Method for extracting nucleic acid, and method for analyzing gene expression

Abstract

To provide a technology for easily and simply performing nucleic acid extraction from a sample containing skin tissue as well as analysis using the nucleic acid.SOLUTION: A method for extracting a nucleic acid includes: a preparation step of preparing a sample of skin tissue dried under room temperature conditions; and an extraction step of adding a nucleic acid extraction reagent to the sample, thereby extracting a nucleic acid from the sample.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for extracting nucleic acids and a method for analyzing gene expression. [Background technology]

[0002] In recent years, techniques have been developed for diagnosing and evaluating the skin condition of a subject by analyzing the expression of nucleic acids, proteins, etc. in a biological sample. As the biological sample used in such evaluation, skin tissue with a diameter of about 2 to 4 mm obtained by punch biopsy or the like is generally used. As a non-invasive method, a method of extracting RNA from the stratum corneum or sebum has also been adopted (Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2018-000206 A [Patent Document 2] JP 2016-95185 A Summary of the Invention [Problem to be solved by the invention]

[0004] In order to grasp the skin condition of a subject more accurately, it is desirable to analyze the skin tissue itself. However, in the punch biopsy of the prior art, the volume of skin to be obtained is large and suturing is required after collection, which places a heavy burden on the subject when obtaining the tissue. In addition, the skin sample needs to be stored in a frozen state or in a stabilizing reagent to suppress the decomposition of nucleic acids, and there is a problem that it is time-consuming to manage the storage conditions during transportation and to secure a storage space.

[0005] In view of the above, an objective of the present invention is to provide a technique for easily extracting nucleic acid from a sample containing skin tissue and performing analysis using the nucleic acid. [Means for solving the problem]

[0006] The present invention for solving the above problems and its preferred embodiments are as follows.

[0007] [1] A preparation step of preparing a sample of skin tissue dried under room temperature conditions; A method for extracting nucleic acid, comprising the step of adding a nucleic acid extraction reagent to the sample and extracting nucleic acid from the sample. According to the present invention, since the decomposition of nucleic acids is suppressed from a skin tissue sample dried at room temperature, high-quality RNA can be efficiently extracted.

[0008] [2] The volume of said sample immediately after removal from the subject is 0.05 mm 3 The method according to claim 1, A sample having a volume within the above range is dried quickly immediately after being obtained from a subject, and therefore degradation of nucleic acids can be more effectively suppressed.

[0009] [3] The method according to [1] or [2], wherein the skin tissue includes an epidermis region including a stratum corneum and a dermis region located at a depth of 0.2 to 0.5 mm from the outermost layer of the skin. By including an epidermal region and a dermal region in the sample, nucleic acids in the epidermis and dermis can be extracted.

[0010] [4] The method according to any one of [1] to [3], wherein the sample is obtained by biopsy.

[0011] [5] The method according to any one of [1] to [4], which does not include a step of subjecting the sample to a nucleic acid degradation inhibiting treatment and / or a step of treating the sample with a protease prior to the extraction step. By adopting the above-mentioned configuration, the sample processing steps can be simplified, and the total time required to complete the extraction can be shortened.

[0012] [6] The method according to [5], wherein the step of subjecting the sample to a nucleic acid decomposition inhibiting treatment is a treatment of contacting the sample with a nucleic acid decomposition inhibiting agent and / or a treatment of freezing the sample.

[0013] [7] The method according to [5], wherein the protease is dispase.

[0014] [8] The method according to any one of [1] to [7], wherein the preparing step includes a step of storing the sample under room temperature conditions. In the above embodiment, the sample can be stored at room temperature during the period from sample acquisition to nucleic acid extraction.

[0015] [9] The method according to any one of [1] to [8], wherein the preparation step is a step of preparing the sample by placing skin tissue in a storage container and drying it under room temperature conditions. By adopting the above-mentioned embodiment, the drying process of the sample after acquisition can be easily carried out.

[0016]

[10] A preparation step of preparing a sample of skin tissue dried under room temperature conditions; an extraction step of adding an RNA extraction reagent to the sample and extracting RNA from the sample; A method for analyzing gene expression, comprising an analysis step of performing RNA expression analysis using the extracted RNA. According to the present invention, RNA analysis in a skin tissue sample can be carried out simply and easily.

