Kit for detecting mRNA in hair follicles and personalized management method for hair loss using same

Abstract

The present invention relates to a kit for quantitatively examining mRNA of hair loss–related genes from hair, preferably from hair follicles, and a method for diagnosing or predicting causes of hair loss using same, and a method for alleviating, preventing, or treating hair loss. According to the present invention, testing from RNA extraction of hair loss–related genes to quantitative PCR can be performed using a small amount of hair, thereby enabling simple and rapid identification of the cause of hair loss and allowing for personalized hair health management. In addition, since no separate laboratory is required for on-site testing and anyone can easily perform testing and result acquisition through simple training, a user can easily perform the testing even in small clinics or facilities without clinical laboratories.

Description

Hair follicle mRNA detection kit and customized hair loss management method using the same

[0001] The present invention relates to a kit for analyzing hair loss-related mRNA from hair follicle tissue attached to collected hair. The kit is used to perform RNA extraction by attaching a metal ring to the top of the hair follicle to allow it to sink for ultrasonic treatment, or by fixing the hair to the front of an ultrasonic probe container with a fixing pin, thereby lysing the cells of the hair follicle tissue attached to the hair using ultrasound and a cell lysate, automatically extracting RNA using magnetic particles, and performing real-time RT-PCR without delay. By using the kit of the present invention to obtain relative quantitative values ​​of hair loss-related mRNAs, hair loss can be managed at a molecular biological level by applying a composition containing siRNA that reduces hair loss-inducing mRNA expressed higher than normal to the hair loss area, or by applying a composition that increases the expression of mRNA involved in hair growth. The present invention relates to a hair loss-related mRNA analysis kit comprising a cartridge containing magnetic particles, cell lysate, washing solution, eluent, and freeze-dried reverse transcription enzyme desalination required for reverse transcription PCR, wherein continuous ultrasound is applied to the hair follicle area to efficiently extract mRNA of hair follicle cells from collected hair, and wherein the cartridge contains a primer and probe capable of amplifying mRNA of hair loss-related genes. The invention relates to a method for diagnosing or predicting hair loss, a method for providing a personalized hair loss management service, and a method for preventing or managing hair loss, wherein the growth cycle and the condition of the extracted hair follicle tissue are determined by microscopic examination of the shape around the collected hair follicle and the mRNA of hair loss-related genes from hair follicle cells attached to the hair are quantified in real time.

[0002]

[0003] Hair loss has a very high incidence rate, affecting 30% of the total population. Although hair loss is not a disease that threatens human life or restricts human activity, it lowers an individual's quality of life due to the psychological problems it causes. The stress caused by hair loss involves significant negative elements, such as a lack of confidence and social phobia, and the resulting loss in social opportunity costs is also considerable.

[0004] Although the mechanisms of hair loss vary depending on individual genetic characteristics and the degree of influence of the hair follicle environment, currently available hair loss management products are general-purpose rather than targeting the specific causative factors of each individual. Consequently, there are limitations in finding products suitable for each user. While significant research has been conducted on hair follicle cells related to the causes of hair loss, a method has not yet been developed to accurately measure gene expression in these cells at the molecular biological level to precisely diagnose the cause of hair loss and provide personalized management based on this diagnosis. Therefore, there is an urgent need for a field-based molecular diagnostic system for hair loss that can precisely measure the diverse gene expression states of hair follicle cells in the affected area and provide customized hair care solutions tailored to the specific cause of hair loss.

[0005] Previously, results have been reported regarding the causes of hair loss by studying the expression of hair loss-related genes in hair follicle cells at a cellular biological level using molecular biological research tools. The most common type, androgenic alopecia, occurs due to not only an excess of male hormones but also the overexpression of androgen receptors. In cases of male hormone excess, substances such as Finastride and Dutastride, which inhibit the activity of alpha-reductase (α-reductase) that produces DHT—a male hormone with a potency approximately 10 times higher than testosterone—are widely used and are known as the most effective treatments. However, it has been reported that these drugs work in about 80% of cases but are ineffective in 20%. It is also known that hair loss can occur in individuals with normal male hormone levels if they are sensitive to androgens. In this case, hair loss occurs due to the overexpression of androgen receptors and is known to occur particularly frequently in women. In a study published by F. Richeti et al., ten hair follicles were extracted from the crown and occipital regions of women with androgenetic alopecia and normal women, immersed in Trizol Reagent (Invitrogen), ground using a grinder, and RNA extracted using a centrifuge. The androgen receptor mRNA was then analyzed via reverse transcription PCR (Increased androgen receptor messenger RNA in frontal-parietal hair follicles of women with androgenetic alopecia, Genetics and Molecular Research 12 (2): 1834-1840 (2013)). They reported that while there was no significant difference in the amount of androgen receptors between the crown and occipital regions in normal individuals, the expression of androgen receptors was increased in the crown region of alopecia patients. However, this experimental method can only be performed in specialized laboratories and is time-consuming, making it difficult to apply generally in the molecular diagnosis of hair loss.

[0006] Meanwhile, to reduce these overexpressed androgen receptors, hair tonics containing SAMiRNA (Cosmerna, Bionia) that decrease androgen receptor mRNA have been developed. However, even with the reduction of androgen receptors, the effect remains minimal in approximately 10% of alopecia patients. This is because hair loss progresses through various other mechanisms. According to reports to date, in addition to the increase of hormones and their receptors in the hair follicle region, various causes of hair loss have been identified, including micro-inflammation within the follicle, fibrosis, inhibition of WNT signaling, a decrease in follicle stem cells, and anagen regulators. Therefore, to accurately diagnose these various mechanisms at the follicle cell level, a diagnostic kit capable of rapidly, conveniently, and accurately analyzing hair loss-related mRNA to identify the cause of hair loss and provide customized management is highly required for the scientific management of alopecia.

[0007] Hair repeats the growth, regression, and resting phases of the hair follicle. Since the growth state of follicular tissues varies according to the follicle's growth cycle, the shape of the follicle changes accordingly, and the expression of various genes in follicle cells also varies. Maintaining healthy hair requires the normal expression of genes that regulate this follicle growth cycle at each stage. (Paus, Ralf et al. Trends in Molecular Medicine, Volume 20, Issue 10, 559 - 570) However, if certain genes regulating this growth cycle are overexpressed or not expressed normally, hair in the growth phase decreases while follicles in the regression and resting phases increase, leading to hair loss. Therefore, analyzing gene expression in each follicle cycle and providing fundamental management based on this analysis can be a fundamental method for managing hair loss. However, to date, no molecular diagnostic test kit has been developed that can easily and quantitatively analyze the expression levels of various hair loss-related mRNAs expressed within the follicle on-site within one hour. Therefore, current hair loss medications and cosmetics are generally used without causal analysis at the follicle cell level, resulting in ineffective treatments that cause hair loss and financial losses. To manage individuals suffering from various types of hair loss economically and efficiently, it is essential to accurately analyze the various causes affecting hair loss at a molecular biological level and provide appropriate management accordingly. However, existing diagnostic methods based on follicle mRNA analysis are not widely used because they involve invasive procedures like scalp biopsies, which lead to low patient compliance; furthermore, they require specialized laboratories and take a long time to complete the analysis.Therefore, there is a great need for a kit that can examine the condition of hair follicles related to hair loss at the molecular and cellular level by accurately testing the hair follicle cycle and the expression levels of major mRNAs from a minimum amount of hair, preferably one hair, collected by a non-invasive method within one hour at a dermatology clinic, hair loss clinic, or examination center.

