JP7498738B2 - Data homogenization method for sebum RNA information - Google Patents

Description

The present invention relates to a data homogenization method for analyzing RNA expression information obtained from lipids on the surface of human skin.

In recent years, technology has been developed to investigate the current and future physiological state of the human body by analyzing nucleic acids such as DNA and RNA in biological samples. Analysis using nucleic acids has the advantages of being able to obtain a wealth of information in a single analysis because comprehensive analysis methods have been established, and that it is easy to functionally link the analysis results based on the many research reports on single nucleotide polymorphisms and RNA functions. Nucleic acids derived from living organisms can be extracted from body fluids such as blood, secretions, tissues, etc., but it has recently been reported that RNA contained in skin surface lipids (SSL) can be used as a sample for analyzing living organisms, and that marker genes for the epidermis, sweat glands, hair follicles, and sebaceous glands can be detected from SSL (Patent Document 1).

RNA sequencing (RNA-Seq) analysis, which directly quantifies RNA sequences expressed in cells, is currently attracting attention as an analytical method that enables the detection of low-expressing genes that are difficult to quantify using microarrays that use signal intensity ratios, and allows for the acquisition of highly accurate expression profiles. In gene expression analysis, the concentration and/or relative or absolute amount of a specific RNA in a sample is determined, and the specific RNA is quantified (quantified); in this case, a highly accurate method is required that allows good comparison of RNA expression data in samples with different RNA contents derived from two or more individuals.

International Publication No. 2018/008319

The present invention relates to providing a data homogenization method for improving analytical accuracy when analyzing RNA expression information obtained from human SSL.

The inventors have found that by using RNA contained in human SSL as a template and the amount of amplification product obtained by RT-PCR targeting the human 18S rRNA gene as an index, it is possible to indirectly quantify the minute amount of human-derived RNA in SSL, which was previously difficult to directly quantify due to the presence of a large amount of RNA derived from resident skin bacteria, and that by using a predetermined amount of human-derived RNA based on the amount of human-derived RNA thus quantified, RNA expression information obtained from SSL can be analyzed with high accuracy.

That is, the present invention relates to the following.
A data homogenization method for analyzing RNA expression information obtained from skin surface lipids collected from a plurality of subjects as biological samples, comprising the steps of:
performing a nucleic acid amplification reaction using RNA contained in each of the biological samples as a template and the human 18S rRNA gene as a target, and quantifying the amount of human-derived RNA contained in each of the biological samples using the amount of the obtained amplification product of the human 18S rRNA gene as an index; and regulating the amount of human-derived RNA to be subjected to the analysis between the biological samples based on the amount of the quantified human-derived RNA.
The method includes:

According to the present invention, when analyzing RNA expression information obtained from multiple human SSL samples, it is possible to reduce the sample-dependent variation in RNA expression data caused by the variation in the amount of human-derived RNA contained in the SSL, thereby improving the analytical accuracy.

(A) Heat map of sequence data when only the volume of human RNA-containing liquid was made uniform between SSL samples (control). (B) Heat map of sequence data when the volume of human RNA-containing liquid and the human RNA concentration in the liquid were made uniform between SSL samples (standardized concentration).

All patents, non-patent literature, and other publications cited herein are hereby incorporated by reference in their entirety.

In this specification, "skin surface lipids (SSL)" refers to the fat-soluble fraction present on the surface of the skin, and is sometimes called sebum. In general, SSL mainly contains secretions from exocrine glands such as sebaceous glands in the skin, and exists on the skin surface in the form of a thin layer that covers the skin surface.

In this specification, unless otherwise specified, "skin" is a general term for an area including tissues such as the stratum corneum, epidermis, dermis, hair follicles, sweat glands, sebaceous glands, and other glands.

In this specification, the "human 18S rRNA gene" refers to a gene that encodes 18S rRNA, a component of the 40S subunit of the human ribosome. Preferably, the human 18S rRNA gene is a gene consisting of the base sequence shown in SEQ ID NO: 1. Hereinafter, the human 18S rRNA gene may be simply referred to as the 18S rRNA gene.

In this specification, "data homogenization" refers to reducing or suppressing the variation in sample-dependent RNA expression data caused by the variation in the amount of human-derived RNA contained in the SSL.