[0017]

[11] The method according to

[10] , further comprising a quality confirmation step of confirming that the proportion of RNA fragments having 200 bases or more is 60% or more after the extraction step and before the analysis step. Effect of the Invention

[0018] According to the present invention, it is possible to provide a technique for easily extracting nucleic acid from a sample containing skin tissue and performing analysis using the nucleic acid. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the results of measuring DV200 of extracted RNA in Test Example 1. [Diagram 2] FIG. 1 shows the results of measurement of the RNA Integrity Number (RIN) of extracted RNA in Test Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention relates to a method for extracting nucleic acid from a sample containing skin tissue, and to a method for performing gene expression analysis using the nucleic acid extracted by the method. The present invention is not limited to the embodiments described below, and can be modified as appropriate within the scope of the invention.

[0021] <Nucleic acid extraction method> The method for extracting nucleic acid (hereinafter referred to as extraction method) of the present invention includes a preparation step of preparing a sample of skin tissue dried under room temperature conditions, and an extraction step of extracting nucleic acid from the obtained sample.

[0022] (1) Preparation process The preparation step is a step of preparing a sample for use in nucleic acid extraction. In the preparation step, a skin tissue sample (hereinafter simply referred to as a sample) is prepared.

[0023] The sample preferably includes an epidermis region including the stratum corneum, and a dermis region. The epidermis may include the stratum granulosum, stratum spinosum, and stratum basale in addition to the stratum corneum.

[0024] The sample skin tissue may contain epidermal cells, such as keratinocytes, epidermal stem cells, melanocytes, and pigment stem cells. The skin tissue sample may also contain dermal cells, such as dermal fibroblasts, nerve cells, microvascular endothelial cells, dermal lymphatic endothelial cells, mesenchymal stem cells, hematopoietic stem cells, blood cells, mast cells, and granular cells. Furthermore, the sample skin tissue may include cells of skin appendages. Examples of hair follicle cells include hair follicle keratinocytes, hair germinal center matrix cells, hair papilla cells, hair follicle outer root sheath cells, hair follicle inner root sheath cells, hair follicle stem cells, hair root stem cells, hair matrix cells, as well as sebaceous gland cells. Examples of sweat gland cells include myoepithelial cells, tonsil cells, serous cells, mucous cells, etc. Examples of arrector pili muscle cells include smooth muscle cells, etc. Examples of nail cells include nail keratinocytes, nail matrix cells, etc. Additionally, the sample skin tissue may contain skin cancer cells, including carcinoma, adenoma, sarcoma, melanoma, melanoblastoma, melanosarcoma, neuroblastoma, and the like.

[0025] The dermis region preferably includes a region immediately below the basement membrane. The dermis region included in the sample includes a region located at a depth (depth) from the outermost layer (stratum corneum) of the skin of preferably 0.2 mm or more, more preferably 0.22 mm or more, even more preferably 0.25 mm or more, and particularly preferably 0.28 mm or more. The dermis region also includes a region located at a depth of preferably 0.75 mm or less, more preferably 0.50 mm or less, even more preferably 0.4 mm or less, and particularly preferably 0.38 mm or less. The depth can be preferably 0.2 to 0.5 mm, more preferably 0.25 to 0.45 mm, and further preferably 0.28 to 0.4 mm. By positioning the dermal region to be obtained within the above range, a sample including the epidermal region and the dermal region can be obtained while reducing the burden on the subject during collection.

[0026] In a preferred embodiment, the sample contains only the epidermis region and the dermis region located within the above-mentioned depth range as skin tissue. Since the sample is obtained by collecting only the superficial layer of the skin, it is minimally invasive and can reduce the burden on the subject when collecting the sample.

[0027] In addition, the incision area of ​​the subject's skin tissue surface layer during sample acquisition is preferably 0.10 mm from the viewpoint of reducing the burden on the subject.2 Less than or equal to 0.08 mm, more preferably 2 Less than or equal to 0.05 mm, more preferably 2 Less than or equal to 0.03 mm, more preferably 2 Less than 0.01 mm, more preferably 2 Less than 0.005 mm, particularly preferably 0.005 mm 2 The following is the result. Specifically, the incision area is 0.001 to 0.10 mm 2 , preferably 0.001 to 0.05 mm 2 , more preferably 0.001 to 0.01 mm 2 , and more preferably 0.001 to 0.005 mm 2 It can be said that: When the acquired skin tissue is cylindrical or approximately cylindrical, the incision area corresponds to the size of the bottom area of ​​the skin tissue. By keeping the incision area within the above range, samples can be obtained in a minimally invasive manner, and the time it takes for wound healing in the subject after skin tissue is obtained can be shortened.