[0008] Previous studies on hair follicle mRNA expression conducted using invasive methods at the molecular biology laboratory level have been reported. The Meta-analysis for Androgenetic Alopecia Novel determinants (MAAN) Consortium reported a study in which researchers plucked dozens of hairs from areas of hair loss and the occipital region where hair loss does not occur, examined mRNA and miRNA expression levels, and found differences in expression levels (Journal of investigative dermatology. 2019 Jan;139(1):235-238. “Insights into Male Androgenetic Alopecia: Differential Gene Expression Profiling of Plucked Hair Follicles and Integration with Genetic Data” (2019)). In this study, mRNA and miRNA were extracted using a Qiagen extraction kit and analyzed using Illumina HT-12v4 Bead Arrays and Affymetrix GeneChip®. This study is difficult to apply to hair testing for people with hair loss because it requires extracting dozens of hairs from them, which causes significant discomfort. Additionally, the extraction and analysis processes require specialized personnel, high costs, and a long time of more than one day, which limits its general use as a hair loss mRNA molecular diagnostic method.

[0009] Japanese Patent JP 7154765B presents a method for measuring mRNA expression levels as a method for predicting hair growth effects. It relates to a method for selecting a therapeutic agent that has a hair growth effect due to actions other than androgen inhibition on the scalp, as a method for predicting the preventive or corrective effect—that is, the hair growth effect—of thinning hair or hair loss caused by any cause within a short period of time. The patent document provides a method of analyzing mRNA from body hair from areas other than the head as a method that enables the exploration of effective treatment methods for preventing and correcting thinning hair and hair loss by utilizing surrounding tissues. However, even if sufficient cells are obtained by removing body hair and mRNA is successfully analyzed, head hair and body hair from the head generally have different properties. It is also known that the opposite phenomenon occurs. Furthermore, head hair and body hair from the head differ in their hair cycles, which are the hair regeneration cycles, so their properties also differ significantly. Therefore, evaluating the hair growth effect on the head using body hair from the head is limited, and it is evident that it is practically difficult to carry out the above process in a hair loss clinic setting.

[0010] Meanwhile, US 20060024705A1 (Molecular analysis of hair follicles for disease) provided a method for analyzing gene expression using RNA from hair follicles. This invention used 10 hairs and presented various general molecular biology laboratory methods for mRNA quantitative analysis, making it difficult to apply in general dermatology clinics or hair loss clinics, similar to the example inventions mentioned earlier. One of the significant problems is that due to the high cost of specialized personnel and equipment required for the extraction and analysis processes, such tests must be entrusted to external specialized laboratories; however, it has also reported issues regarding mRNA degradation caused by time delays during the lengthy extraction stage. Furthermore, since it requires more than a day, there are limitations in terms of cost, efficiency, and accuracy as a field molecular diagnostic method for hair loss-related mRNA.

[0011] Therefore, it can be said that there is an urgent need for a groundbreaking and practical method that can efficiently extract mRNA of hair follicle cells from a single collected hair at the point of diagnosis as in the present invention, perform RT-qPCR automatically without delay, and quantify it within one hour.

[0012] FR 3111917 A (Molecular signature of a common alopecic state, associated with cell junction) describes a step of measuring the expression level of at least one gene selected from CDH1, ACTB, ACTBL2, TUBB, TUBB2A, GSN, MYO3B, MYO5B, MYO6, DSG2, DSG3, DSG4, DSC2, GJB2, GJA1, GJB6, GJA3, TJP2, CLDN8, CLDN10, CLDN19 and optionally CTNNB1 and CTNND2.

[0013] Additionally, FR 3143040 A (Method for prognosis and / or diagnosis of loss of hair density) discloses a method for prognosing hair density loss, comprising measuring the expression level of one or more selected genes among 63 specific genes in a specimen. It relates to a method for cosmetic treatment of a subject's scalp and a method for identifying compounds that prevent hair loss, promote hair growth, or promote an increase in hair thickness. The genes presented in the patent document are typically genes involved in inflammation, immunity, or differentiation of keratinocytes, and are genes involved in the extracellular matrix, the formation or development of stem cells, or hair keratinization. Since these genes are marker genes that are consequently expressed in thinning hair in areas where hair loss is progressing, there are limitations in viewing them as causative genes that induce hair loss. Since the aforementioned genes were obtained from biopsy samples—specifically skin tissue—rather than from hair follicle cells in the collected hair, they differ from the mRNA expression of the collected hair. Furthermore, rather than being causative genes, they include many genes that are ultimately expressed, such as hair keratin-related genes, in the scalp of hair loss. Additionally, the invasive process of collecting a 2.5mm biopsy tissue sample causes pain for patients with hair loss, making sample collection difficult. Moreover, because the biopsy tissue can only be analyzed in laboratories equipped with nucleic acid extraction and various analytical instruments, it is difficult to use in general dermatology hair loss clinics.

[0014] Meanwhile, the applicant has developed next-generation point-of-care (POCT) equipment and a cartridge used therein (Korean Registered Patent 10-2256757 and Korean Published Patent 10-2023-0113032). This equipment and cartridge can analyze various nucleic acids in a short time using fully automated nucleic acid extraction and quantitative PCR. It performs accurate DNA / RNA extraction through a powerful sonicator and performs PCR using a heating block capable of precise temperature control. It is a fully automated device capable of high-multiplexing and syndromic diagnostics that can diagnose up to 40 targets at once using six fluorescence modules. It is a compact, desktop device that recognizes the cartridge type using a barcode scanner and can be easily operated via an LCD touchscreen. The cartridge is an all-in-one kit combining a nucleic acid extraction unit and a PCR reaction plate, allowing the entire process from nucleic acid extraction to amplification to be performed in one go without the need for human hands.

[0015] The inventors intended to accurately quantitatively analyze the expression levels of hair loss-related mRNA in hair follicles to precisely identify the causes of hair loss induced by various factors. This was achieved by attaching a fixation means to the hair to expose the follicle cells, inserting them into a cartridge, extracting mRNA with high efficiency using ultrasound and magnetic particles, mixing the mRNA without delay with a reverse transcription PCR reaction sample, introducing the mixture into a reaction well containing dried primers and probes of hair loss-related mRNA, and simultaneously performing RT-qPCR using a multiplex PCR method to quantitatively analyze the mRNAs, thereby enabling early identification of the precise causes of hair loss and providing a hair loss management method tailored to the findings.

[0016]

[0017] The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that forms prior art already known to those skilled in the art to which the present invention belongs.

[0018]

[0019] Summary of the Invention

[0020] The objective of the present invention is to provide a kit capable of quantitatively and accurately detecting hair loss-related mRNA from hair follicle tissue attached to 1 to 5 hairs, preferably 1 hair, and to provide a hair loss diagnosis or prediction system using the kit, as well as a personalized hair loss management and a method for preventing or treating hair loss.

[0021]

[0022] To achieve the above objective, the present invention provides a kit and apparatus comprising primers / probes capable of extracting RNA from a hair follicle region with high efficiency using 1 to 5 hairs, 1 to 3 hairs, preferably 1 hair, and specifically and accurately amplifying mRNAs of hair loss-related genes using the extracted RNA.

[0023] The present invention also provides a method for providing information and a method for diagnosing or predicting the cause of hair loss using the kit and device.

[0024] The present invention also provides a method for providing a personalized hair loss management service, comprising a method for providing information for diagnosis or prediction.

[0025] The present invention also provides a method for providing information for preventing or treating hair loss using the kit, and a method for preventing or managing hair loss.

[0026] The present invention also provides a primer capable of specifically amplifying the mRNA of a hair loss-related gene, a probe capable of hybridizing complementarily to said gene, and a composition for diagnosing or predicting hair loss comprising the same.

[0027]

[0028] FIG. 1a shows a cross-sectional view of the cartridge of the present invention with a fixing pin inserted, and FIG. 1b shows a cross-sectional view of the cartridge of the present invention with a weight attached.

