27 results about "Rna degradation" patented technology

The invention relates to the technical field of plants, in particular to a paraffin embedding method for reducing plant RNA degradation. In the plant paraffin embedding method provided by the invention, a stationary liquid for fixing plant tissues is prepared from absolute ethyl alcohol and acetic acid in a volume ratio of (2-5): (1-4); buffer I used for dehydration is absolute ethyl alcohol and n-butyl alcohol with the volume ratio of (1-3): (1-2); buffer II used for waxing is n-butyl alcohol and low-temperature paraffin wax with the volume ratio of (1-3): (1-2), and the melting point of the paraffin wax is 20-50 DEG C. According to the method, low-temperature paraffin with the melting point of 20-50 DEG C is adopted, and a reagent for paraffin embedding and a paraffin embedding process are adjusted, so that the plant section with relatively high RNA integrity and relatively high tissue structure integrity is obtained. The paraffin embedding method provided by the invention is suitable for different types of molecular biology experiments of space transcriptome, immunohistochemistry, immunofluorescence and the like.

The invention provides a frozen section method for space transcriptomics analysis of mesentery and regenerative intestinal tract of stichopus japonicus, and belongs to the technical field of biotechnology. The method comprises the steps of sample treatment and embedding, pre-cooling by a freezing slicer, freezing and slicing, and flaking. According to the frozen section method for space transcriptome analysis of the mesentery and the regenerative intestinal tract of the stichopus japonicus, provided by the invention, a fresh material is directly embedded, and a plurality of thin tissue blocks with the same size are integrated into a standard tissue block of 1cm, so that the high coverage rate of a tissue cross section in a space transcriptome chip capture area is ensured; rNA degradation is effectively prevented, and finally, the cDNA library is successfully constructed and is used for subsequent analysis of a space transcriptome. The technical scheme has the characteristics of simplicity and convenience in operation and stable effect, and is particularly suitable for space transcriptomics frozen section preparation of thin film type tissues.

A protection method, a protection sequence, a composition and a kit for preventing RNA degradation, and the use thereof, which can improve the protective effect on viral RNA. After the protection sequence RNase AP is added, the three-dimensional structure of RNA and the structural allosteric characteristics of RNA can be protected; and the protection sequence has a good protection effect on RNA integrity, and a high-level detection rate can be obtained by a one-time amplification, thereby ensuring efficient enrichment of viral RNA and providing guarantee for subsequent reverse transcription and PCR amplification.

This disclosure provides several ribonuclease R variants and their preparation methods. Compared to the parent enzyme, the variants described in this disclosure exhibit increased protease activity per unit and show promising prospects for industrial applications. Furthermore, this disclosure also provides the application of ribonuclease R variants in linear RNA degradation.

The invention discloses a single cell whole genome methylation and transcriptome biomics library construction method, and relates to the technical field of sequencing methods.The method comprises the steps that a single cell is added into a sorting tube with a preset lysis buffer for lysis, a nucleophilic coating is arranged on the inner wall of the sorting tube to enable cell nuclei to be adsorbed, and supernate containing mRNA is obtained; carrying out reverse transcription on supernate to obtain cDNA (complementary deoxyribonucleic acid) and constructing a transcriptome library; splitting the cell nucleus to release DNA (Deoxyribonucleic Acid) and carrying out hydrosulfite conversion; the transformed DNA is connected with a molecular tag-containing joint to obtain a methylated intermediate product, after exonuclease treatment, the intermediate products with different molecular tags are combined, and the methylated library is obtained through magnetic bead purification, single chain, connection with a sequencing joint at the other end, amplification and purification. According to the technical scheme, RNA and DNA are separated in the same tube and a library is established in parallel, separation and transfer steps are reduced, RNA degradation and nucleic acid loss are reduced, and methylation library establishment flux and biomics data consistency are improved.

The invention belongs to the technical field of biomedicine, and particularly relates to a method for preparing a cell suspension through animal spleen dissociation. According to the method, the tissue is fully exposed during contact with enzyme by adopting a secondary crushing mode, the contact area of digestive enzyme and the tissue is increased, the tissue dissociation is more sufficient, and the dissociated cell suspension meets the requirements of single cell sequencing in the aspects of activity, living cell quantity, caking rate and the like in cooperation with the special digestive juice; according to the method, the high-quality cell suspension can be obtained only through one-time digestion, one-time filtration and one-time red blood cell lysis, time waiting, intracellular RNA degradation and related reagent waste caused by secondary digestion are avoided, the effect is obvious, and operation is rapid.

