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WO2018134907A1 - Dispositif et méthode d'extraction de multiples biomolécules d'une cellule unique - Google Patents

Dispositif et méthode d'extraction de multiples biomolécules d'une cellule unique Download PDF

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Publication number
WO2018134907A1
WO2018134907A1 PCT/JP2017/001512 JP2017001512W WO2018134907A1 WO 2018134907 A1 WO2018134907 A1 WO 2018134907A1 JP 2017001512 W JP2017001512 W JP 2017001512W WO 2018134907 A1 WO2018134907 A1 WO 2018134907A1
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carrier
cell
biomolecule
sequence
single cell
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PCT/JP2017/001512
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English (en)
Japanese (ja)
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白井　正敬
友幸 坂井
妃代美 谷口
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株式会社日立ハイテクノロジーズ
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Priority to PCT/JP2017/001512 priority Critical patent/WO2018134907A1/fr
Publication of WO2018134907A1 publication Critical patent/WO2018134907A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to an apparatus and method for simultaneously performing analysis such as genome analysis, gene expression analysis, gene sequence analysis, and protein analysis in a single cell for each single cell.
  • single cell analysis biomolecules in a single cell are individually extracted, and an appropriate sample is prepared to enable analysis, and then the sample is measured.
  • This measurement includes genome sequence analysis, gene sequence analysis, gene expression analysis, protein analysis, and the like. That is, the single cell analysis cannot be directly measured because the amount of the sample is very small, and the analysis needs to be executed by combining sample preparation and measurement.
  • Patent Document 1 discloses that a cell is isolated by capturing a cell in a hole on a flat chip and a DNA probe fixed on the porous material surface immediately below the cell. By capturing the nucleic acid therein, mRNA that is a biomolecule to be measured is extracted.
  • Patent Document 2 discloses that a single cell and a bead with a DNA probe for extracting (capturing) nucleic acid in the cell are confined in an emulsion droplet (a droplet in oil), A method for capturing / extracting the nucleic acid at high efficiency and preparing a sample for sequencing is disclosed.
  • a method of first extracting a nucleic acid sample from a cell sample in a container and then re-extracting the nucleic acid to be measured is generally performed in this technical field.
  • single cell analysis is generally not performed.
  • the amount of nucleic acid sample required for applying this method is 3 ⁇ g (after the genomic DNA is fragmented, it is extracted by the above method. Therefore, the genomic DNA amount can be compared with the mRNA amount. )), And there is a difference of about 6 digits compared to the case of the amount of nucleic acid of about 0.1 to 10 pg which is the amount of nucleic acid in the single cell analysis.
  • Such differences include the process of elution of nucleic acid in the solution from the solid surface without sample preparation on the solid surface where the capture and extraction of nucleic acids from the cells were captured in the vicinity of the cells. This is because not only the ratio of the sample loss above becomes high, but also that the loss due to adsorption to the surface of the container becomes large even if it is moved to another container or the like in a solution state.
  • sample preparation devices for single-cell analysis so far have a small amount of measurement target, so sample processing is performed at or near the same time as cell capture and cell disruption. Since there is one cell, there is one reaction tank. Therefore, only one type of reaction treatment could be performed. That is, it was not possible to analyze a plurality of types of biomolecules for the same cell. On the other hand, if the sample is separated and the reaction vessel is divided into a plurality of samples, the sample is lost at the time of separation / extraction, so a large amount of sample is required before the separation, and a minute sample of a single cell cannot be handled.
  • the present invention Means for separating cells;
  • a single cell analysis device comprising a reaction field disposed in the vicinity of or within the separation means,
  • the reaction field includes a first carrier that captures a first biomolecule extracted from the captured cells, and a second carrier that captures a second biomolecule different from the first biomolecule.
  • the first carrier and the second carrier relate to a single cell analysis device characterized by having different physical properties that can be separated from each other.
  • the present invention also provides the single cell analysis device, wherein the cell is captured by the separation means, and the first biomolecule and the second biomolecule extracted from the captured cell are respectively reacted in the reaction field with the first carrier and Including separately processing the first biomolecule and the second biomolecule by capturing on the second carrier and separating the first carrier and the second carrier based on differences in physical properties
  • the present invention relates to a single cell analysis method.
  • biomolecules with different optimal sample preparation processes or measurement methods existing in a single cell are individually extracted without loss, and information on which single cell is derived is retained.
  • sample preparation individually and performing individual measurement it is possible to measure the state of a complex biological tissue composed of a large number of cells.
  • the simultaneous analysis of DNA genomic mutation analysis and mRNA gene expression analysis enables analysis of how genomic mutations affect gene expression for each single cell. It becomes possible to analyze.
  • the same effect can be expected from simultaneous analysis of epigenome analysis and gene expression analysis, and gene expression analysis and protein analysis.
  • the measurement of the above-mentioned plurality of biomolecules is a single analysis of two types of molecules, that is, an mRNA sequence analysis corresponding to a T cell receptor and an antibody and an mRNA expression analysis related to a cell state. What can be done for each cell is expected to lead to highly accurate cancer diagnosis because information on antigens related to cancer can be obtained from sequence analysis, and the activity state of cells can be obtained from gene expression analysis.
  • FIG. 6 is a diagram illustrating a sample processing flow corresponding to Example 1.
  • FIG. FIG. 3 is a diagram showing the first half of a sample processing flow of second beads (solid support) of Example 1.
  • FIG. 4 is a diagram showing the second half of the sample processing flow of the second bead (solid carrier) of Example 1.
  • FIG. 3 is a diagram showing a first half of a sample processing flow of first beads (solid support) of Example 1.
  • FIG. 4 is a diagram showing the latter half of the sample processing flow of the first beads (solid support) of Example 1.
  • Example 3 shows the device structure of Example 3 as another structural example of the device which concerns on this invention.
  • 10 is a diagram illustrating a sample processing flow of Example 3.
  • FIG. It is a figure which shows one Embodiment of the structure of the device which concerns on this invention.
  • the present invention provides a device, apparatus, and method for preparing a plurality of types of biomolecule samples from each single cell in parallel and rapidly for a plurality of cells and analyzing each sample.