[0028] The method of collecting the sample is not particularly limited, and a conventional method used in a biopsy or the like can be adopted. In the present invention, it is preferable to adopt a minimally invasive collection method in order to reduce the burden on the subject. For example, skin tissue can be collected using a device equipped with a needle having a diameter of 1 mm or less. As such a device, a biopsy punch manufactured by World Precision Instruments or a device disclosed in WO2013 / 155557 can be used.

[0029] The site from which the sample is taken is not particularly limited, but examples include the head, face, neck, chest, hands, fingers, arms, legs, abdomen, back, and buttocks.

[0030] In the preparation step, the sample skin tissue is prepared in a dry state under room temperature conditions. It is preferable that the skin tissue is dried immediately after being collected from the subject. The drying method is not particularly limited, but natural drying is preferable from the viewpoint of simplicity.

[0031] Specifically, the time it takes for the sample to dry (sample drying time) is preferably 30 minutes or less, more preferably 20 minutes or less, more preferably 10 minutes or less, more preferably 5 minutes or less, even more preferably 3 minutes or less, and particularly preferably 1 minute or less. By setting the drying time of the sample within the above numerical range, the decomposition of nucleic acids in the skin tissue can be further suppressed.

[0032] In the present invention, "room temperature" refers to a temperature range of 1 to 30°C, as defined in the 18th Edition of the Japanese Pharmacopoeia.

[0033] In the present invention, the drying temperature for the sample skin tissue is preferably room temperature. In the present invention, "room temperature" refers to a temperature range of 15 to 25°C, as defined in the 18th Edition of the Japanese Pharmacopoeia.

[0034] The temperature at which the sample is dried may be 1° C. or higher, 3° C. or higher, 5° C. or higher, 10° C. or higher, and can be 15° C. or higher, 20° C. or higher, or even 25° C. or higher. By setting the drying temperature of the sample within the above range, the skin tissue can be dried quickly.

[0035] The temperature at which the sample is dried is preferably 30° C. or lower, more preferably 29° C. or lower, and even more preferably 28° C. or lower. By setting the temperature for drying the sample within the above range, it is possible to dry the skin tissue while suppressing decomposition of nucleic acids in the skin tissue.

[0036] Specifically, the temperature at which the sample is dried is preferably 10 to 30°C, more preferably 15 to 30°C, more preferably 15 to 30°C, and further preferably 20 to 28°C.

[0037] In the preparation step, a previously prepared dried skin tissue sample can be prepared, followed by the extraction step, or in the preparation step, the dried skin tissue sample can be prepared by obtaining a skin tissue sample from a subject and drying the sample.

[0038] When the preparation step involves drying the sample, the preparation step may include a step of preparing the sample by placing the skin tissue obtained from the subject in a storage container and drying it under room temperature conditions. By adopting the above-mentioned embodiment, the drying process of the sample after collection can be easily carried out.

[0039] The storage container used in the present invention is not particularly limited, and any container generally used in genetic manipulation can be selected, for example, a tube such as an Eppendorf tube.

[0040] The volume of the skin tissue in the sample is not particularly limited, but from the viewpoint of reducing the burden on the subject, the volume of the skin immediately after being taken from the subject is preferably 0.05 mm 3 Less than or equal to 0.045 mm, more preferably 3 Less than or equal to 0.04 mm, more preferably 3 Less than or equal to 0.038 mm, more preferably 3 Below 0.02mm 3 Below, 0.01mm 3 Below, 0.008mm 3 Below, 0.005mm 3 Below, 0.003mm 3 Below 0.002mm 3 It can be as follows: By keeping the volume of skin immediately after removal from the subject within the above range, samples can be obtained in a minimally invasive manner, the drying time of the sample under room temperature conditions can be shortened, and decomposition of nucleic acids can be suppressed.