The invention discloses a single-cell time transcriptome sequencing method based on RNA (Ribonucleic Acid) bimetabolism markers. The single-cell time transcriptome sequencing method comprises the following steps: (1) obtaining single cells of 6sG and 4sU bimetabolism markers; (2) loading the marked and fixed cells and the coding microspheres onto a double-layer micropore array chip, cracking the cells, and capturing dissociated RNA (Ribonucleic Acid) by the coding microspheres; (3), carrying out TimeLapse chemistry processing on the coding microspheres, and carrying out the TimeLapse chemistry processing on the coding microspheres; (4) sequentially carrying out reverse transcription, cDNA amplification, library construction and sequencing on the material obtained in the step (3), and then carrying out bioinformatics analysis to identify the mutation marker; and (5) for the state or the cell type of each cell, calculating the RNA degradation rate according to a curve that the proportion of reads carrying G-to-A mutation decreases, and deriving the RNA synthesis rate according to a curve that the proportion of reads carrying T-to-C mutation increases. By combining a 6sG and 4sU double-pulse labeling technology and high-throughput single cell sequencing, synchronous measurement of RNA synthesis and degradation dynamics on the single cell level is realized.

Disclosed herein are methods of producing transcribed RNA product with increased yield and reduced RNA degradation.

The invention belongs to the technical field of microorganism preservation, and particularly relates to normal-temperature preservation liquid for group B streptococcus (GBS) in a vaginal swab as well as a preparation method and application of the normal-temperature preservation liquid. The invention provides a GBS normal-temperature preservation solution. The formula of the GBS normal-temperature preservation solution comprises glycerol, glucose, sodium citrate, EDTA, FBS, compound amino acid, NaCl and the like. The preservation solution ensures that GBS can be stably preserved for 30 days at normal temperature, the activity of GBS and the stability of RNA are effectively maintained, the problems of RNA degradation and bacterial activity loss in an existing preservation method are solved, and reliable sample support is provided for subsequent bacterial culture and RNA detection. The method is suitable for long-time preservation and subsequent detection of clinical vaginal swab samples, and has a good application prospect.

The present invention relates generally to methods of analysing ribonucleic acid (RNA). In particular, the invention relates to methods of analysing RNA to assess levels of degradation of RNA molecules in an RNA sample. The methods use an exonuclease and a means for detection of nucleotides and / or nucleosides. The invention provides a method of analysing ssRNA in a sample, said method comprising: (a) contacting the sample with a probe, wherein the probe binds to a target region of ssRNA in the sample; (b) incubating the sample with a single-strand specific exoribonuclease; and (c) detecting nucleotides and / or nucleosides generated by step (b), wherein the nucleotides and / or nucleosides are derived from one or more RNA regions located outside the target region of ssRNA in the sample. The invention also provides kits for performing said method.

The invention belongs to the technical field of biomedical detection, and particularly relates to a lysis solution for candida albicans, a kit and a gene detection method. The cleavage efficiency and background interference are balanced by regulating and controlling the concentration of saponin, nucleic acid can be wrapped by too many saponin micelles, and cleavage is incomplete if too few saponin micelles exist; citric acid buffers to adjust the acidic environment, although RNA degradation may be promoted, nuclease is cleared through high-salt rapid cracking and protease K, and negative effects are counteracted; the pre-activated protease K is synchronous with membrane rupture, so that the enzyme activity peak is matched with the exposure time of the target protein, the degradation efficiency is improved, and a cracking technology which can simultaneously realize high-yield and high-function nucleic acid output and is adaptive to a rapid detection process is developed.

The invention discloses a method for detecting an abnormal mRNA removal mechanism in mammal oocytes and a mechanism for activating the channel, and the method comprises the following steps: step 1, amplifying a complete GFP sequence and a GFP sequence without a termination codon (TAA), and further constructing a reporter plasmid lacking the termination codon; 2, carrying out in-vitro transcription on plasmids, adding Poly A tails, carrying out microinjection in GV-stage oocytes and fertilized eggs, and carrying out immunofluorescent staining; and step 3, injecting reporter plasmids into the oocytes in the GV stage, and treating the oocytes by using CHX at the same time. Step 4, inserting different numbers of A basic groups into the front end of a termination codon expressing GFP, translating the termination codon into different numbers of lysine, similarly performing in-vitro transcription on the plasmid, adding a Poly A tail, performing microinjection on an oocyte in a GV period, and detecting abnormal RNA clearance through the brightness of the GFP; the method can be used for detecting the activation level of an abnormal RNA removal mechanism and the abnormal RNA degradation degree in the oocytes.

