59 results about "Rna degradation" patented technology

A method for isolating nucleic acid molecules having a repeating nucleotide sequence or a homopolymeric nucleotide sequence, e.g. a poly A stretch, is described. In particular, the method uses oligomeric capture probes spiked with various amounts of locked nucleic acid (LNA). The invention further describes methods for the isolation of RNA molecules, for example polyadenylated mRNA molecules, which overcome the problems of rapid RNA degradation during isolation and analysis of such nucleic acid molecules. This is of major clinical and diagnostic importance, especially when dealing with RNA viruses, such as retroviruses or when analyzing rare or low-abundant mRNAs or mRNAs from biopsies or tissues enriched with RNases.

The present invention relates to the field of synthesis of short double-stranded RNAs. An in vitro transcription method using bacteriophage polymerases and target sequence-specific single-stranded DNA oligonucleotides as templates is disclosed. The present invention finds particularly advantageous use in the synthesis of short interfering RNAs (siRNAs) that have been shown to function as key intermediates in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates and vertebrate systems.

The invention provides an RNA protective agent, a kit containing the protective agent, application and a preservation method of RNA. The protective agent contains a trehalose solution with a concentration of 0.27M-1M. The protective agent provided by the invention can achieve the effects of long preservation time, temperature insensitivity and wide range of use temperature, an extracellular RNA sample treated by the protective agent has no influence to cDNA synthesis, PCR reaction and enzyme activity in sequencing and bank building. The protective agent provided by the invention can efficiently solve the RNA degradation, pollution and other problems encountered during long-term preservation or use of exRNA, and especially has important significance for preservation, repeated use and long-distance transportation of extracellular microRNA.

This invention relates to the determination of RNA quality, or RNA integrity, in a biological sample. The extent of RNA degradation, or retention of RNA integrity, is determined based upon the comparison of the relative amount of two sequences of a representative RNA molecule in the sample. Compositions and methods related to the determination are provided to assess RNA quality.

The invention discloses a method for detecting lncRNA in plasma. Through steps of TrizolLS pretreatment of theplasma, chloroform and isoamyl alcohol mixed liquid extraction, isopropanol precipitation, ethanol washing, RNA RT (reverse transcription), PCR (polymerase chain reaction) detection and the like, a TrizolLSreagentcan well remove impurities such as polysaccharide, mucoprotein and the like in blood, RNA degradation is less, the phenomenon thattrichloromethane is easy to emulsify under the TrizolLS strong acid condition is effectively eliminated by a chloroform and isoamyl alcohol mixed liquid, high-quality and high-content extraction of RNA in the plasma is realized, fluorescence quantitative RT-PCR detection is performed on the quality of lncRNA, and meanwhile, a reference Ct (cycle threshold) value of the total RNA (that is, cDNA) mass can be detected.

The invention in particular relates to a method for the quantification of in vivo RNA from a biological sample comprising the steps of: collecting said biological sample in a tube comprising a compound inhibiting RNA degradation and / or gene induction; forming a precipate comprising nucleic acids; separating said precipate from the supernatant; dissolving said precipitate using a buffer, forming a suspension; isolating nucleic acids from said suspension using an automated device; dispersing / distributing a reagent mix for RT-PCR using an automated device; dispersing / distributing the isolated nucleic acids within the dispersed reagent mix using an automated device, and determining the in vivo levels of transcripts using the nucleic acid / RT-PCR reagent mix in an automated setup. The present invention also relates to the quantitatification of DNA from a biological sample. The present invention further elucidates a kit for isolating quantifiable nucleic acids from a biological sample. Applications of the method according to present invention are aldo disclosed.

The invention provides a virus sample inactivation cracking test kit. The virus sample inactivation cracking test kit comprises ammonium salt, guanidine isothiocyanate, lithium chloride, ethylenediamine tetraacetic acid and the like. The invention also provides a target nucleic acid magnetic capture reagent and a target nucleic acid capture test kit. The target nucleic acid magnetic capture reagent and the target nucleic acid capture test kit comprise magnetic polymer microspheres, an intermediate probe, a capture probe and the like. The invention also provides a primer pair and a real-time fluorescent nucleic acid isothermal amplification detection test kit for amplifying and detecting target nucleic acid, and a test kit used for virus inactivation, capture and real-time fluorescent isothermal amplification detection. The inactivation cracking test kit disclosed by the invention can realize quick inactivation at a room temperature and reduce RNA degradation, and is high in sensitivity; the target nucleic acid magnetic capture reagent has stronger specificity, and can obtain templates with good quality and large quantity; and a real-time fluorescent nucleic acid isothermal amplification technology enables reverse transcription and amplification to be carried out together, so the reaction time is shortened, and the sensitivity of a detection reagent is improved.

