JPWO2017056251A1 - Analytical method and analytical device - Google Patents

Abstract

The present invention is a technique for normalizing individual gene expression measurement values in single-cell gene expression analysis that realizes individual cell measurement in the same container. Specifically, an analysis method for separating a cell group as a sample into individual cells, capturing a biological material of each cell in a capture region, and measuring a physical quantity resulting from the captured biological material, the cell group Prior to the separation, a normalization step is performed in which a known substance that is a standard for physical quantity normalization is captured in the capture region.

Description

  The present invention relates to a technique for standardizing sample data in a technique for analyzing a single cell.

  For basic understanding of regenerative medicine, genetic diagnosis, and life phenomena, not only the average gene expression level but also the contents of individual cells that make up the tissue are quantitatively analyzed. It is beginning to be important. Such analysis of individual cell characteristics one by one is called single cell analysis (see, for example, Patent Document 1).

In conventional analysis, DNA, RNA, and protein are extracted and analyzed as a single sample from a large number of cells (10 ³ to 10 ⁶ or more cells) sampled from living tissue (hereinafter “bulk analysis”) And obtained average analysis information in the sample. On the other hand, single cell analysis can detect specific behavior of a small number of cells, and is considered to be an important technique for cancer research and iPSC (induced pluripotent stem cells) research.

  Since the amount of biomolecules in a single cell is extremely small, single cell analysis has so far been used only for analysis of some biomolecules such as proteins on cell membranes. However, in recent years, with the development of technology, it has become possible to quantitatively evaluate a small amount of biomolecules in a single cell. For example, a small amount of mRNA in a single cell is converted to cDNA by reverse transcription, and after nucleic acid amplification using PCR (polymerase chain reaction) amplification, the base sequence of the amplified product is determined using a large-scale DNA sequencer. A method of estimating the number of mRNA molecules by counting the number of DNA sequences after amplification by counting the sequencing results has begun to be used.

  As shown above, a great deal of progress has been made in techniques for treating a small amount of biomolecules derived from a single cell as an analysis target. When the purpose of “analyzing individual cell characteristics one by one” is further examined here, it is obvious that there is a precondition that “the measured individual cell data can be compared with each other”. . This is because it is possible to extract the features and commonality of specific cells by comparing individual cell measurement values, and a technique for standardizing data derived from individual cells is necessary to perform the comparison. .

  In gene expression comparison by conventional bulk analysis, as a standardization technique of measurement values, global normalization method that corrects measurement values assuming that gene expression profiles between samples are almost equal, and foreign known gene sequences are analyzed The gene spike method to be added to the nucleic acid sample is used. Global normalization is a mathematical standardization technique and is convenient for measurement. However, accuracy is difficult due to the assumption that gene expression profiles are approximately equal. In particular, single cell analysis is an analysis technique performed for the purpose of grasping individuality of diverse individual cells, and application of the global normalization method is not recommended.

  On the other hand, the gene spike method is recognized as a means to realize accurate inter-comparison, and an international consortium (ERCC) that makes it possible to mutually compare measurements made by different research institutions by sharing known gene sequences to be added. External RNA Control Consortium) has also been established. An example of normalization using the gene spike method in single cell analysis has been reported (Non-Patent Document 1), and its effectiveness has been shown.

International Publication WO / 2014/020657

Nucleic Acids Research, Vol. 34, no. 5 (2006) Nature Method, Vol. 6, no. 7 (2009), p. 503 J. Biosci. Bioeng., Vol. 119, no. 4 (2015), pp. 492.

  For example, a method has been devised in which almost all mRNA is changed to cDNA, and a cDNA library (cDNA aggregate containing all cDNAs) retained on beads is prepared and used for quantitative analysis. It has been shown that it is possible to accurately measure the expression level of a plurality of genes contained in one cell by eliminating a measurement error of a small amount of expressed gene by dividing a sample by repeatedly using a cDNA library (Non-patent Document 2). ).

  In addition, the group including the inventors has developed a cDNA library using a porous membrane in place of beads in order to realize low-cost gene expression analysis. A two-dimensional device has been developed that can obtain a distribution and simultaneously realize single-cell gene expression analysis of a large number of cells (Patent Document 1). This two-dimensional device can perform many single cell analyzes in parallel in the same reaction vessel (see FIG. 10 of Patent Document 1). Single cell analysis in the same container can reduce the difference between analysis trials compared to the method of preparing and analyzing a reaction solution for each cell as in Non-Patent Document 1, and thus more accurate biomolecules. It is effective for realizing the comparison. In addition, the inside of the same container described here is a container which has a continuous space, and includes the container which can supply a reagent in common.