The application discloses a spherical nucleic acid preparation method based on a cyclic template strategy and application, and comprises the following steps: A) using controllable free radical polymerization (ATRP) and intramolecular click chemistry of copper-catalyzed alkyne and azido, a cyclic polymer is prepared c -P(HEMA) 30 B) using continuous esterification and substitution reaction, the cyclic polymer side chain terminal hydroxyl group is substituted by azido, and a cyclic template c -P(HEMA-N3) 30 C) using copper-free click chemistry reaction of a dibenzo cyclooctyne group (DBCO) and azido, nucleic acid is covalently coupled to the cyclic template, and a cyclic brush macromolecule c -P(HEMA-RNA) 30 The obtained cyclic brush macromolecule is subjected to water dialysis, and spherical nucleic acid is prepared through self-assembly. The spherical nucleic acid prepared by the preparation method has good reproducibility, good in-vitro stability, can better protect the nucleic acid from degradation of RNA degradation enzyme, and can greatly improve the nucleic acid uptake capacity of cells.

The application discloses a kit and a method for extracting nuclei in plant fruit kernels. The kit comprises a lysis solution, a washing solution and a purification solution. The lysis solution comprises 0.04%-0.4% w / v Triton X100, 0.2-0.4 mM spermine, 0.5-1 mM spermidine, 0.4-1.6 U / muL RNase inhibitor, 1-2.56 mM DTT, 10-15 mM NaCl, 1X PBS and 200-350 mM sucrose. The washing solution comprises 2% w / v BSA, 1.25-2.5 mM DTT and 0.2-0.4 U / muL RNase inhibitor. The purification solution comprises methanol and at least two Percoll solutions with different concentrations. The kit with specific components and the optimized extraction method can quickly obtain a high-purity nucleus suspension without extracting protoplasts, effectively remove impurities and secondary metabolites, reduce the risk of RNA degradation, and are simple to operate and low in cost.

Provided herein are compositions, systems, and methods for the generation, identification, characterization, and use of domains for modulating RNA levels and / or gene expression, e.g., inducing RNA degradation or downregulating mRNA translation.

This invention discloses a method for constructing a dual-omics library based on single-cell whole-genome methylation and transcriptomics, belonging to the field of sequencing technology. The method includes: lysing single cells in a sorting tube pre-filled with lysis buffer; applying a nucleophilic coating to the inner wall of the sorting tube to allow cell nuclei to adsorb and obtain a supernatant containing mRNA; reverse transcribing the supernatant to obtain cDNA and constructing a transcriptome library; lysing the cell nucleus to release DNA and converting it to bisulfite; ligating the converted DNA with a molecularly tagged adapter to obtain methylation intermediates; treating with exonucleases; merging intermediates with different molecular tags; purifying with magnetic beads, single-stranding, ligating the other end with a sequencing adapter, and amplifying and purifying to obtain a methylated library. This invention achieves simultaneous separation of RNA and DNA in the same tube for library construction, reducing separation and transfer steps, minimizing RNA degradation and nucleic acid loss, and improving the throughput of methylation library construction and the consistency of dual-omics data.

The invention provides a universal tissue single-cell suspension preparation kit and a use method thereof. The kit provides six common tissue dissociation enzymes and auxiliary materials required by the whole process of single-cell suspension preparation such as dissociation, cleaning, red blood cell lysis, fragment removal and cell mass removal, and can be used for rapidly preparing single-cell suspensions for at least 12 tissues (including difficult tissues such as skin, muscle, fat, pancreas and intestines). The components are complete, extra reagents are not needed when the single-cell suspension is applied to a conventional scene of single-cell suspension preparation, and the compatibility to personnel and a platform system is good. According to the kit, domestic raw materials are used, the cost is only one fifth of that of imported raw materials, and the single-cell suspension prepared by the kit is high in cell yield, good in activity, few in impurities, low in caking rate and high in RNA integrity, so that high-quality single cells can be conveniently and rapidly obtained for downstream capture and analysis; therefore, data waste and gene transcriptome expression information distortion caused by poor cell state or RNA degradation are reduced.

To provide a method for synthesizing RNA that enables efficient and low-cost RNA synthesis by providing a readily and economically producible substrate for RNA synthesis, and to provide an RNA synthesis kit usable in the synthesis method.SOLUTION: This invention has been completed based on the finding that: that a nucleoside polyphosphate, obtained by adding polyphosphate to an RNA degradation product, can serve as a substrate for DNA-dependent RNA polymerase.SELECTED DRAWING: Figure 1

Disclosed herein are methods of producing transcribed RNA product with increased yield and reduced RNA degradation.

