novel method called Sequential IMmunoPeroxidase Labeling and Erasing (SIMPLE) that enables the simultaneous visualization of at least five markers within a single tissue section. Utilizing the alcohol-soluble peroxidase substrate 3-amino-9-ethylcarbazole (AEC), combined with a rapid non-destructive method for antibody-antigen dissociation, the present application discloses the ability to erase the results of a single immunohistochemical stain while preserving tissue antigenicity for repeated rounds of labeling. The present invention also provides methods for visualizing multiple antigens simultaneously.

The invention aims to provide a reagent capable of detecting the expression condition of a target protein in a paraffin-embedded tissue, and particularly provides a kit used for evaluating the prognosis information of a patient suffering from the esophageal squamous cell carcinoma. The kit comprises antibodies of the three target proteins, namely Annexin II, Kindlin-2 and Myosin-9, and further comprises goat serum, 0.01 M citrate repair liquid, 3% H2O2, a Polymer reinforcing agent, a Polymer, a DAB color reagent and a PBS solution. The kit can be used for evaluating the prognosis of the patient; compared with the international TNM staging, the kit is simpler and easier to use, and higher in both sensitivity and specificity, and is more suitable for the prognosis evaluation of Chinese suffering from the esophageal squamous cell carcinoma.

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.

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.

This invention relates to methods for the detection of one or more mRNA transcripts in paraffin-embedded tissue by “mRNA liberation in fixed-treated tissue or ‘MLIFTT’”. This method includes treating the tissue with ammonia-ethanol and sodium borohydride combined with pressure cooking of the tissue. The chemical treatments reduce the tissue autofluorescence and the physical treatments overcome the interference created by the fixation-induced chemical bonds. The methods of the present invention can be utilized to identify a plurality of mRNA transcripts in a microarray format.

The invention discloses a method for extracting total ribonucleic acid (RNA) from paraffin-embedded tissues, which is an improvement method provided on the basis of a kit. The method comprises the following steps: deparaffinating, digesting, extracting and purifying. By adopting the method, the RNA in a tissue sample stored by being embedded by paraffin can be extracted, wherein, the longest storage time reaches up to 6 years; the RNA is analyzed by a nucleic acid trace tester and an internationally recognized Agilent 2100bioanalyzer; the extraction concentration is 211-3116ng / mu l, and the extraction purity is A260 / 280:1.75-2.04, which are all very ideal; and the expression research of microarray high-abundance genes of qRT-PCR and miRNA in mRNA and miRNA level can be carried out, which provides an experimental basis for developing a large amount of research on formalin fixed paraffin-embedded (FFPE) genes, and has wide application value.

The present invention relates to predictive methods for use in medicine, in particular cancer chemotherapy. The purpose of the present invention is to provide a method for evaluating the expression level of ERCC1 in fixed or fixed and paraffin-embedded tissues by detecting the amount of ERCC1 mRNA in tumor cells of a patient and comparing it with a predetermined threshold expression level, and determining the platinum-based Methods of chemotherapy. More particularly, the present invention provides oligonucleotide primer pairs for ERCC1, and methods for detecting ERCC1 mRNA levels using them.

The invention discloses a detection kit and detection method for KRAS gene mutations, and belongs to the technical field of B2D10 currently first developed high-tech industrialization important field guide / biology / novel medical precise diagnosis and treatment equipment. The detection kit is characterized by comprising eight pairs of specifically amplified primers for KRAS gene mutation loci, two efficient blocking probes of a wild sequence and two KRAS gene specific TaqMan fluorescent probes. The kit can detects specimens of which the number of the mutant copies is as low as 5 to 10, and the mutation content is as low as 0.1%. The detection kit can detect five gene mutations of a KRAS gene simultaneously, the sensitivity is high, operation is easy, detection is low in price, the clinical application range is wide, samples can adopt fresh pathological tissue or paraffin-embedded tissue or a pleural fluid or serum or plasma, the detection speed is high, and only 90 minutes are needed for completing the detection process.

The invention discloses a novel method and a kit for extracting high-quality RNA (Ribose Nucleic Acid) from a paraffin-embedded tissue. The method comprises the following steps of: firstly, removing paraffin from a sample by using a nontoxic and non-volatile paraffin removing agent to improve the sensitivity of the paraffin-embedded tissue to lysate; secondly, cracking a tissue sample with SDS (Sodium Dodecyl Sulfate)-containing lysate and protease K, wherein the lysate provides a pH environment suitable for cell cracking, so that proteins are further denaturalized and water solubility is enhanced; thirdly, fully denaturalize the proteins with phenol-chloroform mixed liquid, separating the RNA and DNA (Deoxyribose Nucleic Acid) from the proteins, and chromatographing the RNA and the DNA from the mixed liquid to a water layer; fourthly, precipitating the RNA under the coaction of sodium acetate and glucogen; and lastly, dissolving the RNA in RNase-free water. By adopting the kit and the extraction method, the aim of obtaining total RNA with high quality, a long segment and high yield from the paraffin-embedded tissue is fulfilled. The obtained RNA can be applied to medical molecular biology researches, clinical examination and the like.

