This disclosure relates to a circulating tumour cell typing and identification kit, comprising a capture probe, an amplification probe, and a labeled probe for each marker gene mRNA, wherein the marker gene mRNA comprises the following two types: at least two epithelial cell marker gene mRNAs selected from the group consisting of EPCAM, E-cadherin, CEA, KRT5, KRT7, KRT17, and KRT20 mRNAs; and, at least two mesenchymal cell marker gene mRNAs selected from the group consisting of VIMENTIN, N-cadherin, TWIST1, AKT2, ZEB2, ZEB1, FOXC1, FOXC2, SNAI1 and SNAI2 mRNAs. This disclosure prevents false-positive results caused by, for example, possible presence of a number of non-neoplastic epithelial cells in peripheral blood, introduction of normal epithelial cells during blood sampling, and the like. Accordingly, it may be assured that cells detected with epithelial cell marker genes and/or mesenchymal cell marker genes are indeed circulating tumour cells, further improving accuracy and reliability of the detection results.

This disclosure relates to a circulating tumour cell typing and identification kit, comprising a capture probe, an amplification probe, and a labeled probe for each marker gene mRNA, wherein the marker gene mRNA comprises the following two types: at least two epithelial cell marker gene mRNAs selected from the group consisting of EPCAM, E-cadherin, CEA, KRT5, KRT7, KRT17, and KRT20 mRNAs; and, at least two mesenchymal cell marker gene mRNAs selected from the group consisting of VIMENTIN, N-cadherin, TWIST1, AKT2, ZEB2, ZEB1, FOXC1, FOXC2, SNAI1 and SNAI2 mRNAs. This disclosure prevents false-positive results caused by, for example, possible presence of a number of non-neoplastic epithelial cells in peripheral blood, introduction of normal epithelial cells during blood sampling, and the like. Accordingly, it may be assured that cells detected with epithelial cell marker genes and/or mesenchymal cell marker genes are indeed circulating tumour cells, further improving accuracy and reliability of the detection results.

The present invention discloses a method for capturing an RNA in situ higher-order structure and interaction. The method includes: fixing protein-mediated RNA-RNA interaction in cell or tissue; performing membrane permeabilization while keeping the cell intact; degrading free RNA; labeling the 3′ end of the RNA with pCp-biotin and performing proximal ligation in situ; purifying the chimeric RNA containing the pCp-biotin after the cell is digested; constructing the strand-specific library; and performing high-throughput sequencing. In the present invention, under the condition of not destroying the cell structure and keeping the integrity of cell, treat the intracellular RNA in situ, and capture RNA intra- and intermolecular interactions in a physiological state; the 3′ end of the RNA is labeled with the pCp-biotin, and in situ ligation is performed under non-denaturing conditions, thereby greatly improving the labeling efficiency and reducing intermolecular specific ligation; and the chimeric RNA labeled with C-biotin is enriched by C1 magnetic beads, so that the fraction of effective sequencing data is increased, and the sequencing cost is reduced.

The present invention discloses a method for capturing an RNA in situ higher-order structure and interaction. The method includes: fixing protein-mediated RNA-RNA interaction in cell or tissue; performing membrane permeabilization while keeping the cell intact; degrading free RNA; labeling the 3′ end of the RNA with pCp-biotin and performing proximal ligation in situ; purifying the chimeric RNA containing the pCp-biotin after the cell is digested; constructing the strand-specific library; and performing high-throughput sequencing. In the present invention, under the condition of not destroying the cell structure and keeping the integrity of cell, treat the intracellular RNA in situ, and capture RNA intra- and intermolecular interactions in a physiological state; the 3′ end of the RNA is labeled with the pCp-biotin, and in situ ligation is performed under non-denaturing conditions, thereby greatly improving the labeling efficiency and reducing intermolecular specific ligation; and the chimeric RNA labeled with C-biotin is enriched by C1 magnetic beads, so that the fraction of effective sequencing data is increased, and the sequencing cost is reduced.

Provided herein is a method and system for analyzing a sample. In some embodiments the method makes use of a plurality of capture agents that are each linked to a different oligonucleotide and a corresponding plurality of labeled nucleic acid probes, wherein each of the labeled nucleic acid probes specifically hybridizes with only one of the oligonucleotides. The sample is labeled with the capture agents en masse, and sub-sets of the capture agents are detected using iterative cycles using corresponding subsets of the labeled nucleic acid probes.

Provided herein is a method and system for analyzing a sample. In some embodiments the method makes use of a plurality of capture agents that are each linked to a different oligonucleotide and a corresponding plurality of labeled nucleic acid probes, wherein each of the labeled nucleic acid probes specifically hybridizes with only one of the oligonucleotides. The sample is labeled with the capture agents en masse, and sub-sets of the capture agents are detected using iterative cycles using corresponding subsets of the labeled nucleic acid probes.

The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.

The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.

The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.

The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.