Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention relates to an internal standard gene for lipid samples on the skin surface and a method for measuring gene expression levels using the same.
• Absolute quantification is a method in which a calibration curve is created from a standard sample with known absolute amounts, such as copy number, and this is used to quantify the absolute amount of expression of the target gene in the sample.
• relative quantification is a method in which the expression level of the target gene in the sample is corrected with the expression level of an internal standard gene, and the relative expression level of the target gene is quantified.
• the internal standard gene is a gene whose expression level does not vary between tissues or experimental systems, and housekeeping genes such as GAPDH and ACTB are generally used. However, it has been reported that even housekeeping genes may vary in expression depending on the tissue, and may not function as an internal standard gene (Non-Patent Documents 1 and 2).
• Non-Patent Document 3 skin surface lipids (SSL) contain RNA derived from skin cells of a subject, that the RNA contained in SSL is useful as a sample for gene expression analysis of a living body, and that marker genes of the epidermis, sweat glands, hair follicles, and sebaceous glands can be detected from SSL.
• SSL skin surface lipids
• Patent Document 1 International Publication No. 2018/008319 (Non-Patent Document 1) Journal of Investigative Dermatology, 2009, 129(3):535-537 (Non-Patent Document 2) Acta Biochem Biophys Sin, 2014, 46(4):330-337 (Non-Patent Document 3) Journal of Investigative Dermatology, 2009, 129(3):770-773
• the present invention relates to the following (1) to (3).
• a method for measuring the expression level of a target gene contained in an SSL sample from an infant comprising using at least one gene selected from the group consisting of genes shown in Table 1 below as an internal standard gene in PCR.
• a kit for measuring the expression level of a target gene contained in an SSL sample from an infant used in the method of (1) comprising an oligonucleotide that specifically hybridizes with the internal standard gene.
• nucleic acid refers to DNA or RNA.
• DNA includes cDNA, genomic DNA, and synthetic DNA
• RNA includes total RNA, mRNA, rRNA, tRNA, non-coding RNA, and synthetic RNA.
• the term "gene” refers to double-stranded DNA including human genomic DNA, as well as single-stranded DNA (positive strand) including cDNA, single-stranded DNA (complementary strand) having a sequence complementary to the positive strand, and fragments thereof, and refers to DNA that contains some biological information in the sequence information of the bases that make up the DNA.
• the "gene” in question includes not only “genes” represented by a specific base sequence, but also nucleic acids encoding their homologues (i.e., homologs or orthologs), mutants such as gene polymorphisms, and derivatives.
• the gene names (Gene Symbols) and Gene IDs disclosed herein correspond to the Official Symbols and Gene IDs listed in NCBI ([www.ncbi.nlm.nih.gov/]).
• the term "internal standard gene” is also referred to as an endogenous control gene or a reference gene, and refers to a gene that serves as a reference for correcting (standardizing) the expression level of a target gene in gene expression analysis. "Correction” is typically performed by dividing the expression level of the target gene by the expression level of the internal standard gene. “Expression level” or “amount” includes the absolute amount of expression and the expression level relative to the expression levels of other genes, etc.
• the term “housekeeping gene” refers to a gene that is essential for cell maintenance and proliferation and is expressed at a certain level in many tissues and cells. Common housekeeping genes include, for example, GAPDH and ACTB (see Genome Analysis, 2003, 19(7):362-365).
• infant broadly refers to a "child” before the onset of secondary puberty, specifically a concept that includes children aged 15 or younger, and preferably refers to children and infants before entering elementary school, specifically infants between 0 and 6 years old, and more preferably infants between 0 and 69 months of age.
• SSL skin surface lipids
• RNA expressed in skin cells see Patent Document 1
• SSL collected from test infants is also referred to as SSL specimens.
• skin is a general term for the area including tissues such as the stratum corneum, epidermis, dermis, hair follicles, sweat glands, sebaceous glands, and other glands.
• the present invention relates to providing an internal standard gene for more accurately measuring the expression level of a target gene contained in an infant's SSL, and a method for measuring the expression level of a target gene contained in an infant's SSL using the internal standard gene.
• the internal standard gene of the present invention has small expression variations between infant SSL samples, and in particular has small expression variations compared to GAPDH and ACTB, which are well-known housekeeping genes that are commonly used as internal standard genes.
• GAPDH and ACTB are well-known housekeeping genes that are commonly used as internal standard genes.
• the 30 genes shown in Table 1 of the present invention are genes that were found to have small variations in the expression levels of SSL-derived RNA between test infants, as shown in the Examples described below.