  • the sample is prepared so as to be able to distinguish between single cell and biomolecule types.
  • “preparation of a biomolecule sample” means that a biomolecule contained in a cell is extracted and separated from other cell components. In particular, a plurality of types of biomolecules can be distinguished. It means that the sample is prepared separately.
  • “analysis of a sample” is to analyze a biomolecule related to the sample, specifically, to analyze the expression of the biomolecule in the sample (cell, tissue section, etc.) quantitatively, Analyzing the expression distribution of biomolecules in a sample, analyzing the presence or absence of biomolecules (eg, genomic DNA having a specific mutation) in a sample, and correlating data between a specific position in a sample and the amount of biomolecule expression It means getting.
  • the biomolecule to be analyzed in the present invention is not particularly limited as long as it is a biomolecule contained in a cell. Nucleic acid (eg, messenger RNA (mRNA), non-coding RNA (ncRNA), microRNA, genome) DNA, and fragments thereof), proteins (eg, enzymes, antibodies, etc.), low molecular compounds, and the like.
  • mRNA messenger RNA
  • ncRNA non-coding RNA
  • microRNA genome DNA
  • proteins eg, enzymes, antibodies, etc.
  • low molecular compounds e.g., low molecular compounds, and the like.
  • a plurality of types for example, two types of biomolecules are analyzed.
  • both the first biomolecule and the second biomolecule can be mRNA.
  • one of the first biomolecule and the second biomolecule can be mRNA, and the other can be protein.
  • one of the first biomolecules can be genomic DNA and the other can be mRNA.
  • the types of biomolecules to be analyzed can be set
  • an individual reaction field for capturing a biomolecule is set for each individual cell, but the cell is separated / captured in a position near the reaction field (for example, an adjacent position) or in the reaction field.
  • the reaction field is filled with a carrier for capturing the first measurement target biomolecule, and in addition, the second measurement target biomolecule can be separated from the support and captured. To be filled with another carrier.
  • the present invention provides: Means for separating cells; A single cell analysis device comprising a reaction field disposed in the vicinity of or within the separation means, The reaction field includes a first carrier that captures a first biomolecule extracted from the captured cells, and a second carrier that captures a second biomolecule different from the first biomolecule. Have an area to One or both of the first carrier and the second carrier comprise a tag sequence for identifying captured cells, The first carrier and the second carrier provide a single cell analysis device characterized by having different physical properties that can be separated from each other.
  • the substrate is provided with a hole, and the reaction tank is disposed immediately below the hole.
  • a reaction tank for allowing the cell extraction solution to pass therethrough is provided immediately below the hole in the substrate provided with a plurality of cell capturing (isolation) holes arranged in a plane, and the first measurement target living body is provided here.
  • a first solid support on which a molecule for capturing a molecule and a first capture probe having a cell identification tag sequence are immobilized is filled, and in addition, the physical properties of the first solid support are different.
  • a second capture probe for capturing the second biomolecule is fixed to another solid support, and the reaction vessel is filled.
  • Patent Document 1 International Patent Application Publication No. WO2014 / It is known in the art as described in US Pat.
  • the substrate is not particularly limited as long as it is made of a material generally used in the technical field.
  • the material include metals such as gold, chromium, platinum, titanium, and nickel; alloys such as stainless steel and duralumin; silicon; glass, quartz glass, fused silica, synthetic quartz, alumina, sapphire, ceramics, forsterite, and photosensitive.
  • Glass materials such as reactive glass; plastics such as polyester resin, polystyrene, polyethylene resin, polypropylene resin, ABS resin (Acrylonitrile Butadiene Styrene resin), dimethylpolysiloxane (PDMS), cyclic polyolefin, nylon, acrylic resin, fluorine resin; agarose Dextran, cellulose, polyvinyl alcohol, nitrocellulose, chitin, and chitosan.
  • the material used for the substrate is preferably a hydrophobic material, which can reduce adsorption of cells and reagents.
  • the substrate may be surface-coated so that other substances (nucleic acid, protein, reagent, etc.) do not adsorb.
  • the reaction tank is formed integrally with the cell trapping hole, or may be formed separately and connected by a flow path.
  • the separation means and the reaction field are a flow path through which a nonpolar solvent flows and a droplet including a single cell formed in the nonpolar solvent.
  • a reagent for crushing the cell and the first biomolecule to be measured are captured in the droplet.
  • a solid support on which a first capture probe having a molecule and a cell identification tag sequence is immobilized, and another solid support having physical properties different from those of the solid support are introduced.
  • emulsion droplets A technique for extracting and capturing biological substances (particularly nucleic acids) in cells from droplets in a nonpolar solvent is known as so-called emulsion droplets, and is described in, for example, Patent Document 2.
  • nonpolar solvent for example, mineral oil, silicone oil or the like
  • emulsion droplets are formed in which single cells, reagents and carriers are sealed. Emulsion formation is well known in the art and can be done by any method.
  • Nonpolar solvents and emulsion droplets are preferably thermally stable for biomolecule extraction and sample processing.
  • the size of the emulsion droplet varies depending on the type and size of the cell to be analyzed, the type and size of the carrier to be used, and is about 100 ⁇ L or less, 50 ⁇ L or less, 10 ⁇ L or less, 5 ⁇ L or less, 1 ⁇ L or less, It can be set appropriately such as 500 pL or less.
  • a plurality of types of carriers having different physical properties that can be separated from each other are used. For example, when two types of biomolecules are captured, a first carrier and a second carrier for capturing each biomolecule are filled or introduced into the reaction field. When capturing three or more types of biomolecules, a carrier for capturing each of these biomolecules is filled or introduced into the reaction field.
  • Each carrier is different in at least one physical property selected from the group consisting of size, weight, specific gravity, magnetism and shape.
  • the carrier having a different shape include beads, a porous structure, and a reaction vessel wall surface.
  • the carrier having a different size and / or weight or specific gravity include beads having different sizes and / or weight or specific gravity.
  • the carrier having different magnetism include magnetic beads and a magnetic porous structure.
  • the carrier it is preferable to use a material having a large surface area as the carrier. For example, it is preferable to adopt a structure filled with a large number of beads, a porous structure, a mesh structure, or the like. From the viewpoint of simplicity of operation, it is preferable to use beads as at least one of the carriers.