  The inventors further examined the use of the gene spike method with the goal of improving the accuracy of comparison between individual cells and between trials. However, the conventional gene spike method is performed by adding a certain amount of a foreign gene to each individual reaction vessel. For this reason, the simple application to the inventors' two-dimensional device that performs single cell analysis in parallel in the same reaction solution has been difficult.

  The subject of this invention is adding the foreign gene comprised by the known base sequence so that dispersion | variation may be reduced with respect to the nucleic acid capture region which exists in the cell capture region juxtaposed in the same container.

  One aspect of the present invention that solves the above problem is to separate a cell group as a sample into individual cells, capture the biological material of each cell in a capture region, and measure a physical quantity caused by the captured biological material. An analysis method, comprising a normalization step of capturing a known substance as a standard for physical quantity in a capture region prior to separation of a cell group.

  In a typical example of the present invention, the biological substance is a substance having one or more types of base sequences, and the known substance is a known substance having one or more types of base sequences.

  In a more preferred embodiment of the present invention, the solution containing the known substance includes contents (pseudocells) having the shape of a droplet, and the droplet is configured to contain the known substance.

  Another aspect of the present invention is an analytical device including a plurality of cell capture regions for capturing cells and a plurality of nucleic acid capture regions for capturing nucleic acids derived from the captured cells. In this device, the cell capture unit and the nucleic acid capture unit are arranged one-to-one, and a known substance is captured in advance in the nucleic acid capture unit.

  In a typical example of the present invention, a known substance is a substance having one or more kinds of base sequences.

  It is possible to improve the homogeneity of a foreign gene having a known base sequence that is added to nucleic acid capture regions juxtaposed in the same container. This makes it possible to normalize data between cell capture regions using the number and frequency of appearance of these foreign genes as indices.

Three views of a reaction cell in an embodiment of the present invention Flow chart of standardization process using a foreign gene as a comparative example Flow chart of normalization process using foreign gene in the embodiment of the present invention The expanded perspective view which shows the detail of the normalization process using the foreign gene in Example 1 of this invention The expanded perspective view which shows the detail of the normalization process using the droplet in Example 2 of this invention Sectional drawing of the array device which is an Example of this invention

  Embodiments will be described in detail with reference to the drawings. However, the present invention is not construed as being limited to the description of the embodiments below. Those skilled in the art will readily understand that the specific configuration can be changed without departing from the spirit or the spirit of the present invention.

  In the structures of the invention described below, the same portions or portions having similar functions are denoted by the same reference numerals in different drawings, and redundant description may be omitted.

  In the present specification and the like, notations such as “first”, “second”, and “third” are attached to identify the components, and do not necessarily limit the number or order. In addition, a number for identifying a component is used for each context, and a number used in one context does not necessarily indicate the same configuration in another context. Further, it does not preclude that a component identified by a certain number also functions as a component identified by another number.

  The position, size, shape, range, and the like of each component illustrated in the drawings and the like may not represent the actual position, size, shape, range, or the like in order to facilitate understanding of the invention. For this reason, the present invention is not necessarily limited to the position, size, shape, range, and the like disclosed in the drawings and the like.

  The inventors diligently studied a method for adding a foreign gene to a two-dimensional cell device, and devised a method for adding a foreign gene to a reaction tank before a cell addition step as a sample. More specifically, this is a technique of adding a foreign gene uniformly composed of a known base sequence to a nucleic acid capture region existing in a cell capture region arranged in parallel on a two-dimensional device.

  In the following, in order to facilitate understanding of the present invention, an expression analysis method and a gene spike method will be described first. Furthermore, the problem in applying the gene spike method to the inventors' two-dimensional device will be described with specific examples. After that, a specific configuration of an apparatus for performing the gene spike method according to the present invention will be described.

  In conventional gene expression analysis methods, samples to be analyzed are added to individual containers, and operations such as cDNA synthesis and gene amplification are performed using these spatially isolated containers. The recent development of massively parallel sequencers has made it possible to acquire a large amount of data, so that multiple samples can be identified by labeling them with individual gene tags, and multiple labeled samples can be mixed and sequenced using the same container. The steps after the reaction may be performed. Even in that case, the steps from the extraction of the nucleic acid to the labeling with the tag sequence are performed using containers with different spaces. That is, in the gene spike method of the prior art, standardization between samples is realized by adding a foreign gene when the sample exists in an individual container.

  In the following embodiments of the present invention, an example will be described in which normalization between samples is realized by adding a foreign gene in single cell analysis of a plurality of cells in the same container.

  First, the gene analysis process using our two-dimensional device will be described.

