78 results about "Cellular transcription" patented technology

The present invention relates to a new method for determining the RNAi mediated transcriptional regulation of a cell by the determination of a pattern of at least 3 miRNA detected simultaneously and quantified in the same cell extract. The determination of a pattern of miRNA comprises the steps of: (i) providing an array onto which are fixed capture probes, said capture probes being arranged on pre-determined locations and reflecting the genomic or transcriptional matter of a cell; (ii) isolating a miRNA pool potentially present from a cell; (iii) elongating or ligating said miRNAs into labeled capture probes, (iv) contacting said labeled polynucleotides with the array under conditions allowing hybridization of the labeled polynucleotides to complementary capture probes present on the array; (v) detecting and quantifying a signal present on the specific locations of the array, wherein the detection of a pattern of at least 3 signals on the array reflects the pattern of miRNAs being involved in the RNAi mediated cellular transcriptional regulation.

The present invention relates to methods of generating amounts of selective nucleic acids. The present invention further relates to selective nucleic acids incorporated within non-coding nucleic acids, capable of binding to or altering a target molecule. Selective nucleic acids may generally refer to, but are not limited to, deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), artificially modified nucleic acids, combinations or modifications thereof. Selective nucleic acids may also generally refer to, but are not limited to, nucleic acid aptamers, aptazymes, ribozymes, deoxyribozymes, nucleic acid probes, small interfering RNAs (siRNAs), micro RNAs (miRNAs), short hairpin RNAs (shRNAs), antisense nucleic acids, diagnostic probes or probe libraries, aptamer inhibitors, precursors of any of the above and/or combinations or modifications thereof. In one aspect, a method for generating amounts of selective nucleic acids includes incorporating a selective nucleic acid sequence into a carrier nucleic acid. In general, the carrier nucleic acid may be transcribed by a cell into a product nucleic acid which may carry an incorporated selective nucleic acid sequence.

The invention discloses a method for establishing a single cell transcriptome sequencing library and application of the method. The method comprises the following steps: performing reverse transcription on RNA in a single cell, thus obtaining cDNA; performing pre-amplification on cDNA by using an amplification primer, thus obtaining amplified cDNA; and performing fragmentation library construction on the amplified cDNA, thus obtaining a transcriptome sequencing library of the single cell, wherein dTTP in the amplification primer is substituted by dUTP. According to the method disclosed by the invention, as the dTTP in the pre-amplification primer is substituted by dUTP, fragments containing the pre-amplification primer in a jointed fragment can be interrupted in an enzymic digestion step after the step of joint connection, and are removed in later high-temperature pre-denaturation and denaturation steps; furthermore, the amplification of fragments with the pre-amplification primer can be reduced, the ratio that the proportion of data with pre-amplification primer pollution in the obtained sequencing data is greatly reduced, and the effective data amount of the obtained data is greatly increased.

The present invention provides a transcriptome sequencing method, wherein primers used during a reverse transcription process from RNA to cDNA comprise a random sequence and a specific sequence, the random sequence is positioned at the 3' terminal of the specific sequence, bases from 0 to 5 exist between the random sequence and the specific sequence, the random sequence comprises 4-12 random bases, the specific sequence is a homopolymer of 5-30 bases, and the base of the specific sequence is any one selected from A, G, C and T. According to the present invention, with the design on the reverse transcription primers, the RNA having the poly (A) tail can be sequenced while the RNA with no poly (A) tail can be sequenced, the rRNA removal step is not required during the cell transcriptome sequencing, and the method is especially suitable for the sequencing on the transcriptome having the low initial RNA amount and the low cell number.

The invention discloses a second-generation sequencing technology based microbe unicell transcriptome analysis method. The analysis method comprises the following steps: (1) separating microbe unicells; (2) extracting total RNA; (3) conducting linear amplification on total RNA; (4) constructing an RNA sequencing library; (5) analyzing the RNA sequencing library. By adopting the analysis method, analysis on gene expression difference among different microbe cells can be realized, and physiological function analysis of the unicell level on microbes which cannot be cultivated in extreme environments can be realized; furthermore, the analysis method can be suitable for analysis on eukaryotic unicells, and has quite wide applications in the fields of important biological and medical researches and the like, such as tumor generation and evolution, and stem cell induction and development.