[0029] Fig. 2a shows a hair image including hair follicle tissue captured with a polarizing microscope, and Fig. 2b shows real-time PCR (IRON-qPCR) of the hair captured in Fig. 2a. TM Displays the result.

[0030] Figure 3 shows an image of a hair sample fixed to a fixing pin so that the hair sample does not float during nucleic acid extraction.

[0031] Figure 4a shows an image of a hair sample with a weight attached so that the hair sample does not float during nucleic acid extraction. Figure 4b shows the results regarding usability depending on the material of the weight.

[0032] Figure 5a is a graph confirming the specificity and detection power of the HF-related 13-gene primer / probe set based on the results of Real-time PCR (Exicycler™) analysis using Human reference RNA. Figure 5b is a graph demonstrating the specificity and efficiency of the presented primer set based on the results of the melting curve and amplification curve analysis of SYBR Green qPCR in Real-time PCR (Exicycler™).

[0033] Figure 6 shows a Real-Time PCR device (IRON-qPCR TM This is a diagram showing the mRNA expression pattern measured in a hair sample in the form of a graph.

[0034] Fig. 7 shows hair collection, microscopy, cartridge insertion, and a Real-Time PCR device (IRON-qPCR). TM It represents the process of analyzing hair samples through operation.

[0035] Figure 8 shows the changes in hair loss after treatment with the Cosmerna cosmetic composition for 24 weeks.

[0036]

[0037] Detailed Description of the Invention and Preferred Embodiments

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0039]

[0040] In order to accurately analyze mRNA from hair follicle cells, it is necessary to be able to extract RNA reproducibly with high efficiency, and to perform reverse transcription quantification PCR (RT-qPCR) specifically on each mRNA with high efficiency immediately without the extracted RNA being degraded. In the prior art mentioned above, hair follicle tissue was crushed using a cell disruptor, and mRNA was extracted from it using commercially available mRNA extraction reagents. In the present invention, the aim was to extract mRNA from a single hair follicle tissue by processing it with ultrasonic disruption and cell lysis solution in a fully automated manner. However, since hair does not absorb water well depending on the degree of oil coating, it may float on water; in particular, when vibration is applied with ultrasound, it floats to the top of the solution, resulting in a problem where ultrasonic disruption is not reproducible.

[0041] Molecular diagnostics is divided into two stages: nucleic acid extraction and quantitative PCR. Accurate diagnostic results can only be obtained if each stage is performed reproducibly and with high efficiency. If nucleic acids are not extracted reproducibly and with high efficiency during the first stage, the nucleic acid extraction stage, the subsequent quantitative PCR results cannot be trusted. We attempted to insert a single hair into a cartridge, break down the tissue using ultrasound, and separate it using magnetic nanoparticles. However, as the hair floated and moved during the ultrasound treatment process, the ultrasound was not properly applied, resulting in incomplete tissue fragmentation. Consequently, there was a problem where the efficiency of DNA or RNA extraction varied significantly, making accurate diagnosis impossible. To solve this problem, methods were devised to ensure the hair remained continuously positioned at the front end of the ultrasound during ultrasonic vibration. One method for attaching a fixation means to the hair was to insert the hair into a capillary tube similar to a hypodermic needle, leaving only the hair follicle exposed. The hair was then secured by clamping the metal capillary tube, and this capillary was fixed so that the follicle portion was positioned at the front end of the ultrasound probe tube. Such metal capillaries firmly hold the hair during ultrasonic treatment, allowing the disruption of hair follicle cells by ultrasound to be efficient and reproducible. Another method involves attaching a weight to the hair. After compressing and fixing the weight at a position approximately 5 to 10 mm, preferably 5 mm, above the hair follicle tissue, the hair with the attached weight is submerged and positioned at the front end of the ultrasonic probe tube into which the ultrasonic probe is inserted. When ultrasound is applied, the hair does not float during ultrasonic treatment but remains positioned at the front end of the ultrasonic probe tube, allowing the hair follicle tissue to be efficiently disrupted by ultrasound, thereby enabling the reproducible extraction of RNA from the hair follicle tissue with high efficiency, thus completing the first step of the present invention.By developing primers and probes that selectively amplify hair loss-related mRNAs in the second step, a kit capable of quantitatively and accurately detecting hair loss-related mRNAs was completed, making it possible to develop a hair loss-related mRNA diagnostic kit.

[0042] As shown in Fig. 3, hair was inserted into a metal tube, such as a syringe needle, to expose the hair follicle, and then the metal capillary tube was compressed and fixed, positioned at the bottom of the ultrasonic probe tube as shown in the cartridge of Fig. 1a. Through this, the hair follicle tissue could be stably fragmented without the hair moving during ultrasonic treatment.

[0043] Another method of attaching weights to the hair was devised. As shown in FIGS. 1b and 4a, a weight is clamped and attached to the hair portion approximately 5 to 10 mm, preferably 5 mm above, the tip of the hair follicle so that the follicle is exposed. As shown in FIGS. 1b, the hair is immersed at a location where magnetic particles and an ultrasonic probe (sonicator) act directly, thereby efficiently severing the tissue by ultrasound and allowing RNA to detach from the cell and attach to the magnetic particles via a chaotropic cell lysate. This cartridge configuration solves the problem. Using this nucleic acid extraction cartridge of the present invention, RNA can be obtained rapidly, with high efficiency, and reproducibility. This method of extracting nucleic acids from hair by attaching a weight to the hair using a hair fixing pin or is a method developed for the first time by the inventors. Since only by using this method can nucleic acids be extracted from the hair follicle tissue with high efficiency and reproducibility within 10 minutes, it can be considered a key component for on-site molecular diagnosis of hair.

[0044] To analyze the various causes affecting hair loss, hormonal changes, alterations in hormone receptors within hair follicles, inflammation, fibrosis, WNT signaling, hair stem cells, and anagen regulators were selected as potential causes. Since hair loss is triggered by various factors, either individually or in combination, affecting hair health, it is crucial to accurately analyze the mRNA expression levels of these diverse causative genes. To this end, a method was employed to enhance reliability by calculating the average value through three repeated reactions for each mRNA.

[0045] Follicular tissue is composed of various tissues, such as the inner root sheath, outer root sheath, and dermal papilla, consisting of a total of about 20 different cell types. The composition of the detailed tissues of the follicle changes depending on the growth, regression, and resting phases of the follicular cycle, and consequently, the shape of the follicular tissue varies. Furthermore, the follicular tissue pulled out can differ depending on the condition of the hair during plucking. In particular, the dermal papilla tissue located at the very bottom may or may not be present. Therefore, if the cell types differ, the amount of mRNA expression varies, which can make it difficult to analyze the mRNAs causing hair loss. In order to solve these problems, the present invention produces a hair mRNA quantification kit that examines the cause of hair loss by extracting a small number (5), preferably 3 or fewer, more preferably 1, hair in the growth phase from an area where hair loss is progressing or expected to progress, obtaining an image of the tissue attached to the hair using a microscope, confirming the condition of the hair in the growth phase and the detailed tissue, and then immersing the hair follicle area with a weight attached in a state where the hair follicle is exposed into a nucleic acid extraction solution to extract RNA from the hair follicle cells, quantifying the hair loss-inducing mRNA within 1 hour using real-time PCR, and comparing the microscope image with the mRNA expression amount.

[0046]

[0047] Accordingly, in one aspect, the present invention relates to a kit capable of specifically amplifying the mRNA of one or more genes selected from the group consisting of AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2, and IGF1.