<1. Structure of reaction cell>
FIG. 1 is a three-sided view showing the configuration of a reaction cell used for gene analysis. The array device 102 installed in the solution tank 101 has cell capture regions 103 arranged in parallel, and has a nucleic acid capture region 104 for capturing nucleic acid derived from each cell below each cell capture region 103. This array device should also be referred to as a two-dimensional array device or a parallel array device.

  The individual cells 105 captured in the cell capture region 103 have their cell membranes destroyed on the array device 102, and nucleic acid molecules released to the outside by the cell destruction are captured in the nucleic acid capture region 104 immediately below the cell capture region 103. Therefore, nucleic acid molecules derived from individual cells can be distinguished by the capture position on the array device. Further, by introducing an individual identification tag into each nucleic acid capture region 104, an identification tag corresponding to the position of the nucleic acid capture region 104 can be introduced into the nucleic acid molecule captured in the nucleic acid capture region 104. When nucleic acid molecules derived from individual cells are peeled off from the array device 102, those nucleic acid molecules lose their positional information on the array device, but after introducing the identification tag into the nucleic acid molecule, each nucleic acid molecule is used as an index for the identification tag. The nucleic acid molecules prepared using the array device 102 can be subjected to single cell analysis using a known gene analyzer such as quantitative PCR or a sequencer. About the said identification tag, there exists description in the above-mentioned patent document 1, for example.

  FIG. 1 (a) shows a cross-sectional view of the cell array device. A cell inlet 106, a reagent inlet 107, an upper outlet 108, and a lower outlet 109 are connected to the solution tank 101. In this embodiment, the array device 102 is manufactured using a semiconductor process. Using the flow of the solution from the cell inlet 106 to the lower outlet 109, in order to arrange the cells 105 injected from the cell inlet 106 in a square lattice, the cells are sucked only in a square area of 15 μm on one side. An array of cell capture regions 103 is formed.

  The array device 102 used here forms a 10 μm thick SiO2 layer on a silicon substrate 110, and uses dry etching to form a nucleic acid trap 104 with a through hole 111 having a diameter of 0.3 μm. After thinning 110, a grating 112 having a period of 50 μm was formed by lithography. A DNA probe can be immobilized on the inner wall of the through-hole 111 of the nucleic acid capture region 104 obtained in this way by silane coupling treatment.

  FIG. 1 (c) shows a cross-sectional view at B-B ′, and different hatching indicates that different cell recognition tag sequences are fixed. FIG. 1B shows a cross-sectional view taken along the line A-A ′. The portion 113 where the through-hole 111 is exposed is subjected to patterning so that cells are stored one by one.

<2. Analysis process>
By using the reaction cell of FIG. 1, the cells 105 can be fixed one by one on the array device 102, and the inside of the through-hole 111 can be filled with the solution. The solution tank 101 fills the array device 102 with the solution. An upper lid (upper electrode) 114 and a lower lid (lower electrode) 115 on which transparent (ITO) electrodes are formed by sputtering are disposed inside the upper and lower sides of the array device 102, and an upper reaction region 116 and a lower portion for filling with a reaction solution. A reaction region 117 is formed. The reason why the electrode is transparent is to allow the cells to be observed with an optical microscope. The ITO transparent electrode used this time has a transmission characteristic of 40% or more in the wavelength range of 400 to 900 nm.

<2-1. Extraction and capture of mRNA>
A buffer solution is injected from the inlet 106 and discharged from the upper outlet 108 and the lower outlet 109 to fill the interior with the solution. Next, a cell group is caused to flow into the reaction cell from the cell flow path (inlet 106) while shaking the reaction cell. The cells 105 are captured in a positively charged portion, but of course, the cells may be captured using a container or compartment in which one cell is contained.

  Next, cells on the sheet using a solution in which a cell lysis reagent is mixed with a low melting point agarose gel (SeaPrep Agarose; gelation temperature 19 ° C, melting point 45 ° C at 2%) that changes phase between gel and liquid depending on temperature MRNA is extracted by destroying the cell membrane and cell tissue in a fixed state.

  4% SeaPrep Agarose (Cambrex Bio Science Rockland, Inc.) solution 250 μL, Lysis Solution 495 μL (TaqMan MicroRNA Cell-to-CT Kit; Applied Biosystems Inc.) and DNase I 5 μL are mixed well at 40 ° C. Next, the temperature of the array device is set to 4 ° C., the solution in the reaction regions 116 and 117 is drawn, and then the cell lysis reagent solution is injected from the inlet 107.

  After confirming that the solution on the array device 102 has gelled, raise the temperature of the array device 102 to 20 ° C, react for 8 minutes, and then add 50 μL of Stopping Solution (a solution that deactivates DNase) onto the gel. Add, react for 5 minutes and cool to 4 ° C.