  • the beads can be produced from a resin material (such as polystyrene), oxide (such as glass), metal (such as iron), sepharose, and combinations thereof. Further, when magnetic beads are used as at least one of the carriers, the carrier can be separated quickly and easily.
  • the probe for capturing the biomolecule to be analyzed is fixed to the carrier.
  • a probe can be designed to specifically bind to a biomolecule depending on the type of biomolecule to be analyzed.
  • a DNA probe containing a poly T sequence can be used.
  • a DNA probe containing a poly T sequence, that is, an oligo (dT) can be synthesized by a conventional method, and the degree of polymerization of the oligo (dT) is hybridized with the poly A sequence of mRNA, and the mRNA is oligo (dT).
  • Any degree of polymerization that can be trapped on a fixed carrier For example, it can be about 10 to 30 bases, 10 to 20 bases, 10 to 15 bases.
  • a DNA probe comprising a random sequence or a DNA probe having a sequence complementary to a specific target sequence
  • a molecule that specifically binds to the biomolecule such as an antibody, a receptor, or an aptamer
  • a first binding molecule such as an antibody or an aptamer
  • a first DNA probe bound to a binding molecule can be used.
  • a second binding molecule that binds to the biomolecule in a sandwich state with the binding molecule preferably a molecule of the same type as the binding molecule, such as an antibody or an aptamer
  • the binding molecule preferably a molecule of the same type as the binding molecule, such as an antibody or an aptamer
  • a second DNA probe bonded to a sex molecule is added and a target biomolecule exists
  • the DNA probe and the second DNA probe are ligated, and a ring probe specific to the biomolecule is It is formed.
  • This method is called a proximity ligation method (Proximity ⁇ ⁇ ⁇ Ligation Method) (for example, Malin Jarvius et al. Molecular & Cellular9Proteomics 6500 (9) p.1500, 2007) and is useful for the construction of DNA libraries corresponding to proteins.
  • the probe is fixed to the carrier by any method known in the art. For example, covalent bond, ionic bond, physical adsorption, biological bond (for example, binding of biotin and avidin or streptavidin, antigen and antibody, etc.
  • the probe can be fixed by using a bond). It is also possible to fix the probe to the carrier via a spacer sequence.
  • a protein or a low molecular weight compound is processed as a biomolecule using the proximity ligation, the first binding molecule can be immobilized on another carrier.
  • any one of the above carriers for example, one or both of the first carrier and the second carrier is provided with a tag sequence for identifying captured cells.
  • a cell identification tag sequence can be introduced into a probe fixed to a carrier.
  • a probe fixed to a carrier eg, a bead
  • it is derived from any cell or position even after separation from the device using a cell identification tag sequence.
  • Such information can be held, which is preferable.
  • Those skilled in the art can design cell identification tag sequences so that they can be distinguished according to the number of cells to be captured and the number of reaction fields.
  • the reason why the problem of the present invention is solved by the configuration as described above is as follows. That is, cell disruption is performed in the vicinity of the reaction field or in the reaction field, and by providing a carrier that captures all the measurement target biomolecules in the reaction field, the cell extract The biomolecule to be measured contained in is captured by a probe for capturing the biomolecule on the carrier before it touches the reaction vessel. Thereby, a biomolecule is hardly adsorbed on a wall surface such as a container and the sample is hardly lost. Next, by separating the captured biomolecules based on the physical properties of the carrier, that is, size, shape, specific gravity, magnetism, etc., the probability of adsorbing to the container wall surface can be reduced even during the separation. As a result, it is possible to separate an extremely small amount of a biomolecule to be measured in a single cell and perform optimum sample preparation individually for the separated sample.
  • the present invention provides a single cell analysis device according to the present invention, in which a cell is captured by a separation means, and a first biomolecule and a second biomolecule extracted from the captured cell are reacted.
  • the first biomolecule and the second biomolecule are captured by the first carrier and the second carrier, respectively, in the field, and the first carrier and the second carrier are separated based on the difference in physical properties.
  • a single cell analysis method is provided that includes processing samples individually.
  • the cell to be analyzed is not particularly limited as long as it is a biological sample containing cells.
  • the living body from which the sample is derived is not particularly limited.
  • the sample When used in the device or method according to the present invention, the sample needs to be in a form in which the cells are separated from each other. Therefore, when the sample is a solid sample (for example, a tissue section), it is preferable to form a liquid sample by dissolving or suspending the solid sample in a solvent. Further, when the sample is a gas sample (for example, air, exhaled air, etc.), it is preferable to suspend the cells contained in the gas sample in a solvent to obtain a liquid sample. Sample preparation methods are routinely performed in the art and can be easily understood by those skilled in the art.
  • the sample cell is introduced into the single cell analysis device according to the present invention.
  • a cell is captured by a separation means (for example, a cell capture hole or a nonpolar solvent), and a biomolecule is extracted from the captured cell.
  • a separation means for example, a cell capture hole or a nonpolar solvent
  • cells can be lysed using a cell lysis reagent known in the art, and nucleic acids contained in the cells can be extracted.
  • RNA RNA degrading enzyme
  • DNase DNA degrading enzyme
  • the carrier is then separated based on the difference in physical properties. For example, when using carriers having different sizes, they can be separated using a filter or the like. In the case of a carrier having a different weight or specific gravity, it can be separated using centrifugation, sedimentation or the like. In the case of a carrier having a different shape, it can be separated using washing, filtering, centrifugation or the like. In the case of a carrier having different magnetism, it can be separated using a magnet. The separation of the carrier can be performed by appropriately combining the above means depending on the difference in physical properties of the carrier used.
  • the biomolecules captured on each carrier are individually sampled.
  • sample processing it is also possible to analyze which cell or position the processed sample is derived from using a cell identification tag sequence.
  • the single cell analysis method In the single cell analysis method according to the present invention, cell disruption is performed in the vicinity of the reaction field or in the reaction field, and all the biomolecules to be measured are captured in the reaction field.
  • the carrier By providing the carrier, the biomolecule to be measured contained in the cell extract is captured by the probe for capturing the biomolecule on the carrier before touching the reaction container. Thereby, a biomolecule is hardly adsorbed on a wall surface such as a container and the sample is hardly lost.