The invention discloses a purpose of a polyether compound in medicine preparation and an anti-tumor medicine screening method. The medicine is used for regulating and controlling at least one of the following signal channels: a calcium ion signal channel, a Wnt signal channel, a Hippo signal channel, a TNF signal channel, a VEGF signal channel, a p53 signal channel, an NF-kappa B signal channel, an Ras signal channel, an mTOR signal channel, an RNA degradation signal channel, a PPAR signal channel, a cAMP signal channel, an HIF-1 signal channel, an Insulin signal channel, an AMPK signal channel, an Rap1 signal channel, an apoptosis signal channel, a cell cycle signal channel, a cGMP-PKG signal channel, a DNA copying signal channel, an MAPK signal channel, a PI3K-Akt signal channel and an autophagy signal channel. The polyether compound provided by the invention can cause the tumor cell death through regulating and controlling various signal channels; and a better anti-cancer effect isachieved.

The invention discloses a highly compatible virus preserving fluid for a paramagnetic particle method virus nucleic acid extracting kit. The highly compatible virus preserving fluid comprises the following components: guanidine hydrochloride, tris (hydroxymethyl) aminomethane hydrochloride, ethylenediamine tetraacetic acid, isopropanol or ethanol. The virus preserving fluid disclosed by the invention belongs to an inactivated type, has the effects of preserving and cracking a virus sample, and can be compatible with various common paramagnetic particle method virus nucleic acid extracting kitsin the market; during nucleic acid extraction, the sample and a lysate can be mixed according to any proportion between the proportion suggested by the kit and 100%; and in other words, less or evenno lysate can be added, so that the use amount of the sample is increased, and more virus nucleic acids and more accurate detection results are obtained. The virus preserving fluid has the advantagesof simple components, easily available raw material and relatively low cost, can preserve a sample at room temperature for a long time, does not need high-temperature inactivation, is very safe to operators and the environment, reduces the possibility of RNA degradation, can obtain relatively more nucleic acids, and can lower the omission ratio.

The present invention relates to mda-5 nucleic acids, MDA-5 proteins, the mda-5 promoter, and related molecules, and the use of each of these elements in protecting against or limiting viral infection, controlling cell proliferation, and promoting apoptosis. It is based upon the discovery that, contrary to earlier hypotheses based on the amino acid sequence of MDA-5, the protein is not a defective ATPase but rather exhibits ATPase activity. This supports the role of MDA-5 as a RNA helicase protein and consequently its use in promoting RNA degradation, for example in the context of antiviral defense, limitation of cell proliferation, or induction of apoptosis.

The present invention discloses a use of cholesterol-modified HuR interference siRNA in preparation of drugs for treating acute lung injury caused by sepsis. According to the present invention, the related HuR interference siRNA is an artificially-synthesized and cholesterol-modified small nucleic acid drug; with intravenous administration, results show that the HuR siRNA can be richly distributed in lung tissue, enter lung cells, inhibit a plurality of 3'-UTR of mRNA of inflammatory factors having HuR binding sites, and promote TNFalpha RNA degradation so as to inhibit acute lung injury induced by bacterial lipopolysaccharide (LPS), wherein TNFalpha expression can be inhibited; and the small nucleic acid drug provides significant release effects for lung inflammation, is applicable for intravenous administration, inhalation administration or intratracheal administration, and is the small nucleic acid drug having application values and prospects and provided for treating acute lung injury under the premise of lack of effective and related acute lung injury treatment nucleic acid drugs in the prior art.

The invention discloses a method for judging blood stain formation time in forensic medicine by detecting RNA degradation degree. According to the invention, 18S rRNA is selected as a basis, and a 44bp short fragment, a 149bp middle fragment and a 305bp long fragment are respectively used as target sequences to design primers. Real-time fluorescent quantitative PCR (qPCR) detection is carried outby using the three primers with short, medium and long fragments. The method comprises the following steps that: qPCR reaction is carried out on the standard substance subjected to gradient dilution to construct a standard curve; a Ct value is obtained through qPCR amplification detection; and the initial template copy number can be quantified by substituting the Ct value into a standard curve. Along with the increase of time, the probability of successful amplification of the long-fragment target sequence is the lowest, and the probability of successful relatively stable amplification of medium fragments and short fragments is high. And the integrity of the RNA in the human blood stain sample is judged by calculating the ratio of the initial copy numbers of the long fragment and the shortfragment and the ratio of the initial copy numbers of the middle fragment and the short fragment. By analyzing the linear relationship between the RNA integrity and the time, a technical means is provided for researching the blood stain retention time, the death time, the injury time and the like.