[0048] Specifically, the present invention relates to a kit for detecting hair loss-related mRNA for quantitative analysis of mRNA in a hair follicle region, comprising: a receiving portion that receives hair fixed by a fixing means to prevent the hair follicle region from settling or floating during ultrasonic treatment, wherein magnetic particle beads are located at the bottom of an ultrasonic probe container; a cartridge containing a nucleic acid extraction solution for extracting RNA from the hair contained in the receiving portion and capable of automatically and sequentially applying each solution to the receiving portion; and a kit comprising primers and probes for selectively amplifying and quantitatively detecting hair loss-related mRNA from the extracted RNA, and comprising a plurality of reaction wells for measuring fluorescence.

[0049] More specifically, the present invention relates to a detection kit for quantitatively analyzing hair loss-related mRNA in a hair follicle region, comprising: a cover including a fixing pin insertion port or a hair input port; a receiving portion in which magnetic particle beads are positioned on the front of an ultrasonic probe container; a cartridge containing a solution for extracting RNA from hair contained in the receiving portion and capable of automatically and sequentially applying each solution to the receiving portion; and a reaction plate including primers and probes for selectively amplifying and quantitatively detecting hair loss-related mRNA from the extracted RNA, and including a plurality of reaction wells for measuring fluorescence; wherein the receiving portion further comprises a fixing pin for fixing the hair so that the hair follicle region is exposed, or a weight attached to the upper part of the hair follicle to sink the hair into the solution.

[0050] In the present invention, the hair fixed by the fixing pin is coupled to the fixing pin insertion port, and the hair with the weight attached is fed into the hair input port, but is not limited thereto.

[0051]

[0052] In the present invention, the gene is a hair loss-related gene, listed in Table 1 below, and its function is described.

[0053]

[0054]

[0055] The aforementioned genes play a crucial role in various biological processes related to hair loss, such as hair growth, hair follicle health, and inflammatory responses. Research on the biological role of gene expression in hair loss provides important information for understanding the causes and mechanisms of hair loss. Hair loss can be affected in various ways by fluctuations in gene expression, and these fluctuations are caused by the increase or decrease in the expression of specific genes.

[0056] First, among the genes with increased expression, AR is a gene encoding the androgen receptor (AR) protein, also known as the dihydrotestosterone receptor, located on the X chromosome. AR transmits signals from androgen hormones to hair follicles, affecting hair growth and maintenance. Increased expression of AR enhances the action of androgens, which can lead to androgenic alopecia.

[0057] TNF, also known as DIF, TNFA, TNFSF2, TNLG1F, and TNF-alpha, is a gene encoding the tumor necrosis factor (TNF) protein, a multifunctional inflammatory cytokine. TNF is an important cytokine for inflammatory responses and immune regulation; increased expression of TNF induces inflammation, which can exacerbate inflammatory hair loss conditions such as alopecia areata.

[0058] TGFB1 (Transforming Growth Factor Beta 1) is a gene encoding the secreted ligand of the TGF-beta (Transforming Growth Factor Beta) superfamily of proteins. TGFB1 is a growth factor that plays an important role in cell growth, differentiation, and wound healing processes. Increased expression of TGFB1 promotes the micronization of hair follicles, which can inhibit hair growth and lead to hair loss.

[0059] DKK1 is a gene encoding Dickkopf-associated protein 1, a member of the Dickkopf family of proteins. Increased expression of DKK1 can interfere with the normal growth and development of hair follicles, potentially leading to hair loss.

[0060] PPARGC1A (PPAR gamma coactivator 1 alpha) is a gene encoding Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). PPARGC1A is a transcription factor that regulates cellular metabolism and energy homeostasis, and increased expression of PPARGC1A can affect metabolic processes and indirectly influence the health of hair follicles.

[0061] FGF5 is a gene that encodes the fibroblast growth factor 5 protein. FGF5 (fibroblast growth factor 5) and FGF5-D (fibroblast growth factor 5s) play a role in regulating the hair growth cycle. The ratio of these genes is used to assess the overall expression of hair growth, and a relative increase in FGF5 may be associated with an increased risk of hair loss.

[0062] IL1B is a gene encoding the interleukin-1 beta (IL-1β) protein, a member of the interleukin-1 cytokine family. Increased expression of IL1B can lead to incomplete regulation of the inflammatory response, which may affect hair loss.

[0063] ACTA2 (alpha smooth muscle actin) is a gene encoding the alpha-smooth muscle actin (α-SMA) protein, one of six different actin proteins. ACTA2 is a protein that contributes to the structural integrity and function of hair follicles; increased expression of ACTA2 can impair the structural stability of hair follicles, potentially leading to hair loss.

[0064] Conversely, there are also genes whose expression decreases. AREG (Ampyregulin) is a gene that encodes ampyregulin, a member of the epidermal growth factor family. AREG is a growth factor that promotes the growth and regeneration of hair follicles; a decrease in AREG expression can impair the regenerative capacity of hair follicles, potentially leading to hair loss.

[0065] FGF2 is a gene encoding fibroblast growth factor-2 (FGF2), which is a growth factor and signaling protein. FGF2 (fibroblast growth factor 2) is an important growth factor that stimulates the growth and development of hair follicles, and a decrease in FGF2 expression can inhibit the growth and regeneration of hair follicles, leading to hair loss.

[0066] IGF1 (Insulin-like Growth Factor 1) is a gene that encodes insulin-like growth factor 1 (IGF1). IGF1 is similar to insulin in function and structure and is a member of a family of proteins that mediate growth and development. IGF1 is a hormone that supports the growth and cellular function of hair follicles, and a decrease in IGF1 expression can slow down hair growth and increase the risk of hair loss.

[0067] CD200 is a cell surface glycoprotein with immunosuppressive functions that plays a crucial role in maintaining immune privilege in the tissues surrounding the hair follicle. This protein functions to protect the hair follicle structure from inflammatory responses and prevent hair loss by suppressing the excessive activation of immune cells. If CD200 expression is reduced, the immune response becomes excessively activated, inducing inflammation around the hair follicle; this accelerates follicle damage and can lead to hair loss. Furthermore, CD200 is selectively overexpressed in hair follicle stem cells located in the bulge region of the hair follicle and is known as a marker associated with the maintenance of the stem cell's undifferentiated state and immune evasion capabilities. As such, CD200 functions as a biomarker reflecting the activity of hair follicle stem cells and can serve as a useful molecular target for the development of stem cell-based hair follicle regeneration or hair loss treatment technologies.

[0068] CD34 (a hair follicle stem cell marker) is a gene encoding the transmembrane phosphoglyprotein CD34. CD34 is a marker indicating the presence of stem cells within hair follicles; a decrease in CD34 expression leads to a reduction in the number of stem cells, which in turn impairs the hair follicle's regenerative capacity and can result in hair loss.

[0069] Beta2-Actin (ACTB) is a housekeeping gene used as a control in the present invention, and according to the present invention, other known housekeeping genes such as B2M (Beta2 microglobulin), HPRT1, U6snRNA, Transcription termination factor 1, RPL13A, and GAPDH may be used in addition to ACTB. According to the present invention, one or more housekeeping genes may be used depending on the hair cycle or region.

[0070] In the present invention, the primer capable of amplifying the gene and the probe capable of detecting it may be characterized by including one or more of the nucleotide sequences represented by SEQ ID NOs 1 to 48 as described in Table 2, but are not limited thereto.

[0071] Preferably, the primer may be characterized by comprising one or more of the nucleotide sequences represented by SEQ ID NOs 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, and 47, and the probe may be characterized by comprising one or more of the nucleotide sequences represented by SEQ ID NOs 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, and 48, but is not limited thereto.

[0072]

[0073] In the present invention, the receiving portion may be characterized by receiving 1 to 5 hairs, preferably 1 hair.