In one aspect, the present invention provides a data homogenization method for analyzing RNA expression information obtained from biological samples of surface skin lipids collected from multiple subjects. The method of the present invention includes: performing a nucleic acid amplification reaction using RNA contained in each biological sample as a template and the human 18S rRNA gene as a target; quantifying the amount of human-derived RNA contained in each biological sample using the amount of the obtained amplification product of the human 18S rRNA gene as an index; and aligning the amount of human-derived RNA to be used in the analysis between each biological sample based on the quantified amount of human-derived RNA.

The subject in the method of the present invention is a human and has SSL on the skin. For example, the subject may be a human who needs or desires analysis of his/her nucleic acid. Alternatively, the subject may be a human who needs or desires gene expression analysis in the skin, or detection of a current or future physiological state of the skin or a site other than the skin using said gene expression analysis.

The skin surface lipids (SSL) collected from a subject contain RNA expressed in the skin cells of the subject, preferably RNA expressed in any of the epidermis, sebaceous glands, hair follicles, sweat glands, and dermis of the subject, more preferably RNA expressed in any of the epidermis, sebaceous glands, hair follicles, and sweat glands of the subject (see Patent Document 1). Therefore, in the method of the present invention, the RNA contained in the SSL is preferably RNA derived from at least one site selected from the epidermis, sebaceous glands, hair follicles, sweat glands, and dermis of the subject, more preferably RNA derived from at least one site selected from the epidermis, sebaceous glands, hair follicles, and sweat glands, even more preferably RNA derived from at least two sites selected from the epidermis, sebaceous glands, hair follicles, and sweat glands, and even more preferably RNA derived from at least three sites selected from the epidermis, sebaceous glands, hair follicles, and sweat glands.

In the method of the present invention, the RNA contained in the SSL may include mRNA, tRNA, rRNA, small RNA (e.g., microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), etc.), long intelligent non-coding (linc) RNA, etc. The SSL in the method of the present invention requires rRNA as an essential component and includes one or more of the other RNAs listed above.

Any means used for recovering or removing SSL from the skin can be used to collect SSL from the skin of a subject. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument for scraping SSL from the skin, as described below, 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. In order to improve the adsorption of SSL, an SSL absorbent material that has been previously impregnated with a highly lipid-soluble solvent may be used. On the other hand, if the SSL absorbent material contains a highly water-soluble solvent or moisture, it is not preferable because the adsorption of SSL is inhibited. It is preferable to use the SSL absorbent material in a dry state. The area of skin from which SSL is collected is not particularly limited, and may be any area of the body, such as the head, face, neck, trunk, hands, or feet, with areas that secrete a lot of sebum, such as the skin of the face, being preferred.

The SSL collected from the subject may be used immediately in the RNA extraction step described below, or may be stored for a certain period of time. The collected SSL is preferably stored under low temperature conditions as soon as possible after collection in order to minimize the 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 of storage of the RNA-containing SSL 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.

In the method of the present invention, a nucleic acid amplification reaction is carried out using the RNA contained in the SSL as a template and targeting the 18S rRNA gene. Preferably, the RNA extracted from the SSL is converted to cDNA by reverse transcription, and the 18S rRNA gene is then amplified using the cDNA.

RNA can be extracted from SSL using methods commonly used for extracting or purifying RNA from biological samples, such as the phenol/chloroform method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, or methods using columns such as TRIzol (registered trademark), RNeasy (registered trademark), or QIAzol (registered trademark), methods using special silica-coated magnetic particles, methods using Solid Phase Reversible Immobilization magnetic particles, and extraction using commercially available RNA extraction reagents such as ISOGEN.

For reverse transcription, a random primer may be used, but from the viewpoint that the reverse transcription reaction and the subsequent gene amplification reaction can be performed continuously in one tube, it is preferable to use a primer targeting the 18S rRNA gene. For the reverse transcription, a general reverse transcriptase or reverse transcription reagent kit can be used. Preferably, 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 a commercially available reverse transcriptase or reverse transcription reagent kit, such as the PrimeScript (registered trademark) Reverse Transcriptase series (Takara Bio Inc.), the SuperScript (registered trademark) Reverse Transcriptase series (Thermo Fisher Scientific K.K.), and the like. SuperScript (registered trademark) III Reverse Transcriptase, SuperScript (registered trademark) VILO cDNA Synthesis kit (both from Thermo Fisher Scientific K.K.), etc. are preferably used.
The temperature of the extension reaction in the reverse transcription is preferably adjusted to 41 to 50°C, more preferably 42.5 to 49°C, and even more preferably 42 to 48°C, while the reaction time is preferably adjusted to 30 minutes or more, more preferably 60 minutes or more, and preferably 120 minutes or less.