  Next, 0.5 mL of 10 mM Tris Buffer (pH 8.0) containing 0.03% PEO (polyethylene oxide) with a molecular weight of 600,000, 0.03% PVP (polyvinylpyrrolidone) with a molecular weight of 1 million, and 0.1% Tween 20 is added. At this time, the distance between the upper electrode 114 and the lower electrode 115 is 2 mm, and the gaps (reaction regions 116 and 117) above and below the array device 102 are completely filled with the Tris buffer. While maintaining the sheet and solution temperature at 4 ° C, + 5V is applied for 2 minutes using the upper electrode 114 as the cathode (GND) and the lower electrode 115 as the anode, and the negatively charged mRNA is electrically directed from the inside of the cell to the direction 117. Run. (Electrophoresis conditions may be pulse electrophoresis with on-level 10 V, off-level 0 V, frequency 100 kHz, duty 50% instead of DC voltage application).

  In this process, the mRNA is almost trapped in the oligo (dT) portion of the DNA probe fixed to the through hole 111 in the array device 102.

<2-2. Cleaning>
Next, the Tris buffer is introduced from the inlet 107 and discharged from the outlet 108, so that the solution and the temperature of the array device are raised to 35 ° C while the agarose gel is dissolved while the solution in the region 116 at the top of the array device is changed Then, unnecessary cell tissues and agarose are washed and removed.

<2-3. Reverse transcription reaction, cDNA library formation>
585 μL of 10 mM Tris Buffer (pH = 8.0) containing 0.1% Tween20, 40 μL of 10 mM dNTP, 5 × RT Buffer (SuperScript III, Invitrogen) 225 μL, 40 μL of 0.1M DTT, 40 μL of RNaseOUT (Invitrogen) and Superscript III (reverse transcriptase, Invitrogen 40 μL is mixed, the solution filling the array device 102 is discharged from the outlets 108 and 109, and the solution containing the reverse transcriptase is immediately injected from the inlet 107.

  Thereafter, the solution and the array device 102 were raised to 50 ° C. and kept for 50 minutes to complete the reverse transcription reaction, and a 1st cDNA strand having a sequence complementary to mRNA was synthesized.

  From the above operation, cDNA immobilized on the surface of many pores for each cell can be obtained as a library. This is a so-called 1-cell cDNA library sheet, which is fundamentally different from the averaged cDNA library obtained from many cells so far.

<2-4. Cleaning>
After synthesizing the first cDNA strand, reverse transcriptase was inactivated by maintaining at 85 ° C for 1.5 minutes, cooled to 4 ° C, and 10 mL of 10 mM Tris Buffer (pH = 8.0) containing RNase and 0.1% Tween20 was injected from inlet 107 Then, by discharging from the outlets 108 and 109, the RNA is decomposed, and the same amount of alkali denaturing agent is flowed in the same manner to remove and wash the residue and decomposition products in the pores.

<2-5. 2nd cDNA strand synthesis>
Next, 690 μL of sterilized water, 100 μL of 10 × Ex Taq Buffer (TaKaRa Bio), 100 μL of 2.5 mM dNTP Mix, 100 μL of each 10 μM PCR amplification primer Mix, and 10 μL of Ex Taq Hot start version (TaKaRa Bio) are mixed together. The filled solution is discharged from the outlets 108 and 109, and the solution containing the DNA synthase is immediately injected from the inlet 107.

  Then, the solution and array device 102 are reacted at 95 ° C for 3 minutes → 44 ° C for 2 minutes → 72 ° C for 6 minutes. After annealing the primer using the 1st cDNA strand as a template, a complementary strand extension reaction is performed, and the 2nd cDNA strand To synthesize.

<2-6. PCR amplification process>
Next, 495 μL of sterilized water, 100 μL of 10x High Fidelity PCR Buffer (Invitrogen), 100 μL of 2.5 mM dNTP mix, 40 μL of 50 mM MgSO4, 100 μL of 10 μM PCR amplification primer, and 15 μL of Platinum Taq Polymerase High Fidelity (Invitrogen) were mixed. The filled solution is discharged from the outlets 108 and 109, and the solution is immediately injected from the inlet 107.

  After that, keep the solution and the array device at 94 ° C for 30 seconds, repeat 40 cycles of 3 steps of 94 ° C for 30 seconds → 55 ° C for 30 seconds → 68 ° C for 30 seconds, and finally keep at 68 ° C for 3 minutes. Cool and perform the PCR amplification step. After the amplification process is completed, the PCR amplification product solution accumulated in the solution inside and outside the through hole 111 of the array device 102 is collected.