[0074] In the present invention, the fixing means may be characterized by preventing the hair from floating during ultrasonic treatment and fixing the hair so that it is immersed in the nucleic acid extraction solution.

[0075] In one embodiment of the present invention, if hair is placed directly into a nucleic acid extraction solution, it floats, resulting in a significant difference in nucleic acid extraction efficiency. To solve this problem, a micro metal tube (weight) or a metal pin (fixing pin) is positioned approximately 5 mm from the hair follicle, and the hair is fixed to the metal tube (weight) or pin (fixing pin) by compressing it with tweezers or pliers. Then, the hair that does not include the follicle is cut off, and the hair including the follicle fixed by the metal tube or pin is inserted into the sample inlet of the nucleic acid extraction part. The sample is then dissolved using a sample lysis buffer and ultrasonic disruption. The nucleic acid of the dissolved sample binds to the surface of magnetic particles. Subsequently, impurities other than the nucleic acid bound to the surface of the magnetic particles are removed using first and second washing buffers. Finally, the nucleic acid of the sample bound to the surface of the magnetic particles is eluted using an elution buffer, and the buffer containing the eluted nucleic acid is transferred to a freeze-drying tube containing enzymes necessary for PCR.

[0076] The hair fixing means of the present invention may use a weight (e.g., metal ring, metal tube, ring bead) or a fixing pin (e.g., metal pin, tweezers) made of a metal such as stainless steel, aluminum, or titanium.

[0077] Real-time PCR is a technique that allows for real-time monitoring of the amount of amplified product by measuring the fluorescence intensity generated during the amplification of a specific sequence at every cycle of the PCR. It exhibits high specificity because it measures the amplification amount of the PCR product using primers specific to the specific sequence being amplified and probes labeled with fluorescent substances. The TaqMan probe, primarily used in Real-time PCR, is labeled with a fluorescent substance at the 5' end and a quencher at the 3' end. When bound to the template DNA, the fluorescent energy is transferred to the quencher upon light irradiation, preventing fluorescence emission. However, as the PCR process progresses, the 5'-3' exonuclease of the DNA polymerase becomes activated, causing the fluorescent substance to detach from the probe and emit fluorescence. By measuring the generated fluorescence in real time, the results can be displayed as a PCR amplification curve for each cycle.

[0078] In the kit according to the present invention, the quantitative analysis of the mRNA can be performed fully automatically, and the primer and probe are preferably stored in a dried, preferably freeze-dried, state, but are not limited thereto.

[0079] In addition, the kit according to the present invention is preferably a single-use kit, but is not limited thereto.

[0080] The kit according to the present invention comprises a cartridge containing a nucleic acid extraction solution and a reaction plate for performing a PCR reaction as an integrated unit, so that the extracted nucleic acid can be transferred directly to the PCR reaction plate. In the present invention, the reaction plate includes a single-use portion of a dried tube containing the enzyme, dNTPs, stabilizer, etc. required for the PCR reaction and is provided as an integrated unit included in the extraction cartridge, and the extracted nucleic acid is automatically transferred to the PCR reaction plate and mixed with the dried primers / probes. The PCR reaction plate contains a dried primer and probe set generated by a sophisticated bioinformatics algorithm.

[0081] Accordingly, the kit according to the present invention requires no separate preparation process after injecting a hair sample, and the mRNA expression level of hair loss-related genes can be analyzed within one hour using real-time reverse transcription polymerase chain reaction (Real-time RT-PCR). The analysis can utilize the P-value based on the mRNA expression level of each gene in healthy hair. According to the present invention, by comparing the mRNA expression level measured from the hair of a subject who has hair loss symptoms or is suspected of having hair loss symptoms with the mRNA expression level measured from the hair of a normal control group, it becomes possible to analyze the cause of hair loss, the degree of hair loss progression, and the prognosis and likelihood of the subject.

[0082]

[0083] In another aspect, the present invention relates to a kit for extracting nucleic acids from hair, comprising a composition for extracting nucleic acids from hair, characterized in that it further comprises a fixing pin for fixing the hair so that the hair follicle portion is exposed, or a weight attached to the upper part of the hair follicle to sink the hair into the composition.

[0084]

[0085] In another aspect, the present invention relates to an mRNA quantitative detection device comprising: a kit for detecting mRNA related to hair loss; and a PCR reading unit, wherein nucleic acid is extracted using a cartridge of the kit, PCR is performed using a reaction plate, and the PCR result is read from the PCR reading unit.

[0086] The device according to the present invention comprises a nucleic acid extraction unit constituting an extraction module and a PCR reaction unit composed of a heat block. Nucleic acids extracted from a sample through the extraction module are transferred to a PCR plate, and the heat block amplifies the nucleic acids within the PCR plate located on the heat block by repeating a specific temperature range. Furthermore, the device of the present invention can detect specific nucleic acids by irradiating the amplified sample onto a light source device and using light of a specific wavelength.

[0087]

[0088] In another aspect, the present invention relates to a method for diagnosing or predicting hair loss with respect to the said gene.

[0089] The present invention identifies that changes in gene expression are closely related to the mechanism of hair loss development, and based on this information, presents a new approach for the prevention and treatment of hair loss.

[0090] After confirming the cause of hair loss by checking the expression level of the hair loss-related target gene mentioned in the present invention, treatment suitable for the cause or a hair loss composition suitable for the cause may be used.

[0091] POCT molecular diagnostic equipment (IRON-qPCR) developed by Bionia TMWhen using (refer to Korean Patents 10-2206856, 10-2256757, 10-2105558, PCT / KR2023 / 005507, etc.), it is possible to analyze the expression of 40 types of genes on-site within 30 minutes to 1 hour. The present invention aims to provide, for the first time, an on-site method for analyzing the gene expression of hair loss causes, which analyzes the main causes of hair loss within 1 hour by simultaneously performing relative quantitative analysis of genes that cause hair loss based on the mRNA of housekeeping genes that are uniformly expressed within cells. For reliability, if three replicate experiments are performed per target, up to 15 genes can be quantitatively analyzed, offering the advantage of being easily usable in hair loss clinics without the need for a separate space or expert personnel for testing. The inventors developed a method for simultaneously measuring mRNA that causes hair loss using a dedicated cartridge and a POCT molecular diagnostic device capable of performing RNA extraction from collected or naturally shed hair and real-time PCR immediately and automatically without time delay. In the present invention, real-time qPCR is the IRON-qPCR TM System (Bionia, KR), Exicycler TM (Bionia, KR) or other Real-Time PCR systems may be used.

[0092] In the present invention, the hair loss-related gene may be characterized as being one or more selected from the group consisting of AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2, and IGF1, but is not limited thereto.

[0093]

[0094] In addition, the present invention aims to develop a kit for quantitatively testing the causative mRNA of hair loss to diagnose the main cause of hair loss, and to propose a method for managing hair loss through a composition capable of regulating the corresponding mRNA based on this diagnosis.

[0095] In the present invention, the wells (tubes) of the PCR reaction plate may be characterized by including the primer and / or probe, but are not limited thereto.

[0096] In the present invention, the nucleic acid extraction cartridge may be characterized by including a fixing means for immersing a hair follicle portion of the hair in a nucleic acid extraction solution. In the present invention, the nucleic acid extraction cartridge may be characterized by accommodating 1 to 5 hairs, preferably 1 hair.

[0097]

[0098] In another aspect, the present invention relates to a method for providing information for diagnosing hair loss, determining hair health status and / or prognosis, and a method for diagnosing or predicting hair loss, comprising the following steps:

[0099] (a) A step of extracting RNA from hair to which a fixation means is attached;

[0100] (b) A step of measuring the expression level of hair loss-related genes selected from the group consisting of AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2, and IGF1 by performing relative quantification using reverse transcription quantification PCR using RNA extracted in step (a);

[0101] (c) A step of determining that hair loss or poor hair health is present if the expression level of the gene measured in step (b) above is increased or decreased compared to a control group.