  • the probability of adsorbing to the container wall surface can be reduced even during the separation. As a result, it is possible to separate a very small amount of a biomolecule to be measured in a single cell and to perform optimum sample preparation for each separated sample.
  • Such analysis can provide information on the distribution of various cells from the viewpoint of molecular biology in biological tissues, and the diversity of the cancer progression and associated immune responses at the individual level can also be analyzed by conventional genetic analysis. More detailed and accurate. Therefore, such research is expected to contribute to the development of new disease diagnostic methods and drug discovery, and in particular, to the study of selection of appropriate treatment methods for each individual.
  • a planar substrate provided with a plurality of cell trapping holes arranged in a plane and a reaction vessel for allowing a cell extraction solution to pass therethrough are provided as reaction fields, and two kinds of beads are filled therein. This is an example of preparing a sample containing two types of biomolecules to be measured.
  • first sequence information of mRNA (first biomolecule) in a region where mutation or recombination occurs in the sequence is measured.
  • the devices and methods that can be realized simultaneously for cells are described.
  • the sequence analysis target part of the variable region of the immune cell is separated from the 3 ′ end (the degree of separation is separated from the length of the sequencing lead for performing the sequence analysis).
  • the second analysis is a quantitative analysis (so-called gene expression analysis) that counts the number of molecules for each gene.
  • FIG. 1 shows a configuration diagram of a reaction device which is a basic configuration of the present embodiment.
  • 1A and 1B are a top view and a cross-sectional view of the device, respectively.
  • the cell trapping holes (3) are arranged in a square lattice pattern at equal intervals on the flat substrate (1), and the reaction vessel (2) is placed immediately below the cell trapping holes.
  • the planar substrate is made of PDMS (polydimethylsiloxane), but resin materials such as polycarbonate, polypropylene, (cyclic) cycloolefin, semiconductor materials such as silicon, and inorganic materials such as glass and alumina, A metal material such as stainless steel may be used.
  • the cell trapping hole in the narrowest part can be selected to have a diameter of about 0.1 ⁇ m to 100 ⁇ m and suitable for the size of the cell, but usually about 2 to 3 ⁇ m is preferable.
  • the interval between the cell trapping holes is preferably about 1 ⁇ m to 1 mm, and particularly about 100 ⁇ m.
  • the size (for example, diameter) of the reaction vessel is preferably about 1 ⁇ m to about 500 ⁇ m, particularly about 80 ⁇ m.
  • the height of the reaction tank is preferably about 1 ⁇ m to about 1 mm, and particularly preferably about 100 ⁇ m.
  • the reaction tank (2) was filled with two types of beads having different diameters and physical properties as two types of solid carriers.
  • a first DNA having a gene-specific sequence complementary to a fixed region in the vicinity of a variable region on a Sepharose bead having a diameter of 30 ⁇ m as a first solid carrier.
  • Streptavidin-fixed Sepharose beads are commercially available (for example, manufactured by GE Health Care). Fix it.
  • a second DNA probe having a poly-T sequence is immobilized in order to capture a gene, which is a second biomolecule, on magnetic beads having a diameter of 1 ⁇ m as a second solid support.
  • magnetic beads having streptavidin immobilized on the surface are commercially available (for example, manufactured by Dynal).
  • the 5 'end is modified with biotin, and the DNA probe can be immobilized by mixing the beads and the DNA molecule under the conditions according to the instructions. Of course, both may be fixed by different mechanisms.
  • FIG. 1 A cell suspension to be measured is dropped on the substrate, a negative pressure (95 kPa) is applied to the back surface of the bead-holding membrane (7), and the cell solution becomes a cell trapping hole (3) and a reaction vessel (2). And flow through the membrane (7).
  • the cells in the cell suspension stop in the form of plugging the cell trapping holes, and the holes blocked by the cells stop the flow of the solution, so the cells that have not yet been trapped have priority.
  • the cells become trapped.
  • This process can be repeated to capture cells at most cell capture holes. In this way, the cells (4) are captured on the substrate (1).
  • the cells are crushed to extract the molecules to be measured in the cells.
  • a lysis solution is dispensed on the device, and a negative pressure (96 kPa) is applied to the back surface of the device in the same manner as in the case of the cell suspension so that the cell extract passes through the reaction vessel.
  • a negative pressure 96 kPa
  • the flow of the solution does not occur in the cell trapping hole blocked with cells, when the cells are crushed, the cell extraction solution flows through the reaction trap through the cell trapping hole.
  • the mRNA to be measured is captured on different beads by the capture DNA probes on the two types of beads (the first solid support 5 and the second solid support 6).
  • cDNA which is a complementary strand of mRNA
  • An enzyme reagent for cDNA synthesis is dropped on the substrate (1) in the same manner as the lysis solution to weaken the application of pressure (about 0 to 5 kPa) and slow down the flow of the solution.
  • the temperature of the device is raised to a temperature suitable for cDNA synthesis (50 ° C.) and reacted for an appropriate time (about 50 minutes).
  • the separation method of the two types of beads utilized both the bead size and magnetic properties. That is, after removing the membrane, the obtained bead solution is dispensed into a separation tube (8) and centrifuged, so that a large sepharose bead (first bead) is formed on the upper part of the separation filter (9). The rest, beads with a small diameter settle at the bottom. Magnets were also used to assist in settling.
  • Steps 1 to 5 in FIG. 3 and FIG. 4 show examples of sample preparation methods up to nucleic acid amplification (PCR) after cDNA synthesis possible with this device.
  • PCR nucleic acid amplification
  • the separation of the beads may be performed after capturing the mRNA before step 1 cDNA synthesis, but here, as shown in step 1, after the cDNA synthesis, the enzyme was thermally inactivated.
  • FIG. 3 shows an enlarged view of the surface of the second bead (6) on which the DNA probe (31) is fixed.
  • the DNA probe (31 (SEQ ID NO: 1)) immobilized on the beads includes a cell recognition tag sequence (302) for identifying the position of the reaction vessel.
  • the 3 'end of the DNA probe (31) has a poly T sequence (301), and captures the mRNA by hybridizing with the poly A sequence at the 3' end of the mRNA.