• the 30 genes shown in Table 1 are genes that were found to be stably expressed after normalizing the expression data of genes with a gene detection rate of 70% or more using the RLE method as a normalization method based on the expression data (read count value) of RNA extracted from the SSL of 261 subjects, both male and female infants.
• the expression stability was evaluated using the CV Z-score as an index.
• the "CV Z-score” indicates the deviation of the measured value from the average value of the distribution, and is a value (Z-score) obtained by standardizing the coefficient of variation (CV) calculated by dividing the difference between the measured value and the average value of the distribution by the standard deviation of the distribution.
• CV Z-score The smaller the CV Z-score value, the smaller the variation in gene expression (the average value of the CV Z-score itself is 0, and the standard deviation is 1).
• the CV Z-score is used as an evaluation index for expression stability, and genes with a CV Z-score of -1.874 or less were determined to be stably expressed genes.
• the above 30 genes have CV Z-scores that are equivalent to or smaller than those of GAPDH (CV Z-score: -1.813) and ACTB (CV Z-score: -1.874), which are known housekeeping genes generally considered to be suitable as internal standard genes, and further, RPLP0 (CV Z-score: -1.859), which is suitable as an internal standard gene in gene expression analysis of epidermal keratinocytes, and it has been confirmed that these genes are stably expressed in SSL.
• a gene selected from the group consisting of these 30 genes can be used as an internal standard gene in qPCR (hereinafter also referred to as the internal standard gene of the present invention) when measuring the expression level of a target gene contained in the SSL of an infant.
• the internal standard gene any one of the genes may be used alone, or two or more genes may be used in combination.
• the internal standard gene of the present invention is preferably at least one gene selected from the group consisting of 16 genes: PCBP2, RPL30, ARPC2, UBA52, RAB7A, RAC1, ARL8B, RPL32, PCBP1, BZW1, H3F3A, RPS8, FAU, RPL4, EEF1G, and ARF1, more preferably at least one gene selected from ARF1, RPL30, and FAU, even more preferably ARF1 or RPL30, and even more preferably ARF1.
• the internal standard gene of the present invention is preferably at least one gene selected from the group consisting of the above 16 genes, more preferably at least one gene selected from the group consisting of the 10 genes, PCBP2, RPL30, UBA52, RAB7A, ARL8B, PCBP1, BZW1, RPS8, RPL4, and EEF1G, and even more preferably RPL30.
• the internal standard gene of the present invention also includes genes having a base sequence substantially identical to the base sequence of the DNA constituting the gene, so long as it can serve as an internal standard in gene expression analysis.
• the internal standard gene of the present invention is preferably used in a method for measuring the expression level of a gene of interest contained in an SSL sample from an infant (hereinafter, also referred to as the method of the present invention).
• the method of the present invention includes correcting the amount of the amplification product of the gene of interest for an SSL sample from an infant with the amount of the amplification product of the internal standard gene, and calculating the expression level of the gene of interest.
• the method of the present invention includes amplifying the gene of interest for an SSL sample from an infant, amplifying the internal standard gene, and correcting the amount of the amplification product of the gene of interest for the amount of the amplification product of the internal standard gene, and calculating the expression level of the gene of interest.
• the method of the present invention includes extracting RNA from an SSL sample collected from a test infant, amplifying the gene of interest based on the extracted RNA, amplifying the internal standard gene based on the extracted RNA, and correcting the amount of the amplification product of the gene of interest for the amount of the amplification product of the internal standard gene, and calculating the expression level of the gene of interest.
• the target gene is not particularly limited and may be one or more genes that can be contained in SSL samples from infants.
• any means used for recovering or removing SSL from the skin can be used.
• SSL absorbent material, SSL adhesive material, or a tool for scraping SSL from the skin can be used.
• the SSL absorbent material or SSL adhesive material is not particularly limited as long as it has an affinity for SSL, and examples thereof include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include a method of absorbing SSL into a sheet-like material such as oil blotting paper or oil blotting film, a method of adhering SSL to a glass plate or tape, and a method of scraping SSL off and collecting it with a spatula, scraper, etc.
• an SSL absorbent material that has been previously impregnated with a highly lipid-soluble solvent may be used.
• the SSL absorbent material contains a low content of highly water-soluble solvents and water, since the adsorption of SSL is inhibited if the SSL absorbent material contains highly water-soluble solvents or water. It is preferable to use the SSL absorbent material in a dry state.
• the part of the skin from which the SSL is collected is not particularly limited, and may be any part of the body, such as the head, face, neck, trunk, hands, or feet, and is preferably from a part that secretes a lot of sebum, such as the skin of the face.