The invention discloses a quick osseous tissue RNA extraction kit. The kit comprises at least a genomic DNA removal column, at least one RNA absorptive column, lysis buffer used cooperatively with the genomic DNA removal column and elution buffer used cooperatively with the RNA absorptive column. The kit has the advantages that the exclusive phenol / chloroform-free lysis buffer is used and a plurality of ingredients are added to remove proteoglycan. A particular buffer system and a bimodal pore silica gel purification system are utilized to solve the problem of remaining DNA, thus osseous tissue RNA with high purity and good integrity can be obtained. The particular genomic DNA removal column can effectively remove remaining genomic DNA (gDNA), thus the extracted RNA can be directly used in reverse transcription PCR and real-time PCR without DNase digestion. Extraction of single samples can be completed in 35 minutes generally, and RNA degradation due to long-time extraction, RNase contamination and other causes is avoided.

The present invention relates to the field of synthesis of short double-stranded RNAs. An in vitro transcription method using bacteriophage polymerases and target sequence-specific single-stranded DNA oligonucleotides as templates is disclosed. The present invention finds particularly advantageous use in the synthesis of short interfering RNAs (siRNAs) that have been shown to function as key intermediates in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates and vertebrate systems.

The present invention relates to mda-5 nucleic acids, MDA-5 proteins, the mda-5 promoter, and related molecules, and the use of each of these elements in protecting against or limiting viral infection, controlling cell proliferation, and promoting apoptosis. It is based upon the discovery that, contrary to earlier hypotheses based on the amino acid sequence of MDA-5, the protein is not a defective ATPase but rather exhibits ATPase activity. This supports the role of MDA-5 as a RNA helicase protein and consequently its use in promoting RNA degradation, for example in the context of antiviral defense, limitation of cell proliferation, or induction of apoptosis.

The invention provides a virus preservation solution, a nucleic acid amplification reaction preparation (which can be in the form of freeze-dried powder), a qPCR-based method for detecting viruses through nucleic acid amplification, and a PCR kit. The virus preservation solution can preserve a virus sample for a long time at room temperature and prevent RNA degradation. The sample solution can bedirectly mixed with a nucleic acid amplification reaction preparation to be used for qPCR amplification, a virus nucleic acid extraction step is not needed, and viruses can be rapidly detected with high sensitivity.

The invention discloses a rapid separation and detection kit and method for fragmented crosslinked DNA. The kit includes a reagent for decrosslinking and separation extraction of fragmented DNA, and the reagent is Chelex 100. The kit can further include a protein degradation reagent, a DNA ladder marker, a sample loading buffer solution and / or an RNA degradation reagent. Chelex 100 can integrate multivalent metal ions, has high metal ion selectivity and binding force, can separate DNA and prevent DNA degradation. The rapid separation and detection method for fragmented crosslinked DNA can determine whether a to-be-detected sample needs further ultrasound or is used for a next step precipitation experiment according to the effect of rapid quality control ultrasound fragmentation, or can be used for ultrasound optimization of a plurality of to-be-detected samples so as to select optimum conditions. The kit and the method provided by the invention can be used for rapid detection of chromatin ultrasound fragmented sample quality in chromatin immunoprecipitation assay (ChIP) and other technologies, and can greatly improve the success rate of ChIP.

The present invention is to provide a pretreatment method that allows RNA to be detected promptly and simply. RNA degradation activity due to lactoferrin present in the human rhinal mucosa is inhibited, for example, by adding iron ion and carbonate ion to a biological sample that contains the human rhinal mucosa. With the pretreated biological sample, an RNA virus gene can be amplified by a reverse transcriptase. Iron ion and carbonate ion can also inhibit reverse transcriptase inhibition due to lysozyme C contained in the human rhinal mucosa. Further, it is preferable to remove the envelope of the RNA virus by adding SDS to the biological sample that contains the human rhinal mucosa.

Provided are methods of determining a response to a chemotherapeutic agent in a subject with ovarian cancer, comprising: determining a RNA integrity value of a sample comprising ovarian cancer cell RNA from the subject after the subject has received one or more doses of the chemotherapeutic agent; wherein a low RNA integrity value and / or RNA degradation of the cancer cell RNA is indicative that the cancer is responding to the chemotherapeutic agent and / or a high RNA integrity value and / or stable RNA integrity of the ovarian cancer cell RNA is indicative that the cancer is resistant to the chemotherapeutic agent.