  • the DNA probe for capturing mRNA (31 (SEQ ID NO: 1)) has a slightly more complicated sequence structure in this example, and as shown in FIG. 3 (step 1), it is used for PCR amplification from the 5 ′ end. It consists of a common sequence (303) (Forward direction (SEQ ID NO: 23)), a tag sequence for cell recognition (302) and a nucleic acid capture sequence (301).
  • a poly-T sequence was used as the nucleic acid capture sequence.
  • the degree of polymerization of the poly T sequence may be any degree of polymerization that can hybridize with the poly A sequence of the mRNA and capture the mRNA on the beads to which the nucleic acid probe containing the poly T sequence is immobilized.
  • the common sequence for PCR amplification into the DNA probe (31)
  • this sequence can be used as a common primer in the subsequent PCR amplification step.
  • a tag sequence for molecular recognition for example, 7 bases
  • a poly-T sequence was used as part of the capture DNA probe (31) to analyze mRNA.
  • a random nucleic acid capture sequence was used instead of the poly-T sequence.
  • a sequence or a sequence complementary to a part of the nucleic acid to be analyzed may be used.
  • 1st cDNA strand (33) is synthesized using mRNA (32) captured by DNA probe (31) on the bead as a template.
  • the void portion of the beads packed with a solution containing reverse transcriptase and a synthetic substrate is filled, and the temperature is slowly raised to 50 ° C. to carry out a complementary strand synthesis reaction for about 50 minutes.
  • the mixture is kept at 85 ° C. for 5 minutes, and the reverse transcriptase is thermally inactivated, and then the beads are separated.
  • the membrane (7) was removed, and as shown in FIG. 1 (c), the upper side of the 5 ⁇ m pore filter tube containing PBS (phosphate buffered saline) buffer together with the substrate (1). Then, the beads are released from the substrate by stirring. Unnecessary chips are removed from the tube and centrifuged to collect large sepharose beads along with the solution. Magnetic beads having a diameter of 1 ⁇ m are collected on the lower side of the tube.
  • PBS phosphate buffered saline
  • the magnetic beads may be recovered using a magnet without using a filter.
  • the collection operation may be performed several times.
  • the collected Sepharose beads are collected again in a tube, and RNase enzyme is added to decompose and remove mRNA (32).
  • a solution containing an alkali denaturant and a washing solution are added, the beads are precipitated at the bottom of the tube by centrifugation, and the supernatant is removed to remove residues and degradation products.
  • a cDNA library array as shown in FIG. 5 (step 2) is constructed on the beads packed in the nucleic acid extraction part by the process so far, reflecting the positions of individual cells captured in the cell capture holes. .
  • a primer (34) containing a plurality (up to several hundreds) of target gene-specific sequences (311) to which a common sequence for PCR amplification (Reverse (SEQ ID NO: 2)) (309) is added is added to the first cDNA strand (33 ) (FIG. 3 (step 2)), and a 2nd cDNA strand (35) is synthesized by a complementary strand extension reaction (FIG. 3 (step 3)). That is, 2nd cDNA strand synthesis is performed under multiplex conditions.
  • a common sequence for amplification (Forward (SEQ ID NO: 23) / Reverse (SEQ ID NO: 2)) is provided at both ends, a cell recognition tag sequence, a molecular recognition tag sequence, and a gene-specific sequence Is synthesized in a double-stranded cDNA.
  • 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27, And 20 ⁇ 5 bases of 109 ⁇ 8 bases upstream from the poly A tail of the target gene of OAZ1) were used as gene-specific sequences (SEQ ID NO: 3-22). This is to unify the product size to about 200 bases.
  • Second DNA probe (ATP5B gene, primer for 2nd cDNA strand synthesis for specific analysis) CCTCTCTATGGGCAGTCGGTGATCCCTAACCCAAAAAGCTTCATT (SEQ ID NO: 3) Second DNA probe (GAPDH gene, primer for 2nd cDNA strand synthesis for specific analysis) CCTCTCTATGGGCAGTCGGTGATCACTGAATCTCCCCTCCTCACA (SEQ ID NO: 4) Second DNA probe (GUSB gene, primer for 2nd cDNA strand synthesis for specific analysis) CCTCTCTATGGGCAGTCGGTGATCGTTTCTGGCCTGGGTTTTG (SEQ ID NO: 5) Second DNA probe (HMBS gene, primer for 2nd cDNA strand synthesis for specific analysis) CCTCTCTATGGGCAGTCGGTGATGATGACTGCCTTGCCTCCTC (SEQ ID NO: 6) Second DNA probe (HPRT1 gene, primer for 2nd cDNA strand synthesis for specific analysis) CCTCTCTATGGGCAGTCGGTGATTAG
  • PCR amplification was performed using amplification primers (36 and 37) that bind to the amplification common sequence (Forward (SEQ ID NO: 23) / Reverse (SEQ ID NO: 2)), and PCR products derived from multiple types of genes ( 38) is prepared (FIG. 4 (steps 4 and 5)). Even if an amplification bias occurs between genes or molecules in this process, it is possible to correct the amplification bias using the molecular recognition tag sequence after acquiring next-generation sequencer data. Can be obtained.
  • the DNA probe (50) fixed to the carrier comprises a T7 promoter sequence (SEQ ID NO: 24) from the 5 ′ end direction, a common sequence for PCR amplification (Forward direction, SEQ ID NO: 23), a cell recognition tag sequence, and a molecule recognition tag. It is composed of an array and a poly-T array.
  • T7 promoter sequence SEQ ID NO: 24
  • IVT In Vitro Transcription
  • the T7 promoter sequence (SEQ ID NO: 24) is recognized by T7 RNA polymerase, and transcription (cRNA (63) amplification) reaction is started from the downstream sequence.
  • a common sequence for PCR amplification it can be used as a common primer in the subsequent PCR amplification step.
  • a cell recognition tag sequence for example, 5 bases
  • 4 5 1024 single cells or regions can be recognized as described above.
  • a molecular recognition tag sequence for example, 15 bases
  • 4 15 1.1 ⁇ 10 9 molecules can be recognized. As described above, it is possible to recognize whether it is derived from a molecule.