[0041] The volume of the skin tissue in the sample is, for example, 0.0005 to 0.05 mm 3 and preferably 0.0008 to 0.04 mm 3, more preferably 0.001 to 0.038 mm 3 , more preferably 0.001 to 0.01 mm 3 , more preferably 0.001 to 0.008 mm 3 , more preferably 0.001 to 0.005 mm 3 , and more preferably 0.001 to 0.003 mm 3 , and particularly preferably 0.001 to 0.002 mm 3 It can also be done like this.

[0042] The preparation step may also include a storage step of storing the collected sample.

[0043] The storage step is a step of storing the collected and dried sample under room temperature conditions. The storage step can be performed before the extraction step. In the storage step, the sample is stored in a dried state, so that decomposition of nucleic acids in the sample can be suppressed even under room temperature conditions.

[0044] The period for storing the sample in the storage step can be, for example, preferably 30 minutes or more, more preferably 1 hour or more, more preferably 2 hours or more, more preferably 5 hours or more, more preferably 10 hours or more, more preferably 20 hours or more, more preferably 24 hours or more, even more preferably 48 hours or more, and particularly preferably 72 hours or more. In the storage process, the samples are stored in a dried state, which allows the samples to be stored for long periods at room temperature.

[0045] The storage conditions in the storage step are not particularly limited as long as the conditions allow the sample to be kept dry, but storage at room temperature and normal humidity is preferred, and storage at room temperature and normal humidity is more preferred. The preferred range of room temperature in the storage step is the same as the temperature range for drying the sample described above.

[0046] In addition, the term "normal humidity" in the present invention refers to a temperature range of relative humidity 45 to 85%, as defined in JIS Z8703 "Standard conditions of test locations".

[0047] Specifically, the humidity during storage in the storage step may be 45% or more, and can be 48% or more, 50% or more, or even 55% or more. Furthermore, the humidity during storage in the storage step is preferably 70% or less, more preferably 65% ​​or less, and even more preferably 60% or less. By keeping the humidity during storage within the above range, the sample can be kept dry and decomposition of nucleic acids in the skin tissue during storage can be suppressed.

[0048] (2) Extraction process The extraction step is a step in which a nucleic acid extraction reagent is added to the sample to extract nucleic acid from the sample.

[0049] The nucleic acid to be extracted is preferably DNA and / or RNA, and in the present invention, is preferably RNA.

[0050] The method for extracting nucleic acids is not particularly limited, and may be, for example, the guanidine thiocyanate-cesium chloride (GT-CsCl) ultracentrifugation method, the acidic guanidine thiocyanate-phenol-chloroform (AGPC) method, or the spin column method. For RNA extraction, the RNeasy Mini Kit (QIAGEN), PicoPure TM Commercially available products such as RNA Isolation Kit (Thermo Fisher Scientific) may be used. The nucleic acid extraction reagent to be used can be appropriately selected depending on the nucleic acid extraction method. TM Reagent (Invitrogen) or the extract provided with the kit can be used.

[0051] In the above embodiment, the present invention uses a small amount of skin tissue as a sample. Therefore, the entire tissue dries quickly by natural drying, so there is no need to carry out a separate nucleic acid decomposition suppression treatment. That is, the present invention can be implemented in a form that does not include a step of subjecting a sample to a nucleic acid degradation inhibiting treatment before the extraction step. More specifically, the present invention can be implemented in a form that does not include a step of subjecting a sample to a nucleic acid degradation step after collection of a sample from a subject until the extraction step.

[0052] The step of subjecting the sample to a nucleic acid decomposition inhibiting treatment includes, but is not limited to, a treatment of contacting the sample with a nucleic acid decomposition inhibiting agent and / or a treatment of freezing the sample. Examples of nucleic acid decomposition inhibitors include RNA stabilization reagents such as RNA later (Thermo Fisher Scientific), NucleoProtect RNA (Machley-Nagel), Gene Keeper RNA & DNA stabilization solution (Nippon Gene), etc. Examples of freezing treatments include flash freezing with liquid nitrogen.

[0053] In the above embodiment, the sample includes the epidermis and dermis regions, but the total volume of the sample is very small, so that nucleic acids can be extracted without treating the sample with protease prior to nucleic acid extraction. That is, the present invention can be in an embodiment that does not include a step of treating with a protease prior to the extraction step.