An ultra-sensitive, specific methodology for detecting PIK3CA mutations in biological samples of cancer patients, comprises a combination of allele-specific, asymmetric rapid PCR and melting analysis in a DNA sample from Circulating Tumor Cells, cell-free DNA in plasma / serum, or Formalin-Fixed Paraffin-Embedded tissues. Using the allele-specific primers for hotspot mutations in exons 9 and 20 (E545K and H1047R), detection can enhance amplification of mutant PIK3CA allele sequence, whereas presence of corresponding competitive blocking unlabeled probes for each exon can avoid non-specific amplification of wild-type PIK3CA sequence increasing the sensitivity and the specificity of method. The mutational detection is completed with melting curve analysis of the unlabeled probe and DNA template of the mutant PIK3CA sequence. Evaluation of PIK3CA mutational status on CTC in peripheral blood and cfDNA in plasma / serum of patients has potential for clinical applications and therapeutic interventions, since presence of PIK3CA mutations is associated with response to molecular targeted therapies.

The invention discloses a kit and a method for rapidly releasing nucleic acid from paraffin-embedded tissue sections. The kit comprises a proteinase K solution (prepared from 10-100g / L protease K, 10-200mM / L Tris with pH value of 7.0-11.0 and 0.01-5mM / L anhydrous calcium chloride), a first nucleic acid release reagent (1-1000 mM / L Tris with pH value of 7.0-11.0, Triton X-100 with a volume ratio of0.1 to 2%, Nonidet P-40 or substitutes thereof with a volume ratio of 0.1 to 2%, and 0.1-10mM / L EDTA disodium salt), and a second nucleic acid release reagent (10-200mM / L Tris with pH value of 7.0-8.0, and Nonidet P-40 or substitutes thereof with a volume ratio of 0.1- 2%). The kit is also preferentially provided with a centrifuge column; when the kit is in use, the paraffin section is placed ina centrifuge tube, the first nucleic acid release reagent and the proteinase K solution are added into the centrifuge tube, the materials in the centrifuge tube are fully and evenly mixed, and incubation is carried out for the first time; after that, the second nucleic acid release reagent is added and is evenly mixed in a way of vortex, and incubation is carried out for the second time, so that asplit product is obtained; the product is then filtered by means of the centrifuge column, so that target nucleic acid can be obtained. After the kit and the method are adopted, the paraffin sectionsdo not need to be subjected to dewaxing, and the complicated purification step is omitted. The kit and the method have the advantages of being safe to use, simple and convenient to operate, high in recovery efficiency and lower in manufacturing cost.

The extraction method for protein from paraffin embedded tissue comprises: cutting the paraffin embedded tissue; dewaxing with xylene, hydrating with ethanol; finally, adding cracking liquid and nuclease for centrifugation. This invention needs short time and low cost with high efficiency.

The invention discloses a primer and a probe for detecting an RET fusion gene. The primer comprises an RET fusion mutation specificity reverse transcription primer, an RET gene fusion mutation detection specificity primer and a probe nucleotide sequence. The invention discloses a detection reagent kit for detecting the RET fusion gene. The detection reagent kit comprises an mRNA reverse transcription cDNA system, wherein the mRNA reverse transcription cDNA system is used for detecting the PCR reaction of the detection reagent. The specific primer and probe technology is adopted, so that the human RET gene fusion mutation can be specifically detected; a real-time fluorescent PCR system is established, and 17 fusion mutations of the RET genes can be simultaneously detected; the sensitivity is high, the mutation of 5 to 10 copies can be detected; the operation is simple, the detection is cheap, and the clinical application range is wide; the specimen detection range is wide, and the specimen can be fresh pathological tissues, paraffin-embedded tissues or pleural fluid; the detection speed is high, and the detection process can be completed in 90 minutes.

The invention discloses a detection kit and detection method for BRAF gene mutations, and belongs to the technical field of B2D10 currently first developed high-tech industrialization important field guide / biology / novel medical precise diagnosis and treatment equipment. The detection kit is characterized by comprising two pairs of specifically amplified primers for BRAF gene mutation loci, an efficient blocking probe of a wild sequence and a BRAF gene specific TaqMan fluorescent probe. The kit can detect specimens of which the number of the mutant copies is as low as 5 to 10, and the mutation content is as low as 0.1%. The detection kit can detect five gene mutations of a BRAF gene simultaneously, the sensitivity is high, operation is easy, detection is low in price, the clinical application range is wide, samples can adopt fresh pathological tissue or paraffin-embedded tissue or a pleural fluid or serum or plasma, the detection speed is high, and only 90 minutes are needed for completing the detection process.

The to-be solved technical problem of the invention is to provide a reagent for detecting the expression situation of target protein in a paraffin-embedded tissue. The technical scheme for solving the technical problem is to provide a kit for evaluating prognosis information of an esophagus cancer patient. The kit comprises four antibodies of target protein, and the four antibodies comprise delta-catenin, Dynamin-2, Erbin and LAP-1, and the kit also comprises goat serum, 0.01 M citrate restoration liquid, 3% H2O2, a Polymer reinforcing agent, a polymer, a DAB chromogenic reagent and a PBS solution. The kit can help to relatively simply practicably evaluate prognosis of esophagus cancer patients compared with an international TNM staging method, and is higher in both sensitivity and specificity compared with the TNM staging method, and is relatively suitable for esophagus cancer prognosis evaluation of countrymen.