<2-7. Purification and analysis>
For the purpose of removing surplus reagents such as PCR amplification primers and enzymes contained in this solution, purification is performed using PCR Purification Kit (QIAGEN). After applying this solution to emPCR amplification or bridge amplification, the sequence is analyzed by applying to a next-generation sequencer of each company (for example, Life Technologies (Solid / Ion Torrent), Illumina (High Seq), Roche 454).

<3. Review of standardization>
The two-dimensional array device realizes parallel analysis by separating a plurality of cells into individual single cells in the same solution. Conventional single cell analysis requires the same number of containers / spaces as the number of cells to be analyzed in order to perform individual cells in individual containers / spaces, and is expensive because many reagents are used. In addition, since reaction reagents are prepared for individual cells, reagent dispensing errors also occur. This array device is an effective means for solving these problems and realizing a homogeneous analysis at low cost. The inventors further examined the realization of a homogeneous analysis and attempted to implement the gene spike method as a means for realizing a more accurate comparative analysis. As described above, the gene spike method in the conventional gene analysis method is a method of mixing a known foreign gene into a nucleic acid solution that is a sample to be examined.

  FIG. 2 schematically shows the method of Non-Patent Document 1 from the viewpoint of the inventors. When Non-Patent Document 1 is analyzed in detail, first, cells are individualized (S201), individual cells are separated into individual reaction vessels, and then nucleic acid extraction is performed (S202) to spatially distribute nucleic acid molecules derived from each cell. Isolated. Then, by adding known gene sequences to these individual reaction vessels (S203), normalization by the gene spike method is realized in the gene analysis stage (S204). Although the sample in the individual reaction vessel is a nucleic acid sample derived from a single cell, the standardization technique was the same technique as the bulk analysis.

  On the other hand, in the analysis process of the inventors' parallel array device 102, the sample added to the device is a solution containing a plurality of cells. Even if a foreign gene is added to this solution, individual nucleic acid molecule groups in single cell analysis Addition of exogenous genes to is not realized. In order to realize the gene spike method in the inventors' parallel array device, it is required to add a uniform foreign gene to each nucleic acid capture region 104 where the nucleic acid molecule of each cell is captured. That is, it is necessary to add a foreign gene as a process independent of the sample solution, instead of adding a foreign gene to an analysis sample in an independent container as in the prior art. It is important to add a uniform foreign gene to any nucleic acid capture region 104 regardless of the position on the array device 102.

  The order of the exogenous gene addition process was divided into i) simultaneously with the cell solution, ii) before addition of the cell solution, and iii) after addition of the cell solution, and the effects and problems were examined.

The simultaneous addition of the cell solution is similar to the conventional gene spike method as the timing of addition of the foreign gene, except that the sample contained in the solution is a cell. In the measurement using the array device 102, individual cells are captured in the cell capture region 103 from the sample solution added to the solution tank 101, and nucleic acid molecules derived from the individual cells released by cell destruction are located below the cell capture region 103. As described above, it is captured by the nucleic acid capturing region 104. These foreign genes have a structure that is partially homologous to the base sequence of the nucleic acid molecule or nucleic acid derivative molecule immobilized on the nucleic acid capture region 104, so that the nucleic acid molecules in the solution are captured by the nucleic acid capture region 104. Cell capture to the array device 102 uses a physical force such as solution flow, and the physical force to the cell capture region 103 changes before and after capturing the cell 105. In single cell analysis, one cell is captured. Considering the time from the sample solution addition until the cells 105 are captured, the time for each cell capture region 103 to be released until each cell capture region 103 captures the cells 105 is different. However, simultaneous cell capture is difficult to achieve. When a foreign gene is added for the gene spike method at the same time as the addition of the cell solution to this array device, the added nucleic acid molecule is before the cell capture rather than after the cell capture when the cell capture region 103 is in an open state. More reaches the nucleic acid capture region 104 below the cell capture region 103. That is, different amounts of foreign genes are captured in the nucleic acid capture region 104 below the cell capture region 103 with different open durations, and as a result, accuracy is not guaranteed as an original standard for normalization. That is, even if a foreign gene is added to the sample solution by simulating the conventional spike method, a sufficient effect cannot be expected for the planning of each cell, which is the purpose of the spike method.

  Next, ii) before cell solution addition was examined. A foreign gene with a known base sequence is added to a solution that does not contain the cell 105 to be inspected, and this solution is injected into the solution tank 101 in which the array device 102 is installed.

  Since the gene spike method aims at standardization of measurement values, it is desirable that foreign genes having a uniform number of molecules be captured in each nucleic acid capture region 104 of the array device 102. When a foreign gene is introduced before the addition of the cell solution, there is no factor that inhibits the flow of the solution containing the foreign gene to the cell capture region 103 and the nucleic acid capture region 104. The uniformity of molecular capture is improved.