The invention relates to a real-time fluorescent quantitative RT-PCR detection primers, probe and kit for Palyamserogroup virus, and belongs to the technical field of animal virus molecular biologicaldetection. The kit comprises an upstream primer, a downstream primer, the probe matched with the primers for use, a negative control template, a positive control template, a standard template and a PCR amplification reagent. The kit is adopted for detection, the reaction speed is high, and the whole amplification process is less than 1 hour; only virus RNA needs to be extracted, reverse transcription is not needed, operation steps are few, easy and convenient, and RNA degradation and pollution can be effectively avoided; meanwhile, after qRT-PCR amplification is completed, whether PALV RNA exists in a sample to be detected or not can be directly judged without agarose gel electrophoresis; and in addition, a standard curve established with the help of the standard template can be used forquantitatively detecting the PALV RNA in the clinical sample, so that the working efficiency is greatly improved, and the detection cost is reduced.

The invention relates to a rapid extraction method for total RNA of an acaudina molpadioides digestive tract. At present, a sample is relatively difficult to process before the total RNA of the acaudina molpadioides digestive tract is extracted, the extraction yield of the total RNA is not high, and the total RNA is easy to pollute. The method comprises the following steps of: grinding a liquid nitrogen frozen acaudina molpadioides digestive tract tissue by using a mortar, putting the powder into TRIZOL lysis solution for lysis and homogenization; homogenizing and centrifuging the tissue, taking supernatant fluid, adding chloroform, shaking, blending, centrifuging and taking the supernatant fluid; adding low temperature isopropyl alcohol, blending, and centrifugating; washing by using 75 percent of ethanol; and dissolving precipitates by using RNA (Ribose Nucleic Acid) enzyme-free deionized water, and preserving at the temperature of between -90 and -70 DEG C. According to the method, the RNA degradation of the sample is effectively avoided, the RNA precipitation effect is improved, the extracting time is short, the RNA integrity is high, and the requirement of real-time quantitative PCR (polymerase chain reaction) detection is completely met.

The invention discloses multiple PCR primers for detecting lily viruses. The lily viruses are cucumber mosaic virus (CMV), lily symptomless virus (LSV) and lily mottle virus (LMoV) respectively. Meanwhile, the invention further provides application of the multiple PCR primers in RNA extraction-free rapid detection method of lily viruses. The multiple PCR primers have the advantages that firstly, an RNA extraction method is simplified, a dissecting needle is adopted for picking trace subcutaneous tissues of lily and rapidly transferring the trace subcutaneous tissues to a reverse transcriptionreaction solution, a sampling method is simple, convenient and easy to operate, intermediate links capable of causing RNA degradation and pollution are omitted, an RNA extraction kit is not needed, the detection cost is reduced, and the working efficiency is improved; and secondly, lily virus CMV, LSV and LMoV primers are independently designed and developed, three pairs of specific primers with clear PCR product electrophoresis bands and good repeatability are screened out, amplified fragments are recycled for sequencing, the sequence alignment homology reaches 98% or above, it is proved thatgene fragments obtained through amplification of the primers designed and developed through the method are lily CMV, LSV and LMoV indeed, and it is guaranteed that a detection result is true and reliable.

The invention relates to an efficient extracting method for Wuta-tsai total RNA. The method comprises the steps that liquid nitrogen is added into Wuta-tsai leaves, grinding is carried out, the centrifugal rotating speed is increased, ethyl alcohol of different concentrations is used for washing, and RNA sedimentation is carried out, so that extraction of high-purity, high-concentration and degradation-free Wuta-tsai total RNA is achieved. The method for scientifically and systematically extracting total RNA from the Wuta-tsai leaves is provided for the first time, the extraction efficiency is improved, and the problem that pollution is produced in the extraction process to cause RNA degradation can be effectively avoided. By the adoption of the method, Wuta-tsai total RNA extraction can be completed within only 50-55 min, the high-purity, high-concentration and degradation-free total RNA is obtained, and deepening of follow-up study is facilitated.