Amplification of the 18S rRNA gene using the cDNA obtained by reverse transcription as a template can be carried out according to the PCR procedure commonly used in the field. Examples of PCR techniques include conventional PCR, real-time PCR (also called quantitative PCR (qPCR)), digital PCR, etc.
For detection of the amplification product, known means capable of specifically recognizing the amplification product can be used.
In the present invention, it is preferable to use real-time PCR, which monitors and analyzes the amount of PCR amplified product in real time, as the PCR technique from the viewpoints of speed, simplicity, and quantitativeness. Methods for detecting the amplified product in real-time PCR include methods commonly used in the art, 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.

The TaqMan probe method is a method in which a target sequence-specific oligonucleotide (TaqMan probe) modified at the 5' end with a fluorescent substance (FAM, etc.) and at the 3' end with a quencher substance (TAMRA, etc.) is made to coexist in a PCR reaction system. In this method, the TaqMan probe hybridizes specifically to the template DNA in the annealing step of the PCR reaction, but in this state, the quencher substance is present on the probe, so that the generation of fluorescence is suppressed even when the excitation light is irradiated. Next, during the extension reaction step, the 5'→3' exonuclease activity of Taq DNA polymerase degrades the TaqMan probe hybridized to the template, and the fluorescent substance is released from the probe, and the inhibition by the quencher substance is released, causing the probe to emit fluorescence. The amount of amplified product can be monitored by detecting the fluorescence.

The PCR conditions are not particularly limited, and the optimal conditions may be determined for each PCR. For example, the following conditions may be mentioned.
1) Heat denaturation of double-stranded DNA to single-stranded DNA: the temperature is preferably 94 to 99° C., and the time is 3 to 60 seconds.
2) 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 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.
Here, the annealing and the DNA extension reaction can be carried out simultaneously without being separated. In this case, the PCR conditions are, for example, as follows.
1) Heat denaturation of double-stranded DNA to single-stranded DNA: the temperature is preferably 94 to 99° C., and the time is 3 to 60 seconds.
2) Annealing and DNA extension reaction: 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 more, preferably 10 seconds or more, 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.
The above reactions 1) and 2) are regarded as one cycle, and this cycle may be carried out usually for 30 cycles or more, preferably 35 cycles or more, and usually for 50 cycles or less, preferably for 45 cycles or less.

Reverse transcription and PCR at the temperatures and times described above can be performed using a thermal cycler generally used for PCR or a device dedicated to real-time PCR 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. For example, 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. On the other hand, the chain length of the PCR amplification product is lower limited to 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 90 bp or more. In addition, the chain length of the PCR amplification product is preferably 30 to 1000 bp, more preferably 50 to 700 bp, and even more preferably 90 to 500 bp.

