174 results about "Housekeeping gene" patented technology

The present invention concerns a method for the quantification of a target nucleic acid in a sample comprising the following steps: (i) determination of the amplification efficiency of the target nucleic acid under defined amplification conditions, (ii) amplification of the target nucleic acid contained in the sample under the same defined reaction conditions, (iii) measuring the amplification in real-time, (iv) quantification of the original amount of target nucleic acid in the sample by correction of the original amount derived from step (iii) with the aid of the determined amplification efficiency. The efficiency correction of PCR reactions according to the invention for the quantification of nucleic acids can be used for absolute quantification with the aid of an external or internal standard as well as for relative quantification compared to the expression of housekeeping genes.

The invention relates to a cyclic RNA molecular marker for diagnosis of gastric cancer, the cyclic RNA molecular marker is characterized in that the cyclic RNA is hsa-circ-0001017, the invention also provides a method for detection of the cyclic RNA molecular marker in plasma, and the method comprises the following steps: (1) collecting blood, and extracting total RNA in the plasma; (2) performing reverse transcription of the total RNA into cDNA; (3) performing droplet digital PCR detection of a cDNA solution of the step (2) by use of specific amplification back-to-back primers and amplification upstream and downstream primers of housekeeping gene GAPDH, after the completion of the reaction, detecting fluorescence signal values of all droplets, setting a threshold, and determining whether the droplets include the cyclic RNA or the housekeeping gene GAPDH, wherein the droplets higher than the threshold are positive droplets, and the droplets below the threshold are negative droplets; and (4) counting the number of the positive droplets, and calculating the copy number of the hsa-circ-0001017 and the housekeeping gene GAPDH in the plasma for quantitative detection of the hsa-circ-0001017 and the housekeeping gene GAPDH in the plasma. Compared with the prior art, the advantages are that the hsa-circ-0001017 can be specifically expressed in plasma in patients with gastric cancer, and can be used as a new molecular marker for diagnosis of the gastric cancer.

The present invention provides compositions and methods for simultaneously detecting mRNA expression levels of hormonal receptors, particularly both estrogen receptor (ER) and progesterone receptor (PR), optionally in combination with growth factor receptors, particularly epidermal growth factor receptor ERBB2 (Her-2), and further optionally in combination with control genes, such as the housekeeping genes NUP214 and/or PPIG. Exemplary embodiments of the invention are useful for determining hormonal receptor and/or growth factor receptor status, particular both ER and PR status and optionally also ERBB2 status, such as for assessing or treating breast cancer.

The invention relates to a microRNA standardization reference gene and an application thereof. Specifically, with Solexa sequencing, real-time fluorescence quantitative PCR detection, literature screening, statistical analysis methods, the invention carries out researches upon a large amount of samples collected from a healthy control group and different disease patients. As a result, microRNAs let-7d, let-7g, let-7i, or combinations thereof can serve as microRNA standardization housekeeping gene. Compared with reference genes (such as U6, RNU44, RNU48 and miR-16, and the like) most commonly used in current microRNA quantification, the microRNA standardization reference gene has high stability and accuracy.

The invention discloses screening and application of fluorescent quantitative reference genes of pear fruits at different development stages, and relates to the field of pear molecular biology researches. Reference genes, which are more stable than common housekeeping genes at different development stages of Cuiguan, Fengshui and Xueqing pear fruits, are pertinently obtained according to dynamic RNA (Ribonucleic Acid)-Seq sequencing results, post-period experience validation and data analysis of pear fruits of various varieties; the difference between samples and the difference in the samples, which are caused by the selection of the reference genes, are reduced to the greatest extent; the correction and standardization of a target gene expression level can be realized, and accurate quantification of functional gene expression of the pear fruits is realized.

Disclosed are a data processing and analysis method of gene expression data for identifying endogenous reference genes and a composition for the quantitative analysis of gene expression, comprising a pair of primers and/or probes useful in the amplification of the identified endogenous reference genes. Introduced with the concepts of “Zero's proportion’ and CV, the me allows different datasets to be integrally analyzed, thereby searching for novel reference genes. By the method, 2,087 genes are first found as housekeeping genes which are expressed in most tissues, and the usefulness thereof in the relative quantification of different target genes is determined by analyzing their expression stability. Out of the 2,087 genes, 13 genes are found to show higher expression stability with lower expression levels across a wide range of samples than traditional reference genes such as GAPDH and ACTB, and therefore are suitable for the normalization of universal genes having relatively low expression levels.