[0102] In the present invention, the gene whose expression level increases when there is hair loss or a risk of hair loss in step (c) is AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, or PPARGC1A, and the gene whose expression level decreases is CD200, CD34, AREG, FGF2, or IGF1.

[0103] In the present invention, the gene expression level may be characterized by using the hair loss-related mRNA detection kit or the mRNA quantitative detection device.

[0104] In the present invention, the hair may be characterized as having been pulled out or fallen out from an area of ​​alopecia or an area suspected of having alopecia, but is not limited thereto.

[0105] In the present invention, the reverse transcription quantitative PCR may be characterized as a multiplex real-time polymerase chain reaction that simultaneously quantifies one or more genes.

[0106] The present invention may further include a step of fixing hair to a fixing means before step (a).

[0107] The present invention may further include the step of photographing the hair follicle portion of the hair collected prior to step (a) with a microscope to obtain an image of the hair follicle tissue and / or scalp attached to the hair portion.

[0108] The present invention may further include a step of comparing the hair follicle growth cycle, hair harvesting site, and / or hair follicle tissue condition obtained from the hair follicle tissue image obtained in the step of obtaining the image with the expression ratio of various mRNAs obtained in step (b).

[0109] The present invention may further include an artificial intelligence (AI)-based analysis step generated by comparing and learning the relationship between the area of ​​the hair follicle region in the image, the number of cells measured, and the detected gene expression level. The invention may be characterized by generating a gene expression level correction model by performing the AI-based analysis step.

[0110] In the present invention, the method may be characterized by being performed fully automatically in the mRNA quantitative detection device.

[0111]

[0112] In another aspect, the present invention relates to a method for providing a personalized hair loss management service based on genetic information, comprising the information providing method described above.

[0113] In the present invention, the service provision method may be characterized by managing hair loss by regulating hair loss-related mRNA in hair follicle cells through the application of a composition (e.g., a cosmetic composition) containing siRNA in the form of micelles with a size of 50-200 nm capable of regulating the mRNA of hair loss-related genes based on mRNA expression information of hair loss-related genes. When detection results are obtained according to the present invention, a composition tailored to the cause can be applied to the scalp. A substance capable of regulating the expression of hair loss-causing genes may be used, and preferably, a composition containing siRNA may be used. For example, COSMERNA TMThe composition disclosed in Bionia (Korean Patent No. 10-2370637) and Korean Application No. 10-2022-0025070 may be used. The Cosmerna (Bionia) product contains an siRNA component targeting the androgen receptor (AR) and can be used for androgenic alopecia symptoms. Additionally, if there are multiple genes causing hair loss, a composition containing components applicable to regulating the expression of multiple genes, such as siRNA or RNAi components, may be used. Therefore, depending on the causative gene, various compositions can be applied to subjects with hair loss symptoms. The composition of the present invention may be a pharmaceutical composition or a cosmetic composition. The cosmetic composition may contain, in addition to the component regulating the expression of the above genes, generally known components applicable to the scalp. The cosmetic composition of the present invention may be formulated in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil powder foundation, emulsion foundation, wax foundation, spray, or filler. Such cosmetics may be manufactured by conventional methods. The cosmetic composition of the present invention may be applied topically to the scalp or areas with hair loss.

[0114]

[0115] Accordingly, in another aspect, the present invention relates to a method for providing a service for improving, preventing, or treating hair loss and a method for preventing or treating hair loss, comprising the following steps:

[0116] (a) a step of measuring the expression level of a hair loss-related gene selected from the group consisting of AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2 and IGF1 from a collected hair sample; and

[0117] (b) a step of providing information to apply a composition capable of inducing and / or stimulating hair growth or slowing down or improving hair loss to the scalp from which hair has been harvested or fallen out, if the expression level of the gene measured in step (a) above is increased or decreased.

[0118] In the present invention, the gene whose expression level is increased in step (b) may be AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, or PPARGC1A, and the gene whose expression level is decreased may be CD200, CD34, AREG, FGF2, or IGF1.

[0119] In the present invention, the gene expression level may be characterized by being measured using the hair loss-related mRNA detection kit.

[0120] In the present invention, the composition may be characterized by including a component for regulating mRNA expression of a hair loss-related gene, and the component may be characterized by including one or more selected from siRNA, miRNA, RNAi agent, antisense DNA or RNA, or IncRNA, but is not limited thereto.

[0121] The present invention may further include a step of examining the hair follicle tissue of the hair sample and the corresponding scalp area under a microscope prior to step (a).

[0122] The present invention may further include a step of comparing the hair follicle growth cycle, hair harvesting site, and / or hair follicle tissue condition obtained from the hair follicle tissue and / or scalp image with the expression ratio of various mRNAs obtained in step (a).

[0123]

[0124] In another aspect, the present invention relates to a primer capable of specifically amplifying the mRNA of one or more genes selected from the group consisting of AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2, and IGF1.

[0125] In the present invention, the primer may be characterized by comprising one or more of the nucleotide sequences represented by SEQ ID NOs 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46 and 47, but is not limited thereto.

[0126]

[0127] In another aspect, the present invention relates to a probe capable of hybridizing complementarily to one or more genes selected from the group consisting of AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2, and IGF1.

[0128] In the present invention, the probe may be characterized by including one or more of the nucleotide sequences represented by SEQ ID NOs 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, and 48, but is not limited thereto.

[0129]

[0130] In another aspect, the present invention provides a kit for extracting nucleic acids from hair, comprising a composition for extracting nucleic acids from hair, characterized by further including a fixing pin for fixing the hair so that the hair follicle portion is exposed, or a weight attached to the upper part of the hair follicle to sink the hair into the solution. The hair nucleic acid extraction kit according to the present invention can be operated fully automatically or manually. Here, the nucleic acid may be DNA or RNA. The hair nucleic acid extraction kit of the present invention is Maglisto TM (Bionia, KR), EcoQPrep TM It is applicable to the (Bionia, KR) kit or other commercially available nucleic acid extraction kits.

[0131]

[0132] In another aspect, the present invention relates to a composition for diagnosing or predicting hair loss comprising the primer or the probe.

[0133] The present invention identifies that changes in gene expression are closely related to the mechanism of hair loss development, and based on this information, can present a new approach for the prevention and treatment of hair loss.

[0134] After confirming the cause of hair loss by checking the expression level of the hair loss-associated target gene mentioned in the present invention, hair loss treatment suitable for the cause or hair loss cosmetic product suitable for the cause can be used.

[0135]

[0136] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0137]

[0138] Example 1: Analysis of Hair Image Data

[0139] When hair is plucked, other hair follicle cells surrounding the hair are pulled out along with it (Fig. 2a). At this time, the amount of mRNA detected by a real-time qPCR device varies depending on how many cells are pulled out along with the hair root, making it difficult to quantitatively compare mRNA expression levels among samples. To solve this problem, the present invention allows for the correction of mRNA expression data by capturing a microscopic image of the hair root to visually confirm the amount of cells collected together and quantifying it. A high-resolution microscope capable of photographing hair and scalp may be used, and in this embodiment, a polarizing microscope was used. Based on the microscopic image, the hair follicle growth cycle, the state of hair loss at the hair collection site, and the condition of the hair follicle tissue can be distinguished or confirmed (Fig. 2a).