The primers or probes used in the above measurement, i.e., primers for specifically recognizing and amplifying the 18S rRNA gene, or probes for specifically detecting the 18S rRNA gene, fall under this category, and can be designed based on the base sequence constituting the 18S rRNA gene. Here, "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 18S rRNA gene is amplified.
Specifically, DNA consisting of a base sequence constituting the 18S rRNA gene or an oligonucleotide containing a certain number of nucleotides complementary to its complementary strand can be used. Here, the term "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. In addition, "complementary" does not necessarily mean that the sequence is completely complementary in the certain number of consecutive nucleotide regions, but rather that the sequence has an identity of preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. The identity of the base sequence can be determined by an algorithm such as BLAST.
When used as a primer, such an oligonucleotide may be capable of specific annealing and chain elongation, and may generally have a chain length of, for example, 10 bases or more, preferably 15 bases or more, more preferably 20 bases or more, and, for example, 100 bases or less, preferably 50 bases or less, more preferably 35 bases or less. When used as a probe, it may be capable of specific hybridization, and may have at least a part or all of the sequence of DNA (or its complementary strand) consisting of the base sequence constituting the 18S rRNA gene, and may have a chain length of, for example, 10 bases or more, preferably 15 bases or more, and, for example, 100 bases or less, preferably 50 bases or less, more preferably 25 bases or less.
In this case, the "oligonucleotide" may be DNA or RNA, and may be synthetic or natural. Furthermore, the probe used in hybridization is usually labeled.
In one example, such a primer may be a primer consisting of a nucleotide sequence shown in any one of SEQ ID NOs: 2 to 7. It is preferable to amplify the 18S rRNA gene using a primer pair consisting of a primer consisting of a nucleotide sequence shown in SEQ ID NO: 2 and a primer consisting of a nucleotide sequence shown in SEQ ID NO: 3, a primer pair consisting of a primer consisting of a primer consisting of a nucleotide sequence shown in SEQ ID NO: 4 and a primer consisting of a nucleotide sequence shown in SEQ ID NO: 5, or a primer pair consisting of a primer consisting of a nucleotide sequence shown in SEQ ID NO: 6 and a primer consisting of a nucleotide sequence shown in SEQ ID NO: 7, and it is more preferable to amplify the 18S rRNA gene using a primer pair consisting of a primer consisting of a nucleotide sequence shown in SEQ ID NO: 4 and a primer consisting of a nucleotide sequence shown in SEQ ID NO: 5.
In another example, the amplification of the 18S rRNA gene is a partial sequence contained in the sequence shown in SEQ ID NO: 1, preferably a partial sequence of the 18S rRNA gene shown in any of SEQ ID NOs: 8 to 10, and more preferably a partial sequence of the 18S rRNA gene shown in SEQ ID NO: 9.

In the method of the present invention, cDNA synthesis by reverse transcription (RT) and PCR may be performed in one step or two steps. Among these, one-step RT-PCR is preferred because it allows a series of RT and PCR reactions to be performed continuously in a single tube, is easy to use, and prevents contamination between the RT and PCR operations. For one-step RT-PCR, commercially available kits can be used, such as Power SYBR Green RNA-to-CT 1-Step Kit (Thermo Fisher Scientific K.K.), PrimeScript (registered trademark) One Step RT-PCR Kit Ver. 2 (Takara Bio Inc.), etc.

In the method of the present invention, a nucleic acid amplification reaction is carried out using RNA contained in SSL as a template and the 18S rRNA gene as a target, and the amount of human-derived RNA contained in SSL is quantified using the amount of the obtained amplification product of the 18S rRNA gene as an index. Here, the amount of human-derived RNA contained in SSL refers to the concentration of human-derived RNA in SSL, or the relative or absolute content of human-derived RNA in SSL. The amount of human-derived RNA contained in SSL can be calculated, for example, according to an absolute quantification method commonly used in the field.
In the absolute quantification method, a dilution series prepared from a standard sample with a known concentration of human-derived RNA is used as a template, and a nucleic acid amplification reaction is performed with the 18S rRNA gene as a target to determine the Ct value. Here, the Ct value means the number of cycles (threshold cycle) when the amount of PCR amplified product reaches a certain amount. In PCR, the amount of amplified product theoretically increases by two times per cycle, so when the human-derived RNA 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. Next, a nucleic acid amplification reaction is performed with the 18S rRNA gene as a target under the same conditions for the SSL sample to determine the Ct value. The concentration of human-derived RNA contained in the SSL sample can be calculated from this Ct value and the calibration curve. In addition, the human-derived RNA content of the sample can be calculated by multiplying the human-derived RNA concentration of the sample by the volume of the sample. The standard sample may be a commercially available total RNA solution of human-derived cells with a known concentration. Examples of such human-derived cells include normal human epidermal keratinocytes (NHEK) and human cervical cancer cells (HeLa), and total RNA solutions of these human-derived cells are commercially available. The concentration range of the dilution series of the standard sample is preferably 100 ag/μL or more, more preferably 1 fg/μL or more, while it is preferably 100 ng/μL or less, more preferably 10 ng/μL or less. The concentration range is preferably 100 ag/μL to 100 ng/μL, more preferably 1 fg/μL to 10 ng/μL. Within the concentration range, a dilution series of about 3 to 10 steps may be appropriately prepared.