The invention relates a method for measuring miRNA absolute expression quantity in biological samples, which comprises the following steps: 1) material selection: 8 weeks and 40 weeks of mouse brain tissues are selected; 2) total RNA extract; 3) RT(reverse transcription)-Polymerase Chain Reaction (PCR); 4) quantifying DNA; 5) drawing specification curve; 6) data processing: loss rate and average loss rate of little RNA is figured out in the process of extracting RNA according to the copy number of spike RNA in the extracted total RNA which is detected by the PCR; the expression quantity of every initial cell of the little RNA at the levels of DNA and RNA is figured out according to the cell number of original sample that is respectively expressed from the levels of DNA and RNA. The method has the advantages of miRNA expression quantity is faithfully transformed into the absolute expression quantity in the original sample through the cell number in the sample homogenate which is worked out from the two levels of DNA and RNA; the disadvantage that relative differentiation can only be reflected by taking housekeeping genes as an internal reference, and the analysis is truer and more reliable from the two levels of DNA and RNA.

The present invention relates to a quantitative detection analysis method of human plasma DNA level. Said method includes the following steps: selecting artificially-synthetic exogenous DNA series whose extraction efficiency is identical to or similar to that of human plasma DNA, but they are non-homologous, selecting housekeeping gene beta actin as determination gene, inserting artificially-synthetic exogenous DNA into carrier pMD18T and placing it in the bacteria to make culture, making blue and white spot screening, colony PCR identification, enzyme-cutting into linearity, placing the above-mentioned material into plasma, adopting double fluorescent quantitative gene amplification, fluorescent groups are respectively FAM and HEX, extracting known exogenous positive plasmid recovery efficiency, using computation formula to make computation so as to obtain the quantity of plasma DNA.

The invention discloses a duplex PCR authentication method of cordyceps sinensis original powder. The method adopts a technical scheme that cordyceps sinensis original powder genome DNA is adopted as a template; with PCR primers designed by the invention, a single-tube duplex PCR reaction is carried out, and characteristic housekeeping genes of a cordyceps sinensis strain and a host hepialus are simultaneously amplified, wherein actual amplified fragments are respectively 320bp and 136bp; and through agarose gel electrophoresis detection, product authenticity is determined. With the method provided by the invention, cordyceps sinensis original powder can be specifically identified, and sample authenticity detection can be completed simply with three steps of DNA extraction, PCR amplification, and electrophoresis detection. The operation is simple and is easy to command, and accuracy is high.

The invention discloses a kit for detecting helicobacter pylori drug resistance gene polymorphism by a multiplex fluorescence PCR melting curve method. The helicobacter pylori drug resistance gene comprises the following three genes: a 23S rRNA gene, a 16S rRNA gene and a gyr A gene, the kit comprises a nucleic acid extraction reagent and a nucleic acid amplification reagent, the nucleic acid extraction reagent comprises superparamagnetic silicon oxide nano magnetic beads, a lysis solution, a washing solution and an eluent; the nucleic acid amplification reagent comprises a primer pair and a probe which respectively correspond to the helicobacter pylori drug resistance gene 23S rRNA gene, the helicobacter pylori 16S rRNA gene, the gyr A gene and an internal standard gene human housekeepinggene beta-globin. According to the invention, three drug-resistant sites and one internal standard gene can be simultaneously detected in a single tube, so that the detection flux of a sample is improved; meanwhile, interference between multiple pairs of primer probes in a detection system is improved, and the sensitivity and specificity of reagent detection are effectively improved.

Disclosed is a method which enables an early stage detection of an adenoma or cancer by gene expression analysis of a biomarker in a readily collectable sample. Specifically disclosed is a method for detecting an adenoma or cancer, which comprises the steps of: measuring the quantity of a sequence constituting at least one housekeeping gene or an expression product thereof contained in a body fluid sample or an excrement sample collected from an examinee; and calculating the concentration of the sequence in the sample.

The invention relates to an illegal cooling oil identification method. The method comprises the following steps of: performing deoxyribonucleic acid (DNA) enrichment extraction of an oil sample to be detected; performing enrichment extraction on the obtained DNA by polymerase chain reaction amplification to obtain an amplified DNA segment of the oil sample to be detected; and judging whether the amplified DNA segment of the oil sample to be detected comprises at least one of a plurality of species specificity housekeeping gene segments or not by taking the plurality of species specificity housekeeping gene segments as a detection index, wherein the plurality of species specificity housekeeping gene segments are species specificity housekeeping gene segments of non-oil plants or species specificity housekeeping gene segments of animals. According to the method provided by the invention, the species specificity housekeeping gene segments of the non-oil plants serve as the detection index, so high specificity of the detection index is guaranteed; the detection sensitivity of the method provided by the invention is improved by the polymerase chain reaction; the DNA extraction efficiency of the oil sample to be detected is obviously improved after DNA enrichment treatment; and compared with the prior art, the illegal cooling oil identification method has the advantages of low equipment cost and simple and convenient detection method.