• the RNA-containing SSL sample collected from the test infant may be used immediately in the RNA extraction process described below, or may be stored for a certain period of time.
• the collected SSL sample is preferably stored under low-temperature conditions as soon as possible after collection in order to minimize degradation of the RNA contained therein.
• the temperature conditions for storing the RNA-containing SSL in the present invention may be 0°C or lower, preferably -20 ⁇ 20°C to -80 ⁇ 20°C, more preferably -20 ⁇ 10°C to -80 ⁇ 10°C, even more preferably -20 ⁇ 20°C to -40 ⁇ 20°C, even more preferably -20 ⁇ 10°C to -40 ⁇ 10°C, even more preferably -20 ⁇ 10°C, and even more preferably -20 ⁇ 5°C.
• the period for storing the RNA-containing SSL sample under the low-temperature conditions is not particularly limited, but is preferably 12 months or less, for example, 6 hours or more and 12 months or less, more preferably 6 months or less, for example, 1 day or more and 6 months or less, even more preferably 3 months or less, for example, 3 days or more and 3 months or less.
• RNA contained in an SSL sample from an infant is extracted and converted to cDNA by reverse transcription, and the target gene and internal standard gene are amplified by PCR using the cDNA as a template, and the amplified product is then measured.
• RNA extraction reagent for extraction of RNA from SSL
• a method that is usually used for extracting or purifying RNA from a biological sample such as the phenol/chloroform method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, or a method using columns such as TRIzol (registered trademark), RNeasy (registered trademark), or QIAzol (registered trademark), a method using special silica-coated magnetic particles, a method using Solid Phase Reversible Immobilization magnetic particles, or extraction using a commercially available RNA extraction reagent such as ISOGEN, can be used.
• a primer targeting a specific RNA to be analyzed may be used, but for more comprehensive nucleic acid storage and analysis, a random primer is preferably used.
• a general reverse transcriptase or reverse transcription reagent kit can be used.
• a highly accurate and efficient reverse transcriptase or reverse transcription reagent kit is used, and examples thereof include M-MLV Reverse Transcriptase and its variants, or commercially available reverse transcriptases or reverse transcription reagent kits, such as the PrimeScript (registered trademark) Reverse Transcriptase series (Takara Bio Inc.), the SuperScript (registered trademark) Reverse Transcriptase series (Thermo Scientific Inc.), and the like.
• the temperature of the extension reaction in the reverse transcription is preferably adjusted to 42° C. ⁇ 1° C., more preferably 42° C. ⁇ 0.5° C., and even more preferably 42° C. ⁇ 0.25° C., while the reaction time is preferably adjusted to 60 minutes or more, more preferably 80 to 120 minutes.
• Amplification of the target gene or the internal standard gene using the cDNA obtained by reverse transcription as a template can be carried out according to a PCR procedure commonly used in the art.
• PCR techniques include conventional PCR, multiplex PCR, real-time PCR (also called quantitative PCR (qPCR)), multiplex real-time PCR, and digital PCR.
• cDNA synthesis by reverse transcription (RT) and PCR may be performed in one step or two steps, and either may be appropriately selected depending on the purpose.
• One-step RT-PCR is a method in which a series of RT and PCR reactions are continuously performed in a single tube, which is simple and prevents contamination between the RT and PCR operations.
• random primers can be used for RT, so that the expression levels of multiple target genes can be measured from a single RNA sample.
• Examples of PCR in RT-PCR include the same as the PCR described above.
• a primer pair targeting a specific DNA to be analyzed may be used to amplify only the specific DNA, or multiple primer pairs may be used to simultaneously amplify multiple DNAs.
• Methods for simultaneously amplifying multiple DNAs include multiplex PCR and multiplex real-time PCR. It is preferable to perform multiplex analysis in which the target gene and the internal standard gene are simultaneously amplified, since it is possible to suppress errors in the amount of template DNA and measure the expression level more accurately.
• Such multiplex analysis can be performed using a commercially available kit (e.g., Ion AmpliSeq Transcriptome Human Gene Expression Kit; Life Technologies Japan, Inc., etc.).
• the amplification product can be detected by a known means capable of specifically recognizing the amplification product, for example, a method of detecting labeled double-stranded DNA produced by PCR using a primer previously labeled with RI, a fluorescent substance, or the like.
• the quantitative PCR (qPCR) method is preferably real-time PCR or multiplex real-time PCR, which monitors and analyzes the amount of PCR amplified product in real time, from the viewpoints of speed, simplicity, and quantitativeness.