[0054] The protease treatment may be a dispase treatment, or may be a form in which no treatment with one or more proteases selected from collagenase, hyaluronidase, and trypsin is performed.

[0055] Thus, according to the extraction method of the present invention, there is no need for nucleic acid decomposition inhibition treatment or treatment with protease, so the sample processing steps can be simplified and the total time required to complete extraction can be shortened.

[0056] Furthermore, by not carrying out protease treatment, it is possible to reduce damage to skin tissue and inhibit the decomposition of nucleic acids.

[0057] Furthermore, as described above, the extraction method of the present invention can suppress the decomposition of nucleic acids (especially RNA) in a sample under room temperature conditions, making it possible to work with and store the sample at room temperature from the time it is obtained until it is extracted. This enables an embodiment in which, for example, a subject obtains a sample, the sample is then transported and stored under room temperature conditions for a certain period of time, and nucleic acid is extracted by a third party at a medical institution, testing institution, etc. In such an embodiment, since it is not necessary to transport the sample in a frozen state, the cost of transporting the sample can also be reduced.

[0058] The extraction method of the present invention can be used to extract nucleic acids for use in the analysis of skin tissues, which can include obtaining a minute skin tissue sample of the above-mentioned size and analyzing skin appendages such as hair follicles, sweat glands, and sebaceous glands and their surrounding cells.

[0059] <Gene expression analysis method> The present invention also relates to a method for analyzing gene expression using RNA extracted by the above extraction method (hereinafter referred to as expression analysis method). The expression analysis method of the present invention includes a preparation step of preparing a sample of skin tissue dried under room temperature conditions, an extraction step of adding an RNA extraction reagent to the sample and extracting RNA from the sample, and an analysis step of performing RNA expression analysis using the extracted RNA.

[0060] The preferred forms of the preparation step and the extraction step are the same as those of the above extraction method.

[0061] The analysis step is a step of performing RNA expression analysis using the RNA obtained in the extraction step. RNA expression may be measured according to the procedure of gene expression analysis commonly used in the field. For example, methods for quantifying nucleic acids or their amplification products, such as PCR, multiplex PCR, real-time PCR, hybridization (DNA chip, DNA microarray, dot blot hybridization, slot blot hybridization, Northern blot hybridization, etc.), and sequencing, may be mentioned. In the present invention, RNA may be reverse transcribed to synthesize cDNA and quantified by qRT-PCR, or comprehensive gene expression analysis may be performed by RNA sequencing.

[0062] It is preferable to include a quality confirmation step of confirming the quality of RNA before the analysis step. The method for confirming the quality of RNA is not particularly limited, but examples include the proportion of RNA fragments of 200 bases or more (DV200), measurement of the RNA Integrity Number (RIN) value, and / or confirmation of the band intensity ratio of the 28S subunit and the 18S subunit of rRNA by electrophoresis. When performing expression analysis by RNA-seq, it is preferable to confirm that the DV200 is 60% or higher.

[0063] In the expression analysis method of the present invention, RNA is not easily decomposed during extraction from the sample, and therefore gene expression analysis can be efficiently performed using a small amount of sample. EXAMPLES

[0064] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0065] <Test Example 1> Study of RNA degradation inhibition treatment of samples (1) Obtaining skin samples Skin samples were taken from the subject (human) skin at a depth of about 0.3 mm from the stratum corneum. The obtained skin samples included the stratum corneum of the epidermis to the dermis just below the basement membrane, and had a volume of about 0.0013 mm. 3The obtained sample was dried under room temperature conditions (room temperature 25° C.) and incubated as is for 4 hours (Example 1). It was confirmed that the sample of Example 1 was naturally dried within about 1 minute after being obtained. Meanwhile, a sample obtained in the same manner was transferred immediately after acquisition to an Eppendorf tube containing RNA later (Thermo Fisher Scientific) and incubated at room temperature (room temperature 25° C.) for 4 hours (Comparative Example 1).

[0066] (2) RNA extraction For RNA extraction, use PicoPure TM An RNA Isolation Kit (Thermo Fisher Scientific) was used. First, the extraction buffer included in the kit was added to the sample, and then RNA was extracted according to the method of Lei et al. (JoVE, 2019, 144, 1-8). The proportion of RNA of 200 bp or more in total RNA (DV200 value) was measured using a Bioanalyzer (Agilent) for the extracted RNA. The results are shown in Figure 1.