The invention discloses an albumin mRNA (messenger Ribonucleic Acid) detection kit for auxiliary diagnosis of liver cancer, which comprises nucleic acid probes with sequences as shown in SEQ ID NO: 1-20 and can be combined with albumin mRNA. The 5'or 3 'tail end of the nucleic acid probe comprises a linker 1 combination region base sequence, the linker 1 combination region base sequences of two adjacent nucleic acid probes are combined to form a linker 1, the linker 1 does not participate in complementary pairing with albumin mRNA, and the nucleic acid probe group is obtained through a cascade amplification process. The albumin mRNA detection kit provides 10 groups of in-situ hybridization probes targeting different areas of albumin mRNA, false negative results caused by RNA degradation can be avoided, mRNA signals can be amplified by hundreds of times through a cascade amplification process, and the sensitivity of an in-situ hybridization detection technology is greatly improved.

This invention belongs to the field of biomedical detection technology, specifically a lysis buffer, kit, and gene detection method for Candida albicans. This invention balances lysis efficiency and background interference by regulating the concentration of saponins; too much saponin micelles will encapsulate nucleic acids, while too little will result in incomplete lysis. Although citrate buffering to regulate the acidic environment may promote RNA degradation, the negative impact is offset by rapid lysis with high salt and proteinase K to clear nucleases. Pre-activated proteinase K synchronizes with membrane rupture, ensuring that the peak enzyme activity matches the exposure time of the target protein, thus improving degradation efficiency. This invention develops a lysis technology that can simultaneously achieve high yield, high-functionality nucleic acid production, and is adapted to rapid detection procedures.

The invention provides an application of a heterologous Argonaute protein in degradation and editing of RNA (Ribonucleic Acid) in mammalian cells, and belongs to a molecular biotechnology, the heterologous Argonaute protein is McAgo with an amino acid sequence as shown in SEQ ID NO.1, the heterologous Argonaute protein and guide RNA are delivered into the mammalian cells in the form of an RNP (Ribonucleic Acid) compound, and degradation and / or site-specific editing of the target RNA are / is realized. According to the invention, heterologous Argonaute protein-mediated RNA degradation and RNA editing in mammalian cells are realized for the first time, and the method has important significance for replacing and solving the off-target problem of CRISPR-Cas technology during editing at the RNA level.

The present invention generally relates to methods of analyzing ribonucleic acids (RNAs). In particular, the present invention relates to a method of analyzing RNA to assess the level of RNA molecule degradation in an RNA sample. The method uses exonuclease and means for detecting nucleotides and / or nucleosides. The present invention provides a method for analyzing ssRNA in a sample, the method comprising: (a) contacting the sample with a probe wherein the probe binds to a target region of ssRNA in the sample; (b) incubating the sample together with a single strand specific ribonucleic acid exonuclease; and (c) detecting the nucleotides and / or nucleosides generated by step (b), wherein the nucleotides and / or nucleosides are derived from one or more RNA regions located outside the target region of ssRNA in the sample. The invention also provides a kit for carrying out the method.

This disclosure provides oligonucleotides, methods, and compositions for degrading RNA via the lysosomal pathway. It also envisions oligonucleotides, methods, and compositions for treating, preventing, or improving diseases, disorders, and conditions associated with EXOC2, Ku80, and Task1 in subjects requiring such treatment.

The invention relates to the technical field of plant microscopic observation, in particular to a plant tissue paraffin embedding method of high-fidelity RNA (Ribonucleic Acid). The invention provides a paraffin embedding method for plant tissue of high-fidelity RNA (Ribonucleic Acid), which comprises the following steps: firstly mixing the tissue and a stationary liquid under negative pressure, and then replacing the stationary liquid for fixing, carrying out ethanol solution gradient dehydration on the fixed tissue; the dehydrated tissue is firstly placed in a transparent agent to be treated, and then xylene is used for treatment; mixing the transparent tissue and the mixed wax solution at 35-39 DEG C, heating to 58-62 DEG C, mixing to obtain a mixture, mixing the mixture with the paraffin solution, and embedding to obtain an embedded sample. According to the method, RNA degradation in the paraffin embedding process is reduced, it is ensured that RNA extracted from an embedded sample has high quality, operation is easy, complex equipment is not needed, and the experiment time is short.

The invention discloses a kit for detecting multiple microRNA levels and a method for screening disease markers by using the kit, and belongs to the technical field of molecular biology. According to the kit, at least 96 microRNAs in a sample can be detected at a time, 376 microRNAs can be detected at a time by utilizing a 384 pore plate, all potential markers are covered, the microRNAs of all targets can be completed only by extracting a single sample once, the detection time is short, and the sample demand is low. The kit has no limitation on sample collection, after the standard substance is introduced, all the samples do not need to be detected at the same time, the problem that the screened markers are not excellent enough due to insufficient sample quantity is effectively prevented, and the phenomenon that RNA is degraded due to waiting for the samples is also avoided. By utilizing the method provided by the invention, different microRNA levels of disease patients and healthy subjects can be analyzed, and reliable markers can be obtained more quickly and more effectively.