  • the amount of mRNA present in the sample can be quantified with high accuracy.
  • the poly T sequence located at the most 3 ′ side hybridizes with the poly A tail added to the 3 ′ side of the mRNA and is used to capture the mRNA.
  • mRNA (52) is captured by an 18-base poly-T sequence (51) which is a sequence complementary to the poly-A sequence (53) at the 3 'end of mRNA as described above.
  • the first cDNA strand (54) is synthesized to construct a cDNA library (FIG. 5 (step 1)).
  • a plurality of (up to several hundred genes) target gene-specific sequence primers (60) corresponding to the gene to be analyzed are annealed to the first cDNA strand (54) (FIG. 5 (step 2)), and 2nd by complementary strand extension reaction.
  • a cDNA strand (61) is synthesized (FIG. 5 (step 2)).
  • 2nd cDNA strand synthesis is performed under multiplex conditions.
  • a common sequence for amplification (Forward (SEQ ID NO: 23) / Reverse (SEQ ID NO: 2)) is provided at both ends, a cell recognition tag sequence, a molecular recognition tag sequence, and a gene-specific sequence Is synthesized in a double-stranded cDNA.
  • 20 types (ATP5B, GAPDH, GUSB, HMBS, HPRT1, RPL4, RPLP1, RPS18, RPL13A, RPS20, ALDOA, B2M, EEF1G, SDHA, TBP, VIM, RPLP0, RPLP2, RPLP27, And 20 ⁇ 5 bases of 109 ⁇ 8 bases upstream from the poly A tail of the target gene of OAZ1) were used as gene-specific sequences (SEQ ID NO: 3-22). This is to unify the amplification product size to about 200 bases.
  • T7 RNA polymerase is introduced into the pore to synthesize cRNA (63) (FIG. 5 (step 4)).
  • cRNA cRNA
  • a target gene-specific sequence primer (64) is hybridized (FIG. 6 (step 5)) to synthesize cDNA (65) (FIG. 6 (step 6)).
  • double-stranded DNA for PCR (66) is synthesized by synthesizing 2nd strand using Forward common primer (SEQ ID NO: 23) (FIG. 6 (step 7)).
  • SEQ ID NO: 23 Forward common primer
  • This amplification product has the same length and can be directly applied to PCR and next-generation sequencers. Even if an amplification bias occurs between genes or molecules in this process, it is possible to correct the amplification bias using the molecular recognition tag sequence after acquiring next-generation sequencer data. It is the same as the above that can be obtained.
  • a cell solution (phosphate buffer pH 7.5) adjusted to a concentration of about 100 cells / ⁇ L is dispensed to each chip, and the cells are captured by applying a negative pressure, and then the cell disruption solution is allowed to flow.
  • PCR amplification step was performed (FIG. 3 (step 4)). Thereafter, PCR Purification Kit (QIAGEN) was used to remove residual reagents such as free PCR-amplified common sequence primers (Forward (SEQ ID NO: 23) / Reverse (SEQ ID NO: 2)) and enzymes contained in this solution. Purify using etc. After applying the PCR amplification or the bridge amplification, this solution is applied to a next-generation sequencer of each company (Life Technologies (Solid / Ion Torrent), Illumina (High Seq), Roche 454) and analyzed.
  • QIAGEN PCR Purification Kit
  • the prepared reagent was dispensed into a tube, the temperature was raised to 37 ° C. and maintained for 180 minutes to complete the transcription reaction, and cRNA amplification was performed.
  • the target portions of the 20 target genes are amplified, but the cRNA amplification product size is almost uniform at 200 ⁇ 8 bases. Since the cRNA amplification product is contained in the supernatant of the bead solution, this solution is recovered.
  • it is purified using PCR Purification Kit (QIAGEN) and suspended in 50 ⁇ L of sterile water.
  • the cell recognition tag is the same by rearranging the obtained data for each cell recognition tag sequence (or for each sequence including those sequences if other tag sequences for identifying the sample are present).
  • the sequence data can be analyzed as data indicating gene expression in the same cell (if the additional tag is included, also by the sequence of the tag). That is, since the mRNA corresponding to the same cell has the same cell recognition tag sequence, it can be identified that the mRNA is derived from the same cell even if samples are prepared from different bead types (Fig. 2).
  • FIG. 7A shows a top view
  • FIG. 7B shows a cross-sectional view
  • the flow cell device 701 includes a plurality of reaction chambers (702), and one chip (715) is disposed in the reaction chamber (702).
  • a plurality of cell trapping holes are provided on the chip, and two types of beads to which DNA probes having different cell recognition tag sequences are fixed are filled immediately below.
  • mRNA for sequence analysis and mRNA for gene expression analysis are simultaneously captured to synthesize cDNA. Nucleic acid capture holes and cell recognition tag sequences are arranged in a one-to-one correspondence.
  • the cells (706) flow from the common inlet (707) toward the common outlet (708) in the common flow path (705) on the flow cell device.
  • the nucleic acid in the cell is individually captured for each hole in the two types of beads (first Sepharose beads and second magnetic beads) filled immediately below the cell capture holes on the chip 715.
  • the solution in the common flow path (705) flows through the reaction area filled with cell trapping holes and beads toward the common suction flow path (709). .
  • mRNA in the cell is captured on the beads, and further, a cDNA synthesis reaction occurs on the beads.
  • the chip For reaction after cDNA synthesis, the chip is taken out from the flow cell device, and two kinds of beads are collected in a tube (container).
  • the flow cell device In order to take out the chip, the flow cell device has a combination of upper and lower parts (upper and lower in FIG. 7B) with the device separation boundary (720) as a boundary, and unscrew the upper and lower parts with this boundary as a boundary.
  • the chip is recovered by separating. After taking out the chip, two kinds of beads are separated in a tube as in Example 1, and two kinds of samples are prepared by executing different reaction processes. To implement.
  • the data analysis method is the same as in Example 1. However, by inserting a different sequence for each chip into the PCR primer, gene analysis data for each chip and further for each cell trapping hole is obtained from the sequence analysis data. Is possible.
  • This example shows an example of simultaneous analysis of gene expression analysis in a single cell and protein analysis by mass spectrometry.