  After the foreign gene capturing step is completed, prior to the step of adding the cells 105 to the array device 102, it is important to wash the solution bottle 101 containing the array device 102 with a solution that does not contain nucleic acid molecules. When the cell solution is added without performing the washing process, the foreign gene remaining in the solution 漕 101 and the cell solution will be mixed, and the cause of the occurrence of defects seen by the simultaneous addition of the cells and the foreign gene described above It is because it becomes. That is, normalization using a foreign gene can be performed by a series of steps of adding a solution containing a foreign gene, washing a foreign gene, and adding a solution containing cells to the solution bottle 101 having the array device 102.

  Next, consider after iii) addition of cell solution. At the stage where the solution containing the cells 105 is added, the individual cells 105 are captured in the cell capturing region 103 while maintaining the cell shape. There are two types of steps for introducing foreign genes: One is the addition of a foreign gene when the cell 105 maintains its shape, and the other is the stage after the cell 105 is destroyed and the nucleic acid derived from the cell is captured by the nucleic acid capturing region 104. When a foreign gene is added while maintaining the cell shape, the arrival of the foreign gene to the nucleic acid capture region 104 is affected by the timing of individual cell disruption. More specifically, the foreign gene reaches the nucleic acid capturing region 104 at the lower part of the rapidly disrupted cell, and the arrival of the foreign gene at the nucleic acid capturing region 104 at the lower part of the cell where the disruption is delayed is delayed.

  This difference in the arrival time of foreign genes is not suitable for normalization using foreign genes. When a foreign gene is added after cell disruption, the difference in arrival time of the foreign gene as described above does not occur, which is suitable for the purpose of normalization. However, when the event occurring in the nucleic acid capture region 104 is examined in detail, a foreign gene capture process is performed on the nucleic acid capture region 104 in which the nucleic acid molecules extracted from the cells by cell disruption are captured.

  In general, nucleic acid capture is performed with complementarity of base sequences, and since the binding is reversible, a nucleic acid molecule once bound can be released. That is, it is assumed that nucleic acid molecules derived from individual cells captured in the nucleic acid capturing region 104 are released by adding a foreign gene capturing process. Single-cell analysis is aimed at measuring a small amount of biomolecules derived from a single cell, and in order to realize normalization, it should be avoided that the detection power of nucleic acid molecules derived from the original cell is reduced. .

<4. Standardization process>
In FIG. The flow of the gene normalization process of the single cell parallel analysis device proposed based on examination of normalization> is shown. The apparatus configuration implemented uses the example shown in FIG. 1, but is not limited thereto.

  First, a standardized solution (foreign gene solution) is prepared by adding a foreign gene with a known base sequence to a solution that does not contain the cell 105 to be examined, and the standardized solution is added to the solution tank 101 in which the array device 102 is installed. Injection is performed (S301).

  For this purpose, the standardized solution is injected from the inlet 106 and discharged from the upper outlet 108 and the lower outlet 109 to fill the inside of the solution tank 101 with the standardized solution. As described above, these foreign genes have a structure partially having homology with the base sequence of the nucleic acid molecule or nucleic acid derivative molecule immobilized on the nucleic acid capturing unit 104, so that the nucleic acid molecules in the solution can be Captured at 104. Since the gene spike method aims at standardization of measurement values, it is desirable that foreign genes having a uniform number of molecules be captured in each nucleic acid capturing unit 104 of the array device 102.

  After the foreign gene capturing step is completed, prior to the step of adding the cells 105 to the array device 102, the solution bottle 101 in which the array device 102 is present is washed with a solution that does not contain nucleic acid molecules (S302). For this purpose, for example, a buffer solution containing no nucleic acid molecules is injected from the inlet 106, and the inside of the inlet 106 and the inside of the solution bottle 101 are sufficiently washed. If the cell solution is added from the inlet 106 without washing the inlet 106, the foreign gene remaining in the inlet 106 and the cell solution will be mixed. This is a cause of the occurrence of a malfunction.

  Thereafter, a sample solution containing a cell group is injected from the inlet 106 into the solution bottle 101 (S303), and an individual cell tag applying step (S304) using a parallel array device is performed. For these processes, <2. This is the same as described in the analysis step>.