• Methods for detecting amplified products in real-time PCR or multiplex real-time PCR include methods commonly used in the field, such as the intercalator method and the TaqMan (registered trademark) probe method.
• the intercalator method involves the coexistence of a substance (an intercalator, such as SYBR (registered trademark) Green I) that emits fluorescence when it penetrates into double-stranded DNA in a PCR reaction system, and the amount of amplified product is monitored by detecting the fluorescence that increases as the amplified product is produced.
• a substance such as SYBR (registered trademark) Green I
• the TaqMan probe method involves the coexistence of a target sequence-specific oligonucleotide (TaqMan probe) modified at the 5' end with a fluorescent substance (e.g., FAM) and at the 3' end with a quencher substance (e.g., TAMRA) in a PCR reaction system.
• a fluorescent substance e.g., FAM
• a quencher substance e.g., TAMRA
• the 5' ⁇ 3' exonuclease activity of Taq DNA polymerase degrades the TaqMan probe hybridized to the template, releasing the fluorescent substance from the probe and releasing the inhibition by the quencher substance, causing it to emit fluorescence.
• the amount of amplified product can be monitored by detecting this fluorescence.
• the PCR conditions are not particularly limited, and the optimal conditions may be determined for each PCR.
• the temperature is preferably 94 to 99° C., and the time is 10 to 60 seconds.
• Annealing The temperature is usually 50° C. or higher, preferably 52° C. or higher, more preferably 55° C. or higher, and usually 65° C. or lower, preferably 63° C. or lower, more preferably 60° C. or lower. Also, it is usually 50 to 65° C., preferably 52 to 63° C., more preferably 55 to 60° C.
• the time is usually 5 seconds or higher, preferably 10 seconds or higher, and usually 2 minutes or less, preferably 1 minute or less. Also, it is usually 5 seconds to 2 minutes, preferably 10 seconds to 1 minute.
• 3) DNA extension reaction The temperature is usually 65° C. or higher, preferably 68° C. or higher, and usually 74° C. or lower, preferably 72° C. or lower. The temperature is usually about 65 to 74° C., preferably 68 to 72° C.
• the time is usually 5 seconds or longer, preferably 10 seconds or longer, and usually 2 minutes or shorter, preferably 1 minute or shorter.
• the time is usually 5 seconds to 2 minutes, preferably 10 seconds to 1 minute.
• the annealing and the DNA elongation reaction can be carried out simultaneously without being separated.
• the reactions 1) to 3) above are regarded as one cycle, and this cycle is usually carried out for 30 cycles or more, preferably 35 cycles or more, and usually 50 cycles or less, preferably 45 cycles or less.
• Reverse transcription and PCR at the above-mentioned temperature and time can be carried out using a thermal cycler generally used for PCR or a dedicated real-time PCR device that combines a thermal cycler with a spectrofluorometer.
• the chain length of the PCR amplification product can be appropriately selected taking into consideration factors such as shortening the PCR amplification time.
• the chain length of the PCR amplification product is preferably 1000 bp or less, more preferably 700 bp or less, and even more preferably 500 bp or less.
• the chain length of the PCR amplification product is at its lower limit of 30 to 40 bp, which is the chain length of the PCR amplification product when a primer of about 15 bases is used to avoid non-specific hybridization in PCR, and is preferably 50 bp or more, and more preferably 100 bp or more.
• the chain length of the PCR amplification product is preferably 30 to 1000 bp, more preferably 50 to 700 bp, and even more preferably 100 to 500 bp.
• the probe or primer used in the above measurement i.e., a primer for specifically recognizing and amplifying a target gene, an internal standard gene, or a nucleic acid derived therefrom, or a probe for specifically detecting the target gene, an internal standard gene, or a nucleic acid derived therefrom, corresponds to this, and these can be designed based on the base sequence constituting the target gene or internal standard gene.
• “specifically recognize” means that the detected substance or product can be determined to be the gene or a nucleic acid derived therefrom, such that, for example, in the RT-PCR method, substantially only the target gene, the internal standard gene, or a nucleic acid derived therefrom is amplified.
• a DNA consisting of a base sequence constituting the target gene or internal standard gene of the present invention or an oligonucleotide containing a certain number of nucleotides complementary to the complementary strand thereof can be used.
• complementary strand refers to one strand of a double-stranded DNA consisting of A:T (U in the case of RNA) and G:C base pairs, with respect to the other strand.
• complementary does not necessarily mean a completely complementary sequence in the certain number of consecutive nucleotide regions, but may mean that the base sequence has an identity of preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more.