In the method of the present invention, based on the thus quantified amount of human-derived RNA in the SSL collected from multiple subjects, the amount of human-derived RNA used in the analysis of RNA expression information is adjusted to a predetermined amount so as to be uniform among the subjects. Here, adjusting to a predetermined amount means adjusting the concentration of the human-derived RNA solution used in the analysis to a predetermined concentration range, and specifically, 10 fg/μL to 50 pg/μL is preferable, 50 fg/μL to 20 pg/μL is more preferable, and 0.1 pg/μL to 10 pg/μL is even more preferable.

The method of analyzing RNA expression information obtained from SSL is not particularly limited, but for example, the RNA used for analysis can be converted to cDNA by reverse transcription, and then the cDNA or its amplified product can be measured. Means for measuring the expression level include DNA chips, DNA microarrays, RNA-Seq, etc., and preferably RNA-Seq. When microarray analysis is used, the amount of RNA expression is quantified by the signal intensity ratio, and when RNA-seq analysis is used, it is quantified by the number of sequence reads mapped to the genome (read count value).

According to the method of the present invention, the amount of human-derived RNA contained in SSL can be indirectly quantified using the expression level of the human 18S rRNA gene as an index, and by using a predetermined amount of human-derived RNA, the variation in sample-dependent RNA expression data caused by the variation in the amount of human-derived RNA contained in SSL can be reduced, the data can be homogenized, and the RNA expression information obtained from SSL can be analyzed with high accuracy. For example, when analyzing RNA expression information for multiple SSLs by RNA-seq, the variation between samples in the concentration of the cDNA library used for sequencing, the gene detection rate of the sequence data, and the number of mapped reads, as well as the variation in the distribution of the sequence data, can be reduced to homogenize the data, making it possible to improve the correlation coefficient between samples and increase the number of genes to be analyzed.

As exemplary embodiments of the present invention, the following substances, manufacturing methods, uses, methods, etc. are further disclosed in this specification. However, the present invention is not limited to these embodiments.

[1] A data homogenization method for analyzing RNA expression information obtained from skin surface lipids collected from multiple subjects as biological samples, comprising the steps of:
performing a nucleic acid amplification reaction using RNA contained in each of the biological samples as a template and the human 18S rRNA gene as a target, and quantifying the amount of human-derived RNA contained in each of the biological samples using the amount of the obtained amplification product of the human 18S rRNA gene as an index; and regulating the amount of human-derived RNA to be subjected to the analysis between the biological samples based on the amount of the quantified human-derived RNA.
The method includes:
[2] The method according to [1], wherein a primer pair consisting of a primer having the base sequence of SEQ ID NO: 2 and a primer having the base sequence of SEQ ID NO: 3, a primer pair consisting of a primer having the base sequence of SEQ ID NO: 4 and a primer having the base sequence of SEQ ID NO: 5, or a primer pair consisting of a primer having the base sequence of SEQ ID NO: 6 and a primer having the base sequence of SEQ ID NO: 7 is used in the nucleic acid amplification reaction targeting the human 18S rRNA gene.
[3] The method according to [1] or [2], wherein a primer pair consisting of a primer having the base sequence of SEQ ID NO: 4 and a primer having the base sequence of SEQ ID NO: 5 is used in the nucleic acid amplification reaction targeting the human 18S rRNA gene.
[4] The method according to [1], wherein the nucleic acid amplification reaction targeting the human 18S rRNA gene is an amplification reaction of a partial sequence of the 18S rRNA gene shown in any one of SEQ ID NOs: 8 to 10.
[5] The method according to [1] or [4], wherein the nucleic acid amplification reaction targeting the human 18S rRNA gene is an amplification reaction of a partial sequence of the 18S rRNA gene shown in SEQ ID NO:9.
[6] The method according to any one of [1] to [5], wherein the nucleic acid amplification reaction is preferably carried out by RT-PCR, more preferably by real-time RT-PCR.
[7] The method according to any one of [1] to [6], wherein the nucleic acid amplification reaction is carried out by a one-step RT-PCR method, more preferably a real-time one-step RT-PCR method.
[8] The method according to any one of [1] to [7], wherein the quantitative range of the amount of human-derived RNA is preferably 100 ag/μL to 100 ng/μL, more preferably 1 fg/μL to 10 ng/μL.
[9] The method according to any one of [1] to [8], wherein the amount of human-derived RNA to be subjected to the analysis is adjusted to a concentration range of preferably 10 fg/μL to 50 pg/μL, more preferably 50 fg/μL to 20 pg/μL, and even more preferably 0.1 pg/μL to 10 pg/μL.
[10] The method according to any one of [1] to [9], further comprising carrying out a nucleic acid amplification reaction using a standard sample with a known amount of human-derived RNA as a template and the human 18S rRNA gene as a target, and creating a calibration curve based on the amount of the obtained amplification product of the human 18S rRNA gene.
[11] The method according to [10], wherein the amount of human-derived RNA contained in the biological sample is quantified from the amount of the amplification product of the human 18S rRNA gene obtained from the biological sample and the calibration curve.
[12] The method according to any one of [1] to [11], wherein the analysis is performed by RNA-Seq.