  • FIG. 8 shows a configuration diagram of the sample preparation chip in this example.
  • the substrate (801) is made of a silicon substrate (other inorganic materials such as glass, silicon nitride, aluminum and copper may be used), and a through-hole (803) for capturing cells is formed on the two-dimensional lattice. It is arranged in a shape.
  • a negative pressure to the membrane (807) side as a bead holding filter, the cells (804) are trapped in the cell trapping holes (803).
  • a reaction tank (802) is provided immediately below the captured cells, and a plurality of types of biomolecules to be analyzed are captured in this region.
  • the antibody (805) that specifically captures the protein that is the first measurement target biomolecule is directly fixed to the reaction vessel inner wall of the substrate (801). Furthermore, the reaction vessel is filled with beads (806) to which a DNA probe for capturing mRNA that is the second measurement target biomolecule is immobilized.
  • the second bead is used as the second bead for capturing mRNA that is the second biomolecule.
  • the fixed probe on the bead is the same as the description corresponding to FIG.
  • the beads are fixed with a poly-T probe to which a cell recognition tag sequence is added using magnetic beads.
  • specific sequences for these sequences may be used instead of poly-T sequences.
  • a random probe of about 6 bases may be used in place of the poly-T sequence in order to perform all nucleic acid sequence analysis.
  • the first biomolecule (protein or the like) is captured by the antibody immobilized on the substrate surface in the reaction vessel (802).
  • Example 1 after dropping the cell suspension station on the substrate (801), a negative pressure is applied to the side of the bead-holding porous membrane (807), so that the solution flow penetrating the cell trapping hole is generated. As a result, the cells (804) are trapped in the cell trapping holes (803).
  • a cleaning solution with an appropriate salt concentration (about 0.1 to 1M) is dropped onto the chip surface, and negative pressure is applied to cause the cleaning solution to flow into the reaction tank (802). Wash away the molecules. Thereafter, the membrane (807) is removed, and the beads and the substrate are separated (FIGS. 8B and 8C).
  • the beads are collected in a tube (808), sample processing is performed in the same manner as the second bead in Example 1, and gene sequence analysis is performed with a next-generation sequencer (NGS). Since the cell recognition tag sequence is different for each reaction tank, gene expression analysis can be performed separately for each cell.
  • NGS. 8B and 8C next-generation sequencer
  • the first biomolecule captured by the antibody on the substrate (801) is individually ionized by laser for each reaction tank and analyzed by a mass spectrometer. That is, the substrate (801) on which the protein is captured is subjected to mass spectrometry (MALDI-TOF-MS analysis) retaining position information.
  • MALDI-TOF-MS analysis mass spectrometry
  • the antibody is immobilized on the inner wall of the reaction tank to capture the protein, but it goes without saying that the molecule to be immobilized may be changed depending on the molecule to be measured.
  • the selectivity of the molecule to be measured is lowered, it is also possible to simply perform a surface treatment suitable for the object to be measured, such as making the surface hydrophobic or hydrophilic.
  • the sample processing flow of this example is summarized in FIG. Next, a specific method of MALDI-TOF-MS analysis at the single cell level will be described. Since the measurement target molecule is a polymer, a 5 mg / mL sinapinic acid ethanol solution was used as a matrix agent. The substrate (801) is placed with the surface where the membrane (807) is in close contact, and the sinapinic acid solution is dropped. The chip with the matrix agent dripped is individually irradiated with a nitrogen laser (343 nm) in the reaction vessel, and the sample suction port of the TOF-MS apparatus is brought close to the chip to perform mass spectrometry. Since an apparatus (mass imaging) that performs different mass spectrometry for each laser excitation position is also commercially available, such an apparatus may be used.
  • the gene expression data from the same position is correlated to analyze two types of biomolecules, protein and mRNA from the same cell. Can be executed in parallel.
  • FIG. 10 shows a diagram of a form of filling beads into the reaction vessel on the chip corresponding to this example.
  • the first beads (5) for capturing a trace amount of mRNA are packed in the reaction tank (2) closer to the through hole (3), and the second beads for capturing a very small amount of mRNA.
  • the beads (6) were filled at positions apart from the through holes in the reaction vessel (2). Thereby, the first biomolecule is preferentially captured by the first bead, and a trace amount of mRNA can be efficiently captured.
  • beads in which a DNA probe is immobilized on polystyrene beads having a diameter of 1 ⁇ m are used (the sequence of the immobilized DNA probe is the case of Example 1). Is the same). Therefore, since the separation according to the size of the beads cannot be performed, the second beads are collected at the bottom of the tube by the magnet 1001 and separated into a supernatant and a precipitate. At this time, since the second bead is recovered toward the precipitate, by resuspending this solution, a second bead solution is obtained, and the supernatant is allowed to settle the second bead with a magnet.
  • the purity of the first beads in the supernatant is gradually increased.
  • the precipitated second bead solution is mixed and the volume of the solution is controlled by precipitating the beads using a magnet.
  • the first and second beads are separated.
  • the steps after this step are the same as those in the first embodiment.
  • the first bead captures mRNA for sequence analysis as sepharose beads (diameter 34 ⁇ m)
  • the second bead captures mRNA for gene expression analysis as magnetic beads (diameter 1 ⁇ m)
  • the third bead is a polystyrene bead having a diameter of 1 ⁇ m (non-magnetic and small in size) and captures microRNA for gene expression analysis (a specific probe for microRNA capture is immobilized on the bead).
  • the separation method for example, the same method as in Example 1 can be used.
  • FIG. 11 shows a structural diagram of a device for trapping cells and beads as a solid carrier in a droplet in a chip and capturing two or more types of biomolecules derived from cells on the beads.
  • the chip (1101) is formed of glass, resin, or the like
  • FIG. 11 (a) is a top view
  • FIG. 11 (b) is a cross-sectional view.
  • the size of the cross section of the flow path (1102) is suitably several ⁇ m to several hundred ⁇ m in both vertical and horizontal directions (this flow path size roughly determines the droplet size), but here the length is 30 ⁇ m and the horizontal is 60 ⁇ m.