• the identity of the base sequence can be determined by an algorithm such as BLAST.
• an oligonucleotide When used as a primer, such an oligonucleotide may have a chain length of, for example, 10 or more bases, preferably 15 or more bases, more preferably 20 or more bases, and, for example, 100 or less bases, preferably 50 or less bases, more preferably 35 or less bases.
• such an oligonucleotide When used as a probe, such an oligonucleotide may have a chain length of, for example, 10 or more bases, preferably 15 or more bases, and, for example, 100 or less bases, preferably 50 or less bases, more preferably 25 or less bases, and, for example, a part or all of the sequence of DNA (or its complementary strand) consisting of the base sequence constituting the target gene or internal standard gene of the present invention, and, for example, 10 or more bases, preferably 15 or more bases, and, for example, 100 or less bases, preferably 50 or less bases, more preferably 25 or less bases.
• the "oligonucleotide" may be DNA or RNA, and may be synthetic or natural.
• the probe used in hybridization is usually labeled.
• nested PCR can be used to increase the yield and specificity of the amplified product.
• a first round of PCR is performed using a first primer pair to amplify the target sequence, and then a second round of PCR is performed using the amplified product as a template with a second primer pair designed inside the region amplified by the first primer pair to obtain an amplified product.
• it is preferable that at least the second round of PCR of the two rounds of PCR is real-time PCR or multiplex real-time PCR.
• the target gene and the internal standard gene are amplified based on RNA extracted from SSL samples from infants, and the expression level of the target gene is calculated based on the amplified product.
• the expression level of the target gene can be calculated according to a relative quantification method commonly used in the field.
• the relative quantification method include a method in which an amplified product of the target gene and an amplified product of the internal standard gene are subjected to electrophoresis, and the densities of the bands on the gel are compared to calculate the relative expression level of the target gene relative to the internal standard gene, as well as the well-known calibration curve method, - ⁇ Ct method, and ⁇ Ct method (comparative Ct method) as methods of relative quantification in real-time PCR.
• the - ⁇ Ct method and ⁇ Ct method are preferred because they do not require a calibration curve.
• the relative quantification may be performed as follows. First, a dilution series of the standard sample is prepared, and the target gene and the internal standard gene are each amplified by PCR using this as a template, and the Ct value is obtained.
• the Ct value means the cycle number (threshold cycle) at which the amount of the PCR amplified product reaches a certain amount.
• the concentration of the standard sample and the determined Ct value are plotted, the relationship between the two is expressed as a straight line, which can be used as a calibration curve.
• PCR is performed on the sample under the same conditions to obtain the Ct value, and the quantification results of the target gene and the internal standard gene in the sample are obtained from this value and the calibration curve. Furthermore, the quantification result of the target gene is divided by the quantification result of the internal standard gene to correct the expression amount of the target gene with the expression amount of the internal standard gene, and the expression amount of the target gene is calculated.
• the expression amount of the target gene may be expressed as a relative amount when the result of a certain sample is set to 1, so that the difference in expression amount between samples can be easily understood.
• the relative quantification may be performed as follows. First, the target gene and the internal standard gene in the sample A are each amplified by PCR to obtain the Ct value, and the difference ( ⁇ Ct) between the Ct value of the target gene and the Ct value of the internal standard gene is calculated.
• ⁇ Ct is a value obtained by correcting the quantification result of the target gene with the quantification result of the internal standard gene.
• PCR is also performed under the same conditions for the sample B to be compared, and the Ct value and ⁇ Ct are obtained.
• the obtained ⁇ Ct is substituted into the formula (2 ⁇ Ct ), and the expression levels between the samples A and B are compared based on the calculated relative expression levels.
• the relative quantification may be performed as follows. First, the target gene and the internal standard gene in the sample A are each amplified by PCR to obtain the Ct value, and the difference ( ⁇ Ct) between the Ct value of the target gene and the Ct value of the internal standard gene is calculated.
• ⁇ Ct is a value obtained by correcting the quantification result of the target gene with the quantification result of the internal standard gene.
• PCR is also performed under the same conditions for the sample B to be compared, and the Ct value and ⁇ Ct are obtained.
• the difference ( ⁇ Ct) between the ⁇ Ct of the sample A and the ⁇ Ct of the sample B is obtained.
• ⁇ Ct is a value that reflects the difference in the expression amount of the target gene between the samples. Furthermore, the obtained ⁇ Ct is substituted into the formula (2 ⁇ Ct ). The value obtained here indicates how many times higher or lower the expression amount of the target gene in the sample A is than the expression amount of the target gene in the sample B. That is, the expression amount of the target gene in the sample A is expressed as a relative amount when the expression amount in the sample B is set to 1.