The present invention will be described in more detail below based on test examples and examples, but the present invention is not limited thereto.

Test Example 1 Collection of SSL, RNA Extraction, and Comprehensive Gene Expression Analysis 1) Subjects Eighteen healthy women (aged 20 to 60 years) served as 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. The oil blotting film from which the sebum was collected was immediately placed in a 20 mL glass vial containing 1 g of molecular sieves (dry-heat treated) RNase-free or a 5 mL tube containing 1 g of molecular sieves (Eppendorf DNA LoBind 5 mL, PCR clean), placed on dry ice, 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. Based on the extracted RNA, reverse transcription was performed at 42°C for 90 minutes using SuperScript VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.) to synthesize cDNA. The random primer included in the kit was used as a primer for the reverse transcription reaction. Of the 5 μL of the obtained cDNA, 2.5 μL was used for the following sequence analysis, and the remaining 2.5 μL was used for quantification by real-time PCR in Test Example 2.
From the obtained cDNA, 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 buffer reconstitution, digestion of primer sequences, adapter ligation and purification, and amplification were performed 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.

Test Example 2: Examination of the relationship between gene expression levels measured by sequence analysis and human RNA concentrations based on real-time PCR quantitative values The relationship between the gene expression levels measured by sequence analysis and the human RNA concentrations derived from the gene expression levels measured by real-time PCR was confirmed as follows.
As the gene expression level measured by sequence analysis, the expression level of β-actin (ACTB), a common internal standard gene, was used from the sequence data obtained in Test Example 1, 3) (Table 1). Meanwhile, the human RNA concentration was measured based on the quantitative value (Ct value) of real-time PCR by absolute quantification method using ACTB as the target gene. As a target template for creating a calibration curve, a dilution series (10, 1, 0.1, 0.01, 0.001 ng/μL) was prepared from a normal human epidermal keratinocyte (NHEK) RNA solution (Takara Bio, Inc., 100 ng/μL) with a known concentration, and reverse transcription was performed at 42° C. for 90 minutes using SuperScript VILO cDNA Synthesis kit (Life Technologies Japan, Inc.) in the same manner as in Test Example 1, 3) to synthesize cDNA. The random primer attached to the kit was used as the primer for the reverse transcription reaction. For the SSL sample, the reverse transcription product obtained in Test Example 1, 3) was used as the template for real-time PCR measurement. Real-time PCR was performed under the following conditions using a Taqman Probe for human ACTB (Hs01060665_g1, Thermo Fisher Scientific K.K.), and the Ct values of ACTB, selected as the target gene, were obtained for each of the NHEK samples and SSL samples (Table 2).
(Real-time PCR conditions)
1. Reagent: TaqMan ^™ Fast Universal PCR Master Mix (2x) (Thermo Fisher Scientific K.K.)
2. Equipment: 7500 Fast Real-Time PCR System (Thermo Fisher Scientific K.K.)
3. Reaction plate: MicroAmp ^™ Fast Optical 96-Well Reaction Plate, 0.1 mL (Thermo Fisher Scientific K.K.)
Reaction volume: 20 μL (2×Fast Master Mix: 10 μL, 20×Taqman probe: 1 μL, H ₂ O: 4 μL, 10-fold diluted SSL or NHEK reverse transcription product: 5 μL)
Thermal cycle: [95°C, 20 seconds → (95°C, 3 seconds → 60°C, 1 minute) × 40 cycles]
The human RNA concentration of the SSL sample was calculated from the obtained Ct value and used as real-time PCR quantitative data (Table 3). As a result of checking the data correlation between the real-time PCR quantitative data and the sequence data (Read count), a positive correlation was confirmed (Pearson's correlation coefficient: 0.764). This result made it clear that the RNA concentration of the sample was reflected in the gene expression level obtained from the sequence analysis of the RNA. In addition, since some SSL samples showed Ct values close to the detection limit, it was shown that it is necessary to select a gene with higher expression as the target gene.