[0067] As shown in Figure 1, in Example 1, in which the sample was dried immediately after acquisition, the DV200 value was high and RNA degradation was suppressed. In other words, it was shown that high-quality RNA can be extracted by using skin tissue that was dried under room temperature (preferably normal temperature) conditions after acquisition from a subject.

[0068] Moreover, the DV200 value was higher in Example 1 than in Comparative Example 1, in which the sample was stored in RNA later immediately after acquisition. This revealed that RNA decomposition can be suppressed more effectively when the skin sample is dried at room temperature (preferably at normal temperature) than when an RNA stabilizing reagent such as RNA later is used.

[0069] <Test Example 2> Examination of sample storage at room temperature A sample was obtained from the skin tissue of a subject in the same manner as in Test Example 1. RNA was extracted from the sample (Reference Example 1) immediately after the acquisition in the same manner as in Test Example 1.

[0070] Some of the samples were stored in Eppendorf tubes at room temperature (25°C) for 72 hours. It was confirmed that the sample of Example 1 was naturally dried within about 1 minute after acquisition due to storage at room temperature. The sample thus obtained was designated as Example 2. RNA extraction was then performed in the same manner as in Test Example 1. The RNA integrity number (RIN) value of the extracted RNA was measured using a Bioanalyzer (Agilent). The results are shown in Figure 2.

[0071] 2, the RIN value of the RNA extracted from the minute skin tissue was 7.0 in Reference Example 1 and 6.5 in Example 2. In other words, it was found that the RNA extracted from the skin tissue stored at room temperature for 72 hours was less likely to decompose and maintained the same level of quality as the sample extracted from the skin tissue immediately after collection.

[0072] As described above, the results of Test Example 2 demonstrated that drying the skin tissue enables long-term storage of 72 hours or more under room temperature (preferably normal temperature) conditions. In addition, the volume is 0.05 mm 3 Less than (for example, 0.0013 mm 3 Skin tissue of about 100mm diameter (approximately 100mm thick) dries quickly at room temperature and can easily maintain a dry state, making it suitable for long-term storage at room temperature. [Industrial Applicability]

[0073] INDUSTRIAL APPLICABILITY The present invention can be used for gene expression analysis in skin tissue, and for the development of medical and cosmetic techniques that utilize said expression analysis.

Claims

1. A preparation step of preparing a sample of skin tissue dried under room temperature conditions; A method for extracting nucleic acid, comprising the step of adding a nucleic acid extraction reagent to the sample and extracting nucleic acid from the sample.

2. The volume of the sample immediately after removal from the subject is 0.05 mm ３ 2. The method of claim 1, wherein:

3. The method according to claim 2, wherein the skin tissue comprises an epidermis region including a stratum corneum and a dermis region located at a depth of 0.2 to 0.5 mm from the outermost layer of the skin.

4. The method of claim 2 or 3, wherein the sample is obtained by biopsy.

5. The method according to claim 1 or 2, which does not include a step of subjecting the sample to a nucleic acid degradation suppression treatment and / or a step of treating the sample with a protease prior to the extraction step.

6. The method according to claim 5 , wherein the step of subjecting the sample to a nucleic acid decomposition inhibiting treatment is a treatment of contacting the sample with a nucleic acid decomposition inhibiting agent and / or a treatment of freezing the sample.

7. The method of claim 5 , wherein the protease is dispase.

8. The method of claim 1 or 2, wherein the preparing step comprises storing the sample under room temperature conditions.

9. The method according to claim 1 or 2, wherein the preparation step is a step of preparing the sample by placing the skin tissue in a storage container and drying it under room temperature conditions.

10. A preparation step of preparing a sample of skin tissue dried under room temperature conditions; an extraction step of adding an RNA extraction reagent to the sample and extracting RNA from the sample; A method for analyzing gene expression, comprising an analysis step of performing RNA expression analysis using the extracted RNA.

11. The method according to claim 10, further comprising a quality confirmation step of confirming that the proportion of RNA fragments having 200 bases or more is 60% or more after the extraction step and before the analysis step.

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