• the kit for measuring the expression level of a target gene contained in an SSL sample from an infant used in the method of the present invention contains a test reagent for measuring the expression level of the internal standard gene of the present invention in the SSL collected from a test infant.
• the kit includes a reagent for nucleic acid amplification and hybridization, which contains an oligonucleotide (e.g., a primer or probe for PCR) that specifically binds (hybridizes) to the internal standard gene of the present invention or a nucleic acid derived therefrom.
• the oligonucleotide contained in the kit can be obtained by a known method as described above.
• the kit may include standard samples for creating a calibration curve, RT reagents, PCR reagents, labeling reagents, buffer solutions, instruments and controls necessary for the test, tools for collecting SSL (e.g., oil-removing films for collecting SSL), reagents for storing the collected SSL, storage containers, and reagents for extracting and purifying RNA from the collected SSL.
• tools for collecting SSL e.g., oil-removing films for collecting SSL
• reagents for storing the collected SSL e.g., storage containers, and reagents for extracting and purifying RNA from the collected SSL.
• the present invention further discloses the following aspects.
• a method for measuring the expression level of a target gene contained in a lipid sample on the skin surface of an infant comprising using at least one gene selected from the group consisting of genes shown in Table 2 below as an internal standard gene in PCR.
• ⁇ 2> The method according to ⁇ 1>, further comprising correcting the amount of the amplification product of the target gene with the amount of the amplification product of the internal standard gene to calculate the expression level of the target gene.
• ⁇ 3> Amplifying the target gene; The method according to ⁇ 1>, further comprising: amplifying an internal standard gene; and correcting the amount of the amplification product of the target gene with the amount of the amplification product of the internal standard gene, thereby calculating the expression level of the target gene.
• RNA from a lipid sample on the skin surface collected from the test infant amplifying the gene of interest based on the extracted RNA;
• the method according to ⁇ 1> further comprising: amplifying an internal standard gene based on the extracted RNA; and correcting the amount of the amplification product of the target gene with the amount of the amplification product of the internal standard gene to calculate the expression level of the target gene.
• RNA from a lipid sample on the skin surface collected from a test infant synthesizing cDNA using the extracted RNA as a template; amplifying the gene of interest using the obtained cDNA as a template; amplifying an internal standard gene using the obtained cDNA as a template; and calculating an expression level of the target gene by correcting the amount of the amplification product of the internal standard gene with the amount of the amplification product of the target gene.
• ⁇ 6> The method according to any one of ⁇ 1> to ⁇ 5>, wherein the internal standard gene is preferably at least one gene selected from the group consisting of PCBP2, RPL30, ARPC2, UBA52, RAB7A, RAC1, ARL8B, RPL32, PCBP1, BZW1, H3F3A, RPS8, FAU, RPL4, EEF1G, and ARF1, more preferably at least one gene selected from ARF1, RPL30, and FAU, even more preferably ARF1 or RPL30, and still more preferably ARF1.
• the internal standard gene is preferably at least one gene selected from the group consisting of PCBP2, RPL30, ARPC2, UBA52, RAB7A, RAC1, ARL8B, RPL32, PCBP1, BZW1, H3F3A, RPS8, FAU, RPL4, EEF1G, and ARF1, more preferably at least one gene selected from ARF1, RPL30, and FAU, even more preferably ARF1 or R
• ⁇ 7> The method according to any one of ⁇ 1> to ⁇ 5>, wherein the internal standard gene is preferably at least one gene selected from the group consisting of PCBP2, RPL30, UBA52, RAB7A, ARL8B, PCBP1, BZW1, RPS8, RPL4, EEF1G, FTL, RPL12, CLIC1, RAB11FIP1, PSAP, and CD63, more preferably at least one gene selected from the group consisting of PCBP2, RPL30, UBA52, RAB7A, ARL8B, PCBP1, BZW1, RPS8, RPL4, and EEF1G, and even more preferably RPL30.
• the internal standard gene is preferably at least one gene selected from the group consisting of PCBP2, RPL30, UBA52, RAB7A, ARL8B, PCBP1, BZW1, RPS8, RPL4, and EEF1G, and even more preferably RPL30.
• ⁇ 8> The method according to any one of ⁇ 1> to ⁇ 7>, wherein the target gene and the internal standard gene are amplified by qPCR, preferably by multiplex qPCR.