  FIG. 4 shows details of the above processing. FIG. 4 shows an enlarged perspective view of the cell capture region 103 and the nucleic acid capture region 104 of FIG. As shown in FIG. 4A, the single cell parallel analysis device 102 has a cell capture region 103 and a nucleic acid capture region 104 in a two-dimensional array. In this state, by adding a solution containing a foreign gene (spike) to the solution tank 101, an ideally uniform foreign gene can be given to the nucleic acid capture region 104. That is, as shown in FIG. 4 (b), it can be said that the nucleic acid capture region 104 has become a nucleic acid capture region 104N that has undergone normalization. Thereafter, as shown in FIG. 4 (c), the individual cells 105 are captured in the cell capture region 103 and analyzed in the same manner as in a normal gene analysis process.

  As is clear from the above description, in this example, when the cell group as a sample is separated into individual cells and the biological material of the individual cells is separated or measured, normalization is performed prior to the separation of the cell groups. A reference process for adding known substances and a process for removing known substances are performed.

  As described above, as a standardization step, the introduction of a foreign gene is performed as uniformly as possible into each nucleic acid capture region 104 by performing the step of adding a foreign gene before adding a cell solution. The gene expression measurement value derived from each cell can be normalized by referring to the measurement value of the foreign gene shown in each cell capture region 104. Therefore, it is possible to compare gene expression between cells and to a test measured on different devices.

  In Example 1 described above, the analysis method including the PCR amplification step has been described. However, the embodiment of the present invention is not limited to this. A reference substance is captured together with a nucleic acid molecule as a sample by the nucleic acid capture unit, and data from a plurality of nucleic acid capture units is obtained by measuring both. What is necessary is just to normalize, and the analysis method can use various well-known methods.

  Next, an example of further examination as means for realizing standardization by the exogenous gene spike method will be described below.

  As a foreign gene addition step, the flow of adding a foreign gene, washing a foreign gene, and adding a cell sample is the same as in Example 1, but as a means to achieve a more homogeneous addition of a foreign gene and sufficient washing, The crystallization solution is prepared as droplets (spiked droplets) having a size similar to that of cells and added to the array device tank. That is, as a pre-stage of cell capture to the array device, the spike droplet functions as a pseudo cell containing a known foreign gene, and this is captured in the cell capture region 103, so that the nucleic acid capture region 104 is uniformly distributed. Realize the introduction of foreign genes.

  Specific processing methods include <2. Before the analysis step described in the “analysis step”, the above-described <2-1. Extraction and capture of mRNA> is equivalent to the above <2-2. Processing equivalent to “Cleaning” is performed. In these steps, however, spike droplets (pseudo cells) are injected from the cell channel (inlet 106) instead of the cells to be analyzed. That is, a measurement obtained from nucleic acid derived from a sample cell by providing a process of destroying spike droplets (pseudocells) containing a known substance and substance capture before the process of sample cell destruction and nucleic acid capture. The value is normalized.

  FIG. 5 shows details of the above processing. FIG. 5 shows an enlarged perspective view of the cell capture region 103 and the nucleic acid capture region 104 of FIG. As shown in FIG. 5A, the single cell parallel analysis device 102 has a cell capture region 103 and a nucleic acid capture region 104 in a two-dimensional array.

  In this state, a spike droplet (pseudo cell) 501 containing a foreign gene (spike) is added to the solution tank 101, and the droplet 501 is captured in the cell capture region 103. The capture of a foreign gene in the nucleic acid capture region 104 can be carried out in the same manner as the capture of a cell-derived nucleic acid. Adds foreign genes more uniformly than adding the solution in Fig. 3 by capturing droplets 501 of uniform size, removing foreign genes from the captured droplets, and capturing the extracted foreign genes in the nucleic acid capture region Can be expected. As a result, as shown in FIG. 5C, the nucleic acid capture region 104 becomes the nucleic acid capture region 104N that has undergone the normalization process. Thereafter, as shown in FIG. 5 (c), the individual cells 105 are captured in the cell capture region 103 and analyzed in the same manner as in a normal gene analysis process.

  As described above, the spike droplets 501 are ideally captured one by one in the cell capture region 103 as in the case of the sample cells. Therefore, the number of foreign genes introduced into the nucleic acid capture region 104 below the cell capture region 103 is limited to the number of molecules derived from one droplet 501, and the uniformity of introduction of the foreign gene into the nucleic acid capture unit is improved.

  The size of the spike droplet 501 should be 50% (half) or more of the short side of the cell capture region 103 in order to avoid capturing two or more droplets 501 in one cell capture region 103. Just hold it. More preferably, a diameter of 90 to 100% is ideal. In comparison with the cell 105 as a sample, ideally the same size as the cell 105 to be examined is good. It is preferable that the cell to be examined and the spike droplet have a size of 90 to 100% of the size of the short side of the cell trapping region 103. These values may be measured using the average value of the sample.