The invention relates to the technical field of biology, in particular to a single cell nucleus extraction method suitable for frozen tissue. According to the method disclosed by the invention, the single cell nucleus is successfully separated and extracted from the frozen tissue in a relatively shorter time by using the NEBCS nucleus separation liquid, and the more pure single cell nucleus is obtained through differential centrifugation. The proportion of cell nucleus RNA degradation is greatly reduced, so that protein coding RNA with a higher proportion can be obtained at the single cell level, and the proportion of mitochondrial genes is effectively reduced. The mononuclear extraction method provided by the invention is suitable for various types of frozen tissues, and further degradation by an RNA method is effectively prevented. The operation process is simple, the extraction process of the cell nucleus can be completed by using common laboratory instruments such as a centrifugal machine and a microscope, and the whole process only needs 30-60 minutes. Special instruments and devices are not needed, so that the extraction cost is effectively reduced.

The invention relates to a method for quickly and stably picking a fragile tissue of an animal for experiments, composed of a rectangular plastic vessel and a sepharose gel female die for the fragile tissue of the animal for experiments. Specifically, a female die formed of sepharose gel is arranged in a plastic container, and capable of fixing the fragile tissues such as brains of experimental animals. The size and height of the plastic container can be selected according to the size of the tissue picked for experiments. Transparent sepharose gel is convenient for observing the picked tissue structure in each direction. A device for quickly and stably picking the fragile tissue of the animal is capable of performing precooling at 4 DEG C, and put on ice for operation, thereby preventing the protein or RNA in the tissue from being degraded. The device is capable of conveniently and completely cutting off the picked tissue, and the hardness of the sepharose gel can be adjusted according to experimental material picking requirements. A plaster male die for the fragile tissue of the animal for experiments can be used repeatedly. The sepharose gel can be prepared at different solution proportions according to the specific experimental purpose, such as PBS buffer solution containing a protease inhibitor, DEPC (Diethylpyrocarbonate) water having inhibition effect on ribozyme. The method provided by the invention is simple in operation, easy to control and low in cost; and the tissue picked is disposable in experiments.

The invention provides an optimized extraction method of buckwheat RNA (ribonucleic acid). According to the method, cetyl trimethyl ammonium bromide is particularly adopted for optimized extraction of RNA of buckwheat roots, stems and leaves; required glassware, a gun head, an EP (epoxy resin) pipe and deionized water are subjected to high-pressure moist heat sterilization and disinfection; substances such as polysaccharides and phenols are removed by CTAB (cetyl trimethyl ammonium bromide); the phenols are removed by polyvinylpyrrolidone and beta-mercaptoethanol; an RNA precipitation effect of isopropanol is obvious; a Tris-HCl buffer solution and NaCl exerts an effect of avoiding RNA degradation. The method can extract RNA of different parts of buckwheat, such as the roots, the stems and the leaves; an extraction concentration can reach 100-500 ng / microliter; the method can be used for subsequent experiments of RT-qPCR (real-time-quantitative polymerase chain reaction), RACE (rapid-amplification of complementary deoxyribonucleic acid ends) and the like. The method is easy, simple and quick to operate and low in cost, and obtained RNA is higher in concentration and purity.

The invention relates to a gene probe for alveolar soft part sarcoma and application of a gene probe kit. Clonal fragments selected and used by the gene probe are RP11-634L10, RP11-51H16 and RP11-475F12 combination as well as CTD-2311N12, RP11-416B14, CTD-2522M13, CTD-2312C1 and CTD-2248C21 combination respectively. According to the gene probe, the defects of an influence on prognostic and postoperative treatment and the like due to a tedious and time-consuming RT-PCR (reverse transcription-polymerase chain reaction) and cell karyotype analysis method, limitation of RT-PCR application by RNA degradation and numerous other factors, and inaccurate diagnosis are overcome; the gene probe is high in accuracy, specificity and success rate, strong in fluorescent signal and easy and convenient to operate, and can be applied to paraffin sections; the specimen detection range is extended, a novel method for accurately, reliably, simply and conveniently diagnosing the alveolar soft part sarcoma is built, and a precedent for detecting the alveolar soft part sarcoma by FISH (fluorescence in situ hybridization) is created.

The invention relates to the technical field of biochemistry, in particular to a method for extracting total RNA (ribose nucleic acid) of a kenaf anther, and solves the technical problems that the kenaf anther RNA is easily degraded, and a product is easily contaminated by kenaf secondary metabolite, polysaccharide polyphenol, in an extraction process in the prior art. According to the invention,saturated guanidinium isothiocyanate is used to replace sodium chloride, and an improved CTAB method is adopted to extract the total RNA of the kenaf anther; by adopting the extraction method providedby the invention, the degradation of the total RNA of kenaf is effectively inhibited, and the purity and the integrity of the total RNA of the kenaf are improved, and the reagent cost is low, and theoperation procedure is simple.