• ⁇ 9> A kit for measuring the expression level of a target gene contained in a lipid sample on the skin surface of an infant, which is used in any one of the methods ⁇ 1> to ⁇ 8>, comprising an oligonucleotide that specifically hybridizes to the internal standard gene.
• ⁇ 10> Use of at least one gene selected from the group consisting of genes shown in Table 2 as an internal standard gene in measuring the expression level of a target gene contained in a lipid sample on the skin surface of an infant using PCR.
• the measurement of the expression level of the target gene further comprises correcting the amount of the amplification product of the target gene with the amount of the amplification product of an internal standard gene to calculate the expression level of the target gene.
• ⁇ 12> The measurement of the expression level of the target gene, Amplifying the gene of interest; The use of ⁇ 10>, further comprising: amplifying an internal standard gene; and correcting the amount of the amplification product of the target gene with the amount of the amplification product of the internal standard gene to calculate the expression amount of the target gene.
• ⁇ 13> The measurement of the expression level of the target gene, extracting RNA from surface skin lipid samples taken from the test infants; amplifying the gene of interest based on the extracted RNA; The use of ⁇ 10> further comprising: amplifying an internal standard gene based on the extracted RNA; and correcting the amount of the amplification product of the target gene with the amount of the amplification product of the internal standard gene to calculate the expression level of the target gene.
• ⁇ 14> The measurement of the expression level of the target gene, extracting RNA from surface skin lipid samples taken from the test infants; synthesizing cDNA using the extracted RNA as a template; amplifying the gene of interest using the obtained cDNA as a template; The use of ⁇ 10>, further comprising: amplifying an internal standard gene using the obtained cDNA as a template; and correcting the amount of the amplification product of the target gene with the amount of the amplification product of the internal standard gene to calculate the expression level of the target gene.
• ⁇ 15> The use of any of ⁇ 10> to ⁇ 14>, wherein the internal standard gene is preferably at least one gene selected from the group consisting of PCBP2, RPL30, ARPC2, UBA52, RAB7A, RAC1, ARL8B, RPL32, PCBP1, BZW1, H3F3A, RPS8, FAU, RPL4, EEF1G, and ARF1, more preferably at least one gene selected from ARF1, RPL30, and FAU, even more preferably ARF1 or RPL30, and still more preferably ARF1.
• the internal standard gene is preferably at least one gene selected from the group consisting of PCBP2, RPL30, ARPC2, UBA52, RAB7A, RAC1, ARL8B, RPL32, PCBP1, BZW1, H3F3A, RPS8, FAU, RPL4, EEF1G, and ARF1, more preferably at least one gene selected from ARF1, RPL30, and FAU, even more preferably ARF1 or RPL30
• ⁇ 16> The use of any of ⁇ 10> to ⁇ 14>, wherein the internal standard gene is preferably at least one gene selected from the group consisting of PCBP2, RPL30, UBA52, RAB7A, ARL8B, PCBP1, BZW1, RPS8, RPL4, EEF1G, FTL, RPL12, CLIC1, RAB11FIP1, PSAP, and CD63, more preferably at least one gene selected from the group consisting of PCBP2, RPL30, UBA52, RAB7A, ARL8B, PCBP1, BZW1, RPS8, RPL4, and EEF1G, and even more preferably RPL30.
• the target gene and the internal standard gene are amplified by qPCR, preferably by multiplex qPCR.
• Example 1 Collection of SSL, RNA Extraction, and Comprehensive Gene Expression Analysis 1) Subjects 261 infants, both male and female (ages 0-69 months), were subjects. 2) Collection of SSL Using an oil blotting film (5.0 cm x 8.0 cm, 3M), skin surface lipids (SSL) were collected from the entire face of the subject. To prevent cross-contamination of sebum, the collector's lab gloves were changed for each subject.
• an oil blotting film 5.0 cm x 8.0 cm, 3M
• SSL skin surface lipids
• the oil blotting film from which the sebum was collected was immediately placed in an RNase-free centrifuge tube containing 1 g of molecular sieves (AGC Technoglass, Centrifuge Tube Mini 25 mL) or a 5 mL tube containing 1 g of molecular sieves (Eppendorf DNA LoBind 5 mL, PCR clean), and placed on dry ice or at -20°C, and then stored at -80°C. 3) RNA extraction and sequencing The oil blotting film from which the sebum was collected in 2) above was cut to an appropriate size, and RNA was extracted using QIAzol Lysis Reagent (Qiagen) according to the attached protocol.