  • Mineral oil (or oil) is allowed to flow through the flow path (1102) in the direction of the arrow (1103) at an appropriate flow rate (several ⁇ m / second to several cm / second).
  • the first beads (the same Sepharose beads as in the example) are suspended in a cell lysate (in this example, 100 mM Tris: HCl, pH 7.5 buffer containing 1% SDS, 500 mM NaCl, and 10 mM EDTA).
  • a cell lysate in this example, 100 mM Tris: HCl, pH 7.5 buffer containing 1% SDS, 500 mM NaCl, and 10 mM EDTA.
  • the cell lysate in which the second beads are suspended is caused to flow in the direction of arrow 1105 at the same speed as the first beads.
  • phosphate buffered saline with suspended cells is introduced in the direction of arrow 1106 at the same rate as the bead suspension.
  • the obtained droplets (1107) are generated in the number necessary for the analysis, and the cell recognition tag array is different for each droplet, and is the same array in the same droplet.
  • the cell recognition tag sequence uses the same sequence in the same drop so that it differs from drop to drop. Such placement of the tag sequence in the droplet is realized by controlling the order of introduction of beads having different cell recognition tag sequences.
  • sequences of the two types of mRNA for each cell are matched by the sequence of the cell recognition tag sequence or its order in gene expression analysis.

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Abstract

Afin de mesurer des quantités de traces de multiples sortes de biomolécules dans une cellule unique, la présente invention concerne un dispositif et une méthode capables de différencier des biomolécules dérivées d'une cellule unique et de séparer et d'extraire des échantillons avec une efficacité élevée. Spécifiquement, la présente invention concerne un dispositif d'analyse de cellule unique, qui a un moyen de séparation de cellule et un champ de réaction disposé à proximité ou à l'intérieur dudit moyen de séparation, le dispositif d'analyse de cellule unique étant caractérisé en ce que: ledit champ de réaction a une zone dans laquelle se trouvent des premiers supports pour piéger une première biomolécule extraite d'une cellule piégée et des seconds supports pour piéger une seconde biomolécule différente de la première biomolécule; les premièrs supports et/ou les seconds supports sont pourvus d'une séquence d'étiquettes d'identification de la cellule piégée; et les premièrs supports et les seconds supports ont des caractéristiques physiques différentes, séparées les unes des autres.
PCT/JP2017/001512 2017-01-18 2017-01-18 Dispositif et méthode d'extraction de multiples biomolécules d'une cellule unique WO2018134907A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021166715A1 (fr) * 2020-02-17 2021-08-26 株式会社Ｊｖｃケンウッド Procédé d'analyse d'échantillon biologique
JPWO2021181467A1 (fr) * 2020-03-09 2021-09-16
JPWO2022009642A1 (fr) * 2020-07-06 2022-01-13
WO2023243152A1 (fr) * 2022-06-16 2023-12-21 株式会社日立製作所 Récipient pour analyse de cellule unique et procédé d'analyse de cellule unique l'utilisant

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000342258A (ja) * 1999-06-09 2000-12-12 Hitachi Ltd Ｄｎａ試料調製方法及びｄｎａ試料調製装置
JP2009284834A (ja) * 2008-05-29 2009-12-10 Hitachi Ltd 大規模並列核酸分析方法
WO2014020657A1 (fr) * 2012-07-30 2014-02-06 株式会社日立製作所 Dispositif de banque d'adnc bidimensionnel lié à une séquence étiquette, et procédé d'analyse d'expression génique et appareil d'analyse d'expression génique l'utilisant
WO2014141386A1 (fr) * 2013-03-12 2014-09-18 株式会社日立製作所 Dispositif à réseau cellulaire bidimensionnel et appareil pour la quantification de gènes et l'analyse de séquences
US20150376609A1 (en) * 2014-06-26 2015-12-31 10X Genomics, Inc. Methods of Analyzing Nucleic Acids from Individual Cells or Cell Populations
WO2016038670A1 (fr) * 2014-09-09 2016-03-17 株式会社日立製作所 Système d'analyse de cellules et procédé d'analyse de cellules l'utilisant
WO2016162997A1 (fr) * 2015-04-09 2016-10-13 株式会社日立製作所 Système d'analyse de gène

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000342258A (ja) * 1999-06-09 2000-12-12 Hitachi Ltd Ｄｎａ試料調製方法及びｄｎａ試料調製装置
JP2009284834A (ja) * 2008-05-29 2009-12-10 Hitachi Ltd 大規模並列核酸分析方法
WO2014020657A1 (fr) * 2012-07-30 2014-02-06 株式会社日立製作所 Dispositif de banque d'adnc bidimensionnel lié à une séquence étiquette, et procédé d'analyse d'expression génique et appareil d'analyse d'expression génique l'utilisant
WO2014141386A1 (fr) * 2013-03-12 2014-09-18 株式会社日立製作所 Dispositif à réseau cellulaire bidimensionnel et appareil pour la quantification de gènes et l'analyse de séquences
US20150376609A1 (en) * 2014-06-26 2015-12-31 10X Genomics, Inc. Methods of Analyzing Nucleic Acids from Individual Cells or Cell Populations
WO2016038670A1 (fr) * 2014-09-09 2016-03-17 株式会社日立製作所 Système d'analyse de cellules et procédé d'analyse de cellules l'utilisant
WO2016162997A1 (fr) * 2015-04-09 2016-10-13 株式会社日立製作所 Système d'analyse de gène

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021166715A1 (fr) * 2020-02-17 2021-08-26 株式会社Ｊｖｃケンウッド Procédé d'analyse d'échantillon biologique
JP2021128128A (ja) * 2020-02-17 2021-09-02 株式会社Ｊｖｃケンウッド 生体試料分析方法
JPWO2021181467A1 (fr) * 2020-03-09 2021-09-16
JP7312313B2 (ja) 2020-03-09 2023-07-20 株式会社日立ハイテク 微粒子充填方法及び微粒子充填装置
JPWO2022009642A1 (fr) * 2020-07-06 2022-01-13
WO2023243152A1 (fr) * 2022-06-16 2023-12-21 株式会社日立製作所 Récipient pour analyse de cellule unique et procédé d'analyse de cellule unique l'utilisant

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