Test Example 3 Selection of target genes used to create a calibration curve In order to select the optimal target gene for quantifying the human RNA concentration of SSL samples, the expression levels of the 18S rRNA gene and common internal standard genes (ACTB, GAPDH, RPLP0) were quantified by real-time PCR quantification using an absolute quantification method, and the Ct values were compared. The sequences of the primers used in this test example are shown in Table 4. The reference column in Table 4 indicates the literature in which such sequences are described, with literature 1 being Proc Natl Acad Sci USA., vol.106, No.9, 3384-3389 (2009), literature 2 being http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_2804.pdf, and literature 3 being Virol J., vol.9, No.230 (2012).
For the target template, a dilution series (10, 1, 0.1, 0.01, 0.001 ng/μL) was prepared from the NHEK RNA solution (NHEK RNA solution), and the RT-PCR reaction was performed using a one-step RT-PCR method in which reverse transcription and PCR reactions are performed consecutively in one tube.
(RT-PCR conditions)
1. Reagent: Power SYBR Green RNA-to-CT 1-Step Kit (Thermo Fisher Scientific K.K.)
2. Equipment: 7500 Fast Real-Time PCR System (Thermo Fisher Scientific K.K.)
3. Reaction plate: MicroAmp ^™ Fast Optical 96-Well Reaction Plate, 0.1 mL (Thermo Fisher Scientific K.K.)
Reaction volume: 10 μL (Fwd_Primer (100 μM): 0.1 μL, Rev_Primer (100 μM): 0.1 μL, RT Enzyme Mix (125×): 0.08 μL, RT-PCR Mix (2×): 5.0 μL, H ₂ O: 3.72 μL, NHEK RNA solution: 1 μL)
Thermal cycle: [48°C, 30 min → 95°C, 10 min → (95°C, 15 sec → 60°C, 1 min) × 40 cycles]

The Ct values of each target gene are shown in Table 5. Compared to the common internal standard genes ACTB, GAPDH, and RPLP0, 18S rRNA had a lower Ct value regardless of which primer pair was used. This means that the expression level of 18S rRNA is high, and if it is selected as the target gene for creating a standard curve, the concentration range of the standard curve can be set to a lower range. Therefore, when indirectly quantifying the human RNA concentration in a very small amount of SSL, 18S rRNA is a target gene that is more suitable for creating a standard curve.

Example 1 Analysis of RNA Expression Information Based on Quantitative Values of Human RNA Concentration in SSL 1) Subjects Thirty healthy women (aged 20 to 60 years) served as 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. The oil blotting film from which the sebum was collected was immediately placed in a 20 mL glass vial containing 1 g of molecular sieves (dry-heat treated) RNase-free or a 5 mL tube containing 1 g of molecular sieves (Eppendorf DNA LoBind 5 mL, PCR clean), placed on dry ice, and then stored at -80°C.

3) RNA Extraction The oil blotting film from which the sebum had been collected in 2) above was cut to an appropriate size, and RNA was extracted using QIAzol Lysis Reagent (Qiagen) according to the attached protocol to prepare an aqueous solution containing RNA.

4) Calculation of human RNA concentration in SSL To calculate the human RNA concentration in SSL, real-time PCR (one-step RT-PCR) was performed with 18S rRNA as the target gene. As primers, 18S_02_for (Fwd_Primer) (SEQ ID NO: 4) and 18S_02_rev (Rev_Primer) (SEQ ID NO: 5) targeting the 18S rRNA gene shown in Table 4 above were used. As the target template for creating a calibration curve, a dilution series (10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001 ng/μL) was prepared from human cervical cancer-derived (HeLa) cell RNA (Takara Bio, Inc., 1 μg/μL) with a known concentration and used (HeLa RNA solution). As the target template for calculating the human RNA concentration, an aqueous solution containing RNA prepared in 3) above for 30 people was used (SSL-derived RNA aqueous solution).
(RT-PCR conditions)
1. Reagent: Power SYBR Green RNA-to-CT 1-Step Kit (Thermo Fisher Scientific K.K.)
2. Equipment: QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific K.K.)
3. Reaction plate: MicroAmp ^™ Fast Optical 96-Well Reaction Plate, 0.1 mL (Thermo Fisher Scientific K.K.)
Reaction volume: 10 μL (Fwd_Primer (100 μM): 0.1 μL, Rev_Primer (100 μM): 0.1 μL, RT Enzyme Mix (125×): 0.08 μL, RT-PCR Mix (2×): 5.0 μL, H ₂ O: 3.72 μL, SSL-derived RNA aqueous solution or HeLa RNA solution: 1 μL)
Thermal cycle: [48°C, 30 min → 95°C, 10 min → (95°C, 15 sec → 60°C, 1 min) × 40 cycles]