• QIAzol Lysis Reagent Qiagen
• RNA reverse transcription was performed at 42°C for 90 minutes using SuperScript VILO cDNA Synthesis kit (Life Technologies Japan, Inc.) to synthesize cDNA.
• the random primers included in the kit were used as primers for the reverse transcription reaction.
• a library containing DNA derived from the 20802 gene was prepared by multiplex PCR. Multiplex PCR was performed using Ion AmpliSeqTranscriptome Human Gene Expression Kit (Life Technologies Japan, Inc.) under the conditions of [99 ° C, 2 minutes ⁇ (99 ° C, 15 seconds ⁇ 62 ° C, 16 minutes) ⁇ 20 cycles ⁇ 4 ° C, Hold].
• the obtained PCR product was purified with Ampure XP (Beckman Coulter, Inc.), and then the buffer was reconstituted, the primer sequence was digested, adapter ligated and purified, and amplified to prepare a library.
• the prepared library was loaded onto an Ion 540 Chip and sequenced using an Ion S5/XL system (Life Technologies Japan, Inc.). Each read sequence obtained by sequencing was genetically mapped to the human genome reference sequence hg19 AmpliSeq Transcriptome ERCC v1 to determine the gene from which each read sequence originated.
• Example 2 Identification of Internal Control Gene Based on the data obtained in Example 1, the following analysis was carried out. Genes with a detection rate of 70% or more were analyzed (Gene Threshold), and normalization was performed using the RLE method.
• the RLE method is a method of calculating a coefficient called the Size factor and normalizing the read count data of each sample using this coefficient. Genes that show a certain percentage of stable expression in the normalized corrected data were selected. Gene expression stability was evaluated using CV Z-score. As a result of extracting genes with higher expression stability than those with a CV Z-score of -1 or less in the corrected data, 1636 types of genes were extracted.
• genes that have been reported as housekeeping genes are marked with * (see Genome Analysis, 2003, 19(7):362-365). These 30 genes were shown to be more stably expressed in RNA derived from infant SSL, and were shown to be usable as internal standard genes when analyzing gene expression in infant SSL as the analysis sample.
• Example 3 Confirmation of correlation between real-time PCR data and NGS data 1 Sebum was collected from 15 infants (3-8 months old), and RNA was extracted and purified. SSL-RNA and RT Mix were mixed and reverse-transcribed, and a library containing DNA derived from the 20802 gene was prepared from the resulting reverse transcription reaction product by multiplex PCR (1st round PCR, Ion AmpliSeqTranscriptome Human Gene Expression Kit). The resulting 1st round PCR product was purified using Ampure XP. The resulting purified product was divided into one for next-generation sequencer analysis and one for real-time PCR measurement.
• One of the purified products was subjected to buffer reconstitution, digestion of primer sequences, adapter ligation, purification, and amplification, and comprehensive gene expression information was obtained by next-generation sequencer analysis, and was used as NGS data.
• the other 1st round PCR purified product was used as a template for real-time PCR measurement, and real-time PCR was performed using a primer (nested primer) designed inside the 1st round PCR amplification region.
• the Ct values of 14 genes selected as the target gene and ARF1 as the internal control gene (control gene) were obtained.
• Example 4 Confirmation of correlation between real-time PCR data and NGS data 2 Sebum was collected from 16 infants (3-24 months old), and RNA was extracted and purified. SSL-RNA was mixed with RT Mix and reverse transcription was performed, and a library containing DNA derived from the 20802 gene was prepared from the obtained reverse transcription reaction product by multiplex PCR (1st round PCR, Ion AmpliSeqTranscriptome Human Gene Expression Kit). The obtained 1st round PCR product was purified using Ampure XP. The obtained purified product was divided into one for next-generation sequencer analysis and one for real-time PCR measurement.
• One of the purified products was subjected to buffer reconstitution, digestion of primer sequences, adapter ligation, purification, and amplification, and comprehensive gene expression information was obtained by next-generation sequencer analysis, and was used as NGS data.
• the other 1st round PCR purified product was used as a template for real-time PCR measurement, and real-time PCR was performed using a primer (nested primer) designed inside the 1st round PCR amplification region.
• the Ct values of 14 genes selected as target genes and 6 genes (ARF1, FAU, RPL30, GAPDH, ACTB, RPLP0) as internal standard genes (control genes) were obtained.
• Ct value Ct_ target gene -Ct_ control gene
• ⁇ ARF1 ⁇ FAU
• ⁇ RPL30 ⁇ GAPDH
• ⁇ ACTB ⁇ RPLP0
• data correlation was confirmed between the qPCR data and the NGS data (Log2 (normalized count+1)).

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