[0140] Specifically, the area where cells exist is extracted using AI-based image analysis technology based on the number of cells captured in a hair image using a high-resolution microscope, preferably a polarizing microscope, and the expression level can be corrected by matching the area information with real-time qPCR results. Microscopic imaging utilizes a blue light background to maximize contrast between the hair and cells, allowing the area to be measured by recognizing only bright-colored cell regions. This method ensures high accuracy and consistency compared to manual methods, and the analysis accuracy can be gradually improved by accumulating multiple sample data and iteratively training the AI. Furthermore, this method can be utilized for the qualitative estimation of hair loss status by combining it with factors such as the hair growth cycle, thickness, and sampling site (hair loss area or non-hair loss area), and can be applied as a foundational technology to enhance the accuracy of hair loss diagnostic kits or gene-based analysis platforms.

[0141] IRON-qPCR analysis was performed on hair in the anagen phase using the primers and probes presented in Table 2 below, with the main target being hair in the growth phase viewed through a microscope. Tweezers, medical pliers, and medical scissors were prepared for sample pretreatment.

[0142] First, a single harvested strand of hair was secured with tweezers, and the ring bead included in the kit was grasped using pliers and attached to the hair. During this process, the rootless end was inserted into the ring bead, and the position was adjusted so that the ring bead was located approximately 0.5 cm away from the root area. Next, while holding the hair with tweezers, the ring bead was compressed using pliers, and this compression was repeated until the hair was sufficiently secured and did not detach from the ring bead. Subsequently, the remaining end of the hair, excluding the root and ring bead, was cut with medical scissors. The cut hair was then subjected to IRON-qPCR TM After inserting the sample into the sample injection port of the cartridge, analysis was performed by operating the device.

[0143] Through this method, hair is injected while stably fixed within the cartridge, allowing for direct hair-based IRON-qPCR TM (Bionia, KR) Analysis is possible. Therefore, this method enabled stable gene expression analysis by efficiently securing nucleic acids from the hair root region while minimizing loss or detachment during the handling of hair samples. Thus, it was confirmed that stable gene expression analysis is possible by efficiently securing nucleic acids from the hair root region through this method. The results of the expression analysis of hair health-related genes secured in this way are shown in Fig. 2b.

[0144]

[0145] Example 2: Preparation, Pretreatment, and Nucleic Acid Extraction of Hair Samples

[0146] The cartridge and kit according to the present invention are products optimized for extracting mRNA from human hair samples. Immediately after collecting the hair sample, an image is captured, and the sample is used for testing by attaching a weight (metal ring bead) or an equivalent hair fixing wire as shown in FIG. 1 or FIG. 4a. mRNA was extracted from the hair sample as disclosed in FIG. 1b, and the extraction performance was verified using the primer / probe set in Table 2. It was confirmed that using a weight or hair fixing wire made of stainless steel, aluminum, or titanium resulted in performance equivalent to that of using hair alone (Fig. 4b). When collecting hair for a short period, it is preferable to use an RNase-free disposable tube (microcentrifuge or conical tube). Tweezers, medical pliers, or medical scissors may be used for hair sample pretreatment. One hair is pulled from an area of ​​hair loss or an area where hair loss is expected. A single strand of hair with the hair follicle attached is grasped using tweezers. Using medical pliers, the weight included in the kit is grasped, and the hair is placed between them. Insert the end without the hair follicle so that the weight is positioned approximately 5 mm from the point where the hair follicle is attached. While holding the hair with tweezers to prevent it from slipping out of the weight, press the medical pliers to compress the weight against the hair until it is secured. As shown in FIG. 3 or 4a, cut the hair on the side opposite the root with medical scissors, remove the sticker from the sample inlet of the cartridge of the present invention containing the nucleic acid extraction solution, and insert the cut hair into the sample inlet by holding it with tweezers. Cover the sample inlet with a sticker to prevent sample contamination. The subsequent process follows the IRON-qPCR disclosed in Korean Registered Patent 10-2256757 and Korean Published Patent 10-2023-0113032. TM IRON-qPCR according to the (Bionia, KR) manual TMThe extraction process was carried out in the Bionia system. The nucleic acid extraction solution contains magnetic particles (beads) capable of binding to nucleic acids. The general workflow of the system is shown in Fig. 7. A barcode reader mounted on the equipment recognizes the cartridge, and the test results can be analyzed after about one hour.

[0147] In another embodiment, Maglisto TM Magnetic Separation Rack (Bionia, KR) or EcoQprep TM RNA from hair follicles was extracted using the RNA Kit (Bionia, KR) according to the manufacturer's manual as follows. First, 1 to 5 strands of hair were collected and prepared. As mentioned above, weights were attached to the hair and placed into a tube for nucleic acid extraction. For cell lysis, 400 μL of lysis buffer was added to the tube and vortexed to mix thoroughly. Subsequently, for nucleic acid binding, 400 μL of binding buffer and 30 μL (200 mg / mL) of magnetic beads were added, followed by vortexing to mix thoroughly. Then, for the washing step, 700 μL of wash buffer was added for the first washing and vortexed, and 700 μL of wash buffer was added for the second washing and vortexed. For extraction, 30 μL of elution buffer was added to a tube, and after vortexing, RNA was collected after incubation at 60°C for 3 minutes.

[0148]

[0149] Example 3: Design of a primer / probe set to determine the expression level (mRNA) of a hair follicle (HF)-related target gene

[0150] To design a primer set capable of verifying mRNA expression levels for 13 types of HF mRNA (AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2, and IGF1), the nucleotide sequences registered in the nucleotide database of the National Center for Biotechnology Information (NCBI) and the whole gene sequences were referenced. The ACTB gene was used as a control. From this, a primer / probe set capable of specifically binding to 14 types (including the control) was designed as shown in Table 2.

[0151] In order to stably detect mRNA expression in the case of a gene having multiple transcript variants, two or more primer / probe oligosets targeting different locations were designed through variant sequence alignment and conservation region analysis (Table 2). These sets consist of a set targeting a common sequence and a set targeting variant-specific sequences, and through parallel application, detection coverage was maximized and even low-expression variants were precisely detected, thereby improving the reproducibility and reliability of quantitative analysis.

[0152]

[0153]

[0154]

[0155] Example 4: Real-time PCR and IRON-qPCR Verification of 13 HF mRNA Types

[0156] In the present invention, Real-time PCR was performed by adding 1 μl of human reference RNA to verify the PCR reaction product for confirming the HF mRNA expression level.

[0157] Real-time PCR was performed on Exicycler™ (Bionia, KR) using primer pairs and TaqMan probes to verify the expression levels of 13 types of HF-related genes. As a result, the specificity of each primer set was secured and the gene expression levels were verified, confirming that this is an effective verification method for gene analysis primer sets applicable to IRON-qPCR devices (Fig. 5a).

[0158] In addition, as shown in Fig. 5b, primer specificity and amplification efficiency were qualitatively examined through the melting curves and amplification curves of each target gene (e.g., AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2, and IGF1) using SYBR Green qPCR with Exicycler™. Verification was performed on a total of 13 genes, and among them, clear single peaks and amplification curves appeared in the 13 genes related to HF, confirming that the primer design is applicable to the present invention. Fig. 5b is a graph showing the results of verification by SYBR Green qPCR for each gene, and each panel includes the amplification curve (left) and melting curve (right) for the corresponding gene. Through this, it was demonstrated that the primer set for gene expression analysis presented in the present invention is substantially suitable for measuring hair follicle-related gene expression.

[0159] Figure 6 is a graph showing the mRNA expression pattern measured in a hair sample using a Real-Time PCR device (IRON-qPCR™) according to an embodiment of the present invention. Hair was collected from the left temporal region (side hair) and crown region of a subject according to the method of Example 1, and the sample containing the hair roots was directly inserted into the cartridge of the device. mRNA was then quantitatively analyzed using IRON-qPCR™ with the sequences in Table 2. For each sample, the expression levels of the target gene and the housekeeping gene were measured, converted into relative expression levels (fold change), and graphed. As shown in Figure 6, it was confirmed that the present invention can precisely detect differences in mRNA expression patterns depending on the hair collection site.