The invention discloses a high-throughput single-cell transcriptome and gene mutation integration analysis method. The high-throughput single-cell transcriptome and gene mutation integration analysismethod comprises the following steps that (1) a high-throughput single-cell encoding chip is provided; (2) single-cell surface protein parting analysis is conducted; (3) single-cell transcriptome mutation analysis is conducted; (4) a database for single-cell surface protein parting and mutation integration analysis is established; and (5) a high-throughput single-cell transcriptome and gene mutation integration analysis model is established. According to the high-throughput single-cell transcriptome and gene mutation integration analysis method, by designing the single-cell encoding chip withtriple encoding technologies of microporous spatial coordinates, cell nucleic acid labels and molecular nucleic acid labels and by combining the modes of single-cell surface protein parting, single-cell transcriptome mutation analysis and gene sequencing, gene mutation information, transcriptome information and protein expression information of single cells can correspond one by one, the completedatabase for high-throughput single-cell transcriptome and gene mutation integration analysis is formed, and the multi-omics integration analysis model is obtained.

The invention relates to the field of gene detection technologies and biomedicine, in particular to a breast cancer patient recurrence risk 20 gene prediction model based on breast cancer single-celltranscriptome sequencing analysis and an establishment method and application thereof. The model is composed of 20 genes of CEBPD, SERPINA1, CD24, ERRFI1, BCL3, DSTN, BTG2, SERTAD1, SPINT1, BAMBI, LIMCH1, NFIA, SKP1, DHRS7, ODF3B, KRT7, ZFP36, CEBPB, BHLHE40 and UGDH. The breast cancer patient recurrence risk 20 gene prediction model provided by the invention provides more accurate judgment for long-term prognosis of a breast cancer patient, and provides a basis for selection of a postoperative adjuvant therapy scheme of the patient, so that individualized accurate treatment is realized, and meanwhile, a new research perspective is provided for research of breast cancer tumor stem cells.

The invention discloses a high-throughput single-cell transcriptome and gene mutation integration analysis coding chip. According to the chip, a substrate is provided with a plurality of microholes, the micropores have a size and a shape which can only accommodate a single cell in a micropore, each micropore has a unique spatial coordinate encoding, the micropores are internally modified with a plurality of known nucleic acid sequences, the nucleic acid sequences successively include Spacer sequences, universal primer sequences, cell labels, molecular labels, and Ploy T, wherein the universalprimer sequences are used as primer-binding regions when a PCR is augmented, the cell labels are used for labeling cells from which the RNA originated, and the molecular labels are used for labeling combinative RNA. The invention provides the chip capable of being used for high-throughput single-cell transcriptomes and gene mutation integration analysis. By adopting a triple coding technique of microporous space coordinates, cell nucleic acid labels and molecular nucleic acid labels, gene mutation, the transcriptomes and protein expression information of a single cell can be matched one by one.

The invention discloses a single cell transcript isomer sequencing analysis method and a kit. By adding tag sequences to the two tail ends of a single mRNA, after trapping and enriching are performed,the whole transcriptome is sequenced, and by accurately judging the head and tail structures of different transcript isomers, full-length transcripts can be judged. Existing technical problems can besolved by the new research strategy. The single cell transcript isomer sequencing analysis method and the kit have the following beneficial effects that 1, the sensitivity of the detection of the full-length transcripts is improved, specifically, due to the targeted detection of the head and tail areas of the transcripts, signals are mostly concentrated at the end edges of the transcripts, so that the detection of the full-length transcripts is higher; 2, simple operation is achieved, and the cost low, specifically, the method is simple to operate, widely applicable and the cost is low; and 3, the accuracy of the detection of the transcripts is improved, specifically, higher accuracy can be achieved by combining the tag sequences of the tail end sequences and the optimization of a data analysis algorithm.

The invention discloses an integrated device for integrated analysis of high-throughput single-cell transcriptome and gene mutation. The integrated device includes a high-throughput single-cell codingchip and an integrated analysis device body, wherein the integrated analysis device body includes a housing and a temperature-control thermal cycle module, a fluorescence imaging module and a data storage analysis module which are disposed in the housing, and the fluorescence imaging module includes a light source component, a microscope objective, a fluorescence splitting component and an imaging detector. One-to-one correspondence of gene mutation, transcriptome and protein expression information of single cells can be achieved through the designed high-throughput single-cell coding chip which is provided with triple-encoding functions of micropore spatial coordinates, cellular nucleic acid tags and molecular nucleic acid tags, and PCR amplification can be realized through the temperature-control thermal cycle module; and the fluorescence imaging module is used for collecting fluorescence images of a sample, and the fluorescence images are stored and analyzed through the data storage analysis module, so that integrated analysis of single-cell transcriptome and gene mutation is achieved.