The invention provides a method for quickly detecting klebsiella pneumoniae triple qPCR in a respiratory tract sample. The method comprises the following steps: collecting and selecting a clinical respiratory tract sample; performing bacterial culture and identifying; extracting and purifying nucleic acid; constructing quality control/interior label plasmid; designing and compounding a sequencing primer; optimizing a clinical isolate high-fidelity system, amplifying and judging a result; sequencing a high-fidelity PCR product and controlling quality; designing a triple qPCR primer and a probe and compounding a marker; and optimizing a triple qPCR system, amplifying and judging the result. The method has the beneficial effects that klebsiella pneumoniae rcsA and 23s RNA housekeeping genes can be simultaneously detected, and the adopted primer and probe are designed in the manner of effectively avoiding a variation area or a mutational site according to a gene sequencing result of popular distribution strain in China; a quick sputum liquefying bactericide is added into a sputum sample, so that the sputum can be quickly liquefied, pathogenic microorganisms can be killed, and an operator can be protected from being infected; and compared with the traditional method, the method provided by the invention can reduce the time by about 1 hour and has the characteristics of simple and convenient operation, high speed, safety and sensitivity.

The invention provides methods to monitor cell free nucleic acids. The method comprises obtaining a plasma sample from a subject known to have a cancer characterized by a pair of mutually exclusive mutations specific to the cancer; isolating cell free nucleic acids from the plasma sample obtained from the subject; measuring the amount a housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the plasma sample to confirm that the amount of housekeeping gene and/or total DNA in the sample is within a selected range; measuring the amount of a first of the pair of mutually exclusive mutations specific to the cancer in the cell free nucleic acids isolated from the plasma sample; and indicating in a report that the subject has the first mutation when (a) the amount of the housekeeping gene and/or total DNA in the cell free nucleic acids isolated from the plasma sample is within the selected range and (b) the amount of the first mutation is increased as compared to a control amount, wherein the control amount is determined by measuring the apparent amount of the first mutation in control cell free nucleic acids isolated from plasma samples obtained from control subjects known to have the second of the pair of mutually exclusive mutations specific to the cancer using measuring conditions substantially the same as those used to measure the amount of the first mutation in the cell free nucleic acids isolated from the plasma sample from the subject.

Recurrent gene fusions in prostate cancer of androgen regulated genes or housekeeping genes and ETS family member genes are described. Compositions and methods having utility in prostate cancer diagnosis, research, and therapy are also provided.

The present invention discloses a method to test the growth rate of the skeletonema costatum, which includes the procedures as follows: a. the extraction of RNA of skeletonema costatum, b. separation and cloning of partial sequences of the PCNA gene of the skeletonema costatum, c. cloning of partial sequences of Cytb gene of skeletonema costatum, d. a standard real-time quantitative PCR curve used to test genes of PCNA and Cytb of skeletonema costatum is developed, based on the gene sequences of both PCNA and Cytb, e. the growth rate of the skeletonema costatum is tested based on the expression of quantity (REQ) of gene PCNA of skeletonema costatum, with PCNA as the target gene and the Cytb as the housekeeping gene. The present invention provides a technical method for accurate estimation on the growth rate on the spot of the skeletonema costatum, which can be used to estimate the productivity of the floating algae, study the variance of the alga species, construct the zoological dynamic model and even forecast the red tide.

The invention discloses a selection method of reference genes in quantitative real-time PCR analysis of jerusalem artichoke, and relates to the field of quantitative PCR. The method comprises the following steps: taking different tissues (radicles, immature stems, leaves, stem blocks and petals) of the jerusalem artichoke as materials, and carrying out expression analysis of the reference genes of18S ribosomal RNA gene (18S rRNA), transcription elongation factor gene (Ef-1a), actin gene (Actin and beta-actin), 3-glyceraldehyde phosphate dehydrogenase (GAPDH), 25S ribosomal RNA gene (25S rRNA)and poly-ubiquitin enzyme gene (UBQ)7 by using a q PCR technology; carrying out statistical assessment on the obtained data and analyzing expression change of all housekeeping genes by utilizing GeNorm and NormFinder software, so as to screen out relatively-stable genes as the reference genes of the jerusalem artichoke, which are used for studying the gene dosage changes of the jerusalem artichoke.