[0160]

[0161] Example 5: Treatment of a patient with alopecia with a cosmetic composition

[0162] Cosmetics from COSMERNA (Bionia) were applied to the scalps of subjects diagnosed with alopecia. As shown in Figure 8, it was confirmed that hair loss symptoms were significantly improved when the COSMERNA product containing siRNA was treated for 24 weeks.

[0163]

[0164] The present invention provides means, preferably a kit and an apparatus, for simultaneously and quantitatively and accurately analyzing mRNAs of various genes related to hair loss from harvested or naturally shed hair, preferably from the hair follicle region of the hair, within a short period of time, for example, within one hour.

[0165] In one embodiment, to quantify hair loss-related mRNAs from hair follicle cells in a single hair, a fixing means is fixed to the upper 5-10 mm portion of the hair follicle tissue so that the hair follicle tissue is always exposed to and submerged in the extract, and the tissue is submerged in the extraction container portion where an ultrasonic probe is directed. Then, a lysis buffer is added and ultrasound is applied to lyse the hair follicle tissue cells, attach the RNA to magnetic particles, and then extract the RNA through washing and elution processes. Afterward, the process proceeds automatically to quantitative PCR without delay so that the RNA is not degraded, thereby allowing the cause of hair loss to be identified quickly, simply, and accurately at the molecular biology level.

[0166] By using the kit according to the present invention, the phenomena occurring within hair follicle cells are analyzed at the mRNA level, thereby enabling more accurate and rapid identification of the causes of hair loss based on a molecular biological understanding of hair loss, and allowing for personalized hair health management.

[0167] Furthermore, the kit and device of the present invention automatically perform tests ranging from RNA extraction of hair loss-related genes to quantitative PCR from a small amount of hair, allowing for the convenient and rapid identification of the causes of hair loss and enabling personalized hair health management. In addition, the kit and method of the present invention are designed for on-site testing and do not require a separate laboratory. Since the tests can be easily performed and mRNA within hair follicles analyzed through simple training, accurate molecular biology-based hair loss management can be carried out even in small hospitals or clinics.

[0168]

[0169] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0170]

[0171] I have attached the electronic file.

Claims

1. A cover including a fixing pin insertion port or a hair insertion port; A receiving portion in which magnetic particle beads are located on the front of the ultrasonic probe tube; A cartridge containing a solution for extracting RNA from hair contained in the above-mentioned receiving portion, capable of automatically and sequentially applying each solution to the above-mentioned receiving portion; and A reaction plate comprising primers and probes for selectively amplifying and quantitatively detecting hair loss-related mRNA from the extracted RNA, and a plurality of reaction wells for measuring fluorescence; A detection kit for quantitatively analyzing hair loss-related mRNA in the hair follicle region containing, A kit characterized by further including a fixing pin that fixes the hair so that the hair follicle area is exposed, or a weight attached to the upper part of the hair follicle to sink the hair into the solution.

2. A kit according to claim 1, characterized in that the hair fixed by the fixing pin is coupled to the fixing pin insertion port, and the hair with the weight attached is fed into the hair input port.

3. A kit according to paragraph 2, characterized in that the fixing pin or weight is made of a metal such as stainless steel, aluminum, or titanium.

4. A kit according to claim 1, characterized in that the primer and probe specifically quantify the mRNA of a hair loss-related gene selected from the group consisting of AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2, and IGF1.

5. A kit according to claim 1, characterized in that the primer or probe comprises one or more of the nucleotide sequences represented by SEQ ID NO. 1 to SEQ ID NO.

48.

6. A hair nucleic acid extraction kit comprising a composition for extracting nucleic acids from hair, A kit characterized by further including a fixing pin for fixing hair so that the hair follicle area is exposed, or a weight attached to the upper part of the hair follicle to sink the hair into the composition.

7. A kit according to any one of claims 1 to 5; and an mRNA quantitative detection device comprising a PCR reading unit, An apparatus characterized by extracting nucleic acid using the cartridge of the above kit, performing PCR using the above reaction plate, and then reading the PCR result in the above PCR reading unit.

8. A method for providing information for the diagnosis of hair loss, assessment of hair health status, and prognosis, including the following steps: (a) A step of extracting RNA from hair to which a fixation means is attached; (b) a step of measuring the expression level of a hair loss-related gene selected from the group consisting of AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2, and IGF1 from the RNA extracted in step (a) by performing relative quantification using reverse transcription quantitative PCR; and (c) A step of determining that hair loss or poor hair health is present if the expression level of the gene measured in step (b) above is increased or decreased compared to a control group.

9. An information provision method according to claim 8, characterized in that the above method is measured using a kit according to any one of claims 1 to 6, or a device according to claim 7.

10. A method for providing information according to claim 8, wherein the reverse transcription quantitative PCR of step (b) above is a multiplex real-time polymerase chain reaction that simultaneously quantitatively detects one or more genes.

11. A method for providing information according to claim 8, further comprising the step of photographing the hair follicle area or scalp collected prior to step (a) with a microscope to obtain an image of the hair follicle tissue or scalp attached to the hair area.

12. A method for providing information according to claim 11, further comprising the step of comparing the hair follicle growth cycle, hair harvesting site, and / or hair follicle tissue condition obtained from the tissue or scalp image with the expression ratio of various mRNAs obtained in step (b).

13. An information provision method according to claim 8, characterized in that the above method is performed fully automatically in a device according to claim 7.

14. Method of providing services for the improvement, prevention, or treatment of hair loss including the following steps: (a) a step of measuring the expression level of a hair loss-related gene selected from the group consisting of AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2 and IGF1 from a collected hair sample; and (b) a step of providing information to apply a composition capable of inducing and / or stimulating hair growth or slowing down or improving hair loss to the scalp from which hair has been harvested or fallen out, if the expression level of the gene measured in step (a) above is increased or decreased.

15. A method of providing a service characterized in that, in claim 14, the gene expression level is measured using the kit of claim 1 or claim 6.

16. A method of providing a service according to claim 14, characterized in that the composition comprises a component for regulating the mRNA expression of a hair loss-related gene.

17. A method for providing a service according to claim 16, characterized in that the above-mentioned component comprises one or more selected from siRNA, miRNA, RNAi agent, antisense DNA or RNA, or IncRNA.

18. A method of providing a service according to claim 14, further comprising the step of examining the hair follicle tissue or scalp of the hair sample under a microscope prior to step (a).

19. A method for providing a service according to claim 18, further comprising the step of comparing the hair follicle growth cycle, hair harvesting site, and / or hair follicle tissue condition obtained from a hair follicle tissue image with the expression ratio of various mRNAs obtained in step (a).

20. Primers capable of specifically amplifying the mRNA of one or more genes selected from the group consisting of AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2, and IGF1.

21. The primer according to claim 20, characterized in that the primer comprises one or more of the nucleotide sequences represented by SEQ ID NOs 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31, 32, 34, 35, 37, 38, 40, 41, 43, 44, 46, and 47.

22. A probe capable of hybridizing complementarily to one or more genes selected from the group consisting of AR, FGF5, ACTA2, IL1B, TNF, TGFB1, DKK1, CD200, CD34, AREG, PPARGC1A, FGF2, and IGF1.

23. The probe according to claim 22, characterized in that the probe comprises one or more of the nucleotide sequences represented by SEQ ID NOs 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, and 48.

24. A composition for diagnosing or predicting hair loss comprising the primer of claim 20 or the probe of claim 22.

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