  Many prior studies including Non-Patent Document 3 have been made on the cell-size droplet forming technique used in this example. By capturing pseudo-cells with uniform size, that is, pseudo cells with a uniform content of foreign genes in each cell capture region, each nucleic acid capture region 104 can capture foreign genes from one pseudo cell. Realized. Although the addition of the standardized solution shown in Example 1 may be difficult to uniformly capture the foreign gene in the nucleic acid capture region 104 depending on the shape of the device and the way the solution flows, a droplet containing the foreign gene By using this, a uniform introduction of a foreign gene into the nucleic acid capture region 104 is achieved.

  Then, <2. By performing the analysis process described in the analysis step>, normalization by a foreign gene becomes possible.

  In Examples 1 and 2, in the same reaction cell, after normalizing the array device, <2. An example in which the processing described in the analysis step> is performed is shown.

  Other practical examples include normalization and <2. It is also possible to perform the processing described in the analysis step> separately. That is, the array device 102 is standardized and sold in advance, and a purchaser incorporates the solution into the solution tank 101 to form a reaction cell. Analysis process> is performed.

  FIG. 6 shows a cross-sectional view of an example of the array device 102 of the third embodiment. The planar shape is the same as that shown in FIG. 1 and has a two-dimensional array configuration. Since the nucleic acid capturing unit 104N has been subjected to the normalization processing described in Example 1 or 2 in advance and has already captured a foreign gene, in the subsequent analysis process, the foreign device already captured by the array device 102 is captured. Data can be normalized using genes.

  According to the embodiment of the present invention described above, by uniformly adding a foreign gene having a known base sequence to a nucleic acid capture region existing in a cell capture region arranged in parallel on a two-dimensional device, the number of appearances of those foreign genes, Data standardization between cell capture regions using frequency as an index becomes possible. In addition, comparison of gene expression analysis data obtained by a plurality of two-dimensional devices is also achieved by data normalization using this technology.

  The present invention is not limited to the embodiments described above, and includes various modifications. For example, a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. Moreover, it is possible to add / delete / replace the configurations of the other embodiments with respect to a part of the configurations of the embodiments.

  It can be used for various gene analyses.

  Solution tank 101, array device 102, cell capture region 103, nucleic acid capture unit 104, cell 105, spike droplet (pseudo cell) 501

Claims (10)

An analysis method for separating a cell group as a sample into individual cells, capturing a biological material of each individual cell in a capture region, and measuring a physical quantity caused by the captured biological material,
Prior to the separation of the cell group, an analysis method comprising a normalizing step of capturing a known substance serving as a standard for normalization of the physical quantity in the capture region. The analysis method according to claim 1,
The biological substance is a substance having one or more types of base sequences,
The known substance is a known substance having one or more types of base sequences.
Analysis method. The analysis method according to claim 1,
Using an array device with multiple cell capture regions to capture cells,
The array device is
A plurality of the capture regions corresponding to the cell capture region,
The capture region is configured as a nucleic acid capture region that captures nucleic acids derived from the cells captured in the cell capture region,
A first step of supplying a solution containing the cell group to the array device;
A second step of separating the cell group into individual cells and capturing them in the cell capturing region;
A third step of causing the nucleic acid capture region to capture nucleic acid derived from the cells captured in the cell capture region;
Before the first step,
Comprising the normalization step,
Analysis method. The analysis method according to claim 3,
The standardization process includes
A first normalization step of placing the array device in a solution tank;
A second normalization step of supplying a solution containing the known substance to the solution tank from a flow path;
A third normalization step of capturing the known substance in the nucleic acid capturing region;
A fourth normalization step of cleaning the solution tank and the flow path;
Comprising
Analysis method. The analysis method according to claim 4, wherein
The solution containing the known substance is
Including a content having the shape of a droplet, the droplet including the known substance,
Analysis method. The analysis method according to claim 5, wherein
The third normalization step includes
Capturing the contents having the shape of the droplets in the cell capture region;
Extracting the known substance from the contents captured in the cell capture region;
Capturing the extracted known substance in the nucleic acid capturing region;
Comprising
Analysis method. The analysis method according to claim 6, comprising:
The average diameter of the contents is 90-100% of the average diameter of the cells,
Analysis method. In a device for analysis comprising a plurality of cell capture regions for capturing cells and a plurality of nucleic acid capture regions for capturing nucleic acids originating from the captured cells,
The cell capturing part and the nucleic acid capturing part are arranged one-to-one,
An analytical device in which a known substance is captured in advance in the nucleic acid capturing unit. The analytical device according to claim 8, comprising:
The cell capture unit and the nucleic acid capture unit are arranged in a two-dimensional array,
Analytical device. The analytical device according to claim 8, comprising:
The known substance is a substance having one or more kinds of base sequences.
Analytical device.

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