The invention relates to a method of analyzing and quantifying thallus quantity of probiotics and pathogenic bacteria by means of a molecular biological technique. The method comprises the following steps: S1, dissolving a test article in sterile water, performing high speed centrifugation to separate thalli, and adding the sterile water to dilute as a to-be-detected bacterial sample; S2, matching the to-be-detected bacterial sample with a target bacterial special primer set with a real-time quantification polymerase chain reaction, wherein the target bacterial special primer set is a conserved gene of the target bacteria, including an inner transcription region or a housekeeping gene sequence fragment of 16S rDNA, 23S rDNA and 16S-23S rDNA as an amplification target, and the target bacteria are probiotics and pathogenic bacteria; and determining whether the to-be-detected bacterial sample contains a non-target culture or not according to the condition that whether a melting curve of the amplification fragment has a specific wave crest or not; and S3, introducing the obtained Ct value of the test article into a regression equation of a standard curve of a standard substance to obtain the thallus quantity of the to-be-detected bacterial sample.

The invention belongs to the technical field of detection kits, and particularly relates to a kit for aided-diagnosis and prognostic evaluation on glioblastoma based on a gene CLCF1 and a use method thereof. The kit comprises a PCR (polymerase chain reaction) primer pair for amplifying the gene CLCF1 and a PCR primer pair for amplifying a housekeeping gene GAPDH. The kit has the advantages that the obvious difference of the gene CLCF1 in transcription expression levels of LGG (brain lower grade glioma) and GBM (glioblastoma multiforme) is found for the first time, the prognostic condition of apatient with the glioblastoma is well judged by the expression level of the gene CLCF1, and the gene CLCF1 can be used as the index for independently judging the prognostic condition of the glioblastoma after determining by Cox multi-factor regression analysis; after the expression condition of the transcription level of the gene CLCF1 is detected, the severity of the glioblastoma can be diagnosed, and the prognostic condition of the patient can be predicted.

The invention provides a method for judging whether cancer cells are present or not in a sample, comprising a obtaining step of obtaining values related to an expression level of a caner marker gene and a housekeeping gene; a first comparing step of comparing the value related to the expression level of the cancer marker gene with a first threshold value; a normalizing step of normalizing the value related to the expression level of the cancer marker gene based on the value related to the expression level of the housekeeping gene; a second comparing step of comparing the normalized value with a second threshold value; and a judging step of judging whether cancer cells are present or not in the sample based on comparison results obtained in the first and second comparing steps, as well as an apparatus for judging whether cancer cells are present or not.

The invention relates to a closed-tube visualized LAMP (loop-mediated isothermal amplification) detection method for Aermonas hydrophila sourced from Anguilla spp. and relates to detection of pathogenic microorganisms of aquatic animals. The method comprises steps as follows: extracting a genome of Anguilla spp.; extracting a genome of pathogenic bacteria; preparing a positive control; preparing a negative control; detecting a sample and determining a result. LAMP primers are designed by taking conserved sequences of housekeeping gene gyrB of Aermonas hydrophila and pathogenic factor hlyA, an LAMP reaction system is established, and whether a target gene is amplified effectively can be determined in a closed-tube visualized manner by adding different pH indicators to an LAMP reaction. With adoption of the closed-tube visualization for rapid detection of pathogenic Aermonas hydrophila sourced from Anguilla spp., a reaction product is not needed to be identified through electrophoresis and/or open-tube addition of fluorescent dyes and the like, so that the pollution risk of the LAMP product is reduced.

The invention discloses a library of artificial promoter regulatory elements with different expression intensities in corynebacterium glutamicum. Firstly, according to the core region of promoters ofcorynebacterium glutamicum housekeeping genes and the conserved sequences of the -10 region and the -35 region, the corynebacterium glutamicum artificial promoter regulatory element library is designed and built. Each regulatory element comprises a promoter region and a ribosome binding site region, and a promoter upstream region exists. Subsequently, the artificial promoter regulatory element library is subjected to a three-step screening method of flow cytometry screening, plate screening and 96-well plate screening, and the artificial promoter regulatory element library with with differentexpression intensities and wide control range is obtained.