The Ct values of the target gene, 18S rRNA gene, obtained for each of the HeLa RNA solution and the SSL-derived RNA aqueous solution are shown in Table 6. The human RNA concentration in the SSL-derived RNA aqueous solution calculated based on the calibration curve created from the Ct values of the HeLa RNA solution is shown in Table 7.

5) Standardization of human RNA concentration in SSL-derived RNA aqueous solution Based on the human RNA concentration obtained in 4) above, the human RNA concentration of each SSL-derived RNA aqueous solution was adjusted as follows. Human RNA concentrations of 5 pg/μL or more were diluted to 5 pg/μL. Human RNA concentrations of 3 pg/μL or more and less than 5 pg/μL were diluted as necessary to adjust to within the range of 3 to 3.5 pg/μL. Human RNA concentrations of less than 3 pg/μL were diluted as necessary to adjust to within the range of 0.1 to 0.7 pg/μL. UltraPure DNase/RNase-Free Distilled Water (Thermo Fisher Scientific K.K.) was used for dilution.

6) Sequencing Sequencing was performed on the aqueous solution of SSL-derived RNA obtained in 3) above. Sequencing was performed in two ways: using the aqueous solution obtained in 3) above as is (control) and using an aqueous solution with the human RNA concentration adjusted in 5) above (uniform concentration). In both cases, 1.5 μL of the aqueous RNA solution was subjected to sequencing.
cDNA synthesis was carried out by reverse transcription at 42° C. for 90 minutes using a SuperScript VILO cDNA Synthesis kit (Life Technologies Japan, Inc.) The random primers included in the kit were used as primers for the reverse transcription reaction.
From the obtained cDNA, 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 buffer reconstitution, digestion of primer sequences, adapter ligation and purification, and amplification were performed 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 Comparison of sequence data distribution The distribution of sequence data obtained in Example 1, obtained by aligning the human RNA concentration of the SSL-derived RNA aqueous solution (hereinafter referred to as SSL sample), was compared with that of sequence data (control) obtained by aligning only the volume without aligning the human RNA concentration. Table 8 shows the cDNA library concentration used in the sequencing of each SSL sample, the gene detection rate of the sequence data (the ratio of the number of genes detected to the number of genes that can be analyzed by sequence analysis), and the number of mapped reads. When the variance of each value was calculated, the variance of each value was lower when the human RNA concentration was aligned. In other words, it was revealed that the variance of each value between samples was reduced and the data was homogenized by aligning the human RNA concentration.

Next, the expression distributions of all samples and all genes were output using the DESeq2 package used for expression analysis of sebum RNA sequence data, and the variances were compared (Table 9). In addition, the median of the expression distribution of all genes in each sample was output, and the variances were compared (Table 9). As a result, it was revealed that for each item, variance was reduced by analyzing with the same human RNA concentration, and the data was homogenized.

Furthermore, the DESeq2 package was used to normalize the data, calculate the Spearman correlation coefficient between samples, and output a heat map (Figure 1). In addition, the number of samples that cleared the cutoff for low sequence quality and the number of genes that cleared the cutoff for low expression genes were calculated (Table 10). As a result, it was confirmed that the correlation was improved by standardizing the concentration compared to the control. It was also revealed that the number of genes analyzed increased by standardizing the concentration.

Claims (7)

A data homogenization method for analyzing RNA expression information obtained from skin surface lipids collected from a plurality of subjects as biological samples, comprising the steps of:
performing a nucleic acid amplification reaction using RNA contained in each biological sample as a template and the human 18S rRNA gene as a target, and quantifying the amount of human-derived RNA contained in each biological sample using the amount of the obtained amplification product of the human 18S rRNA gene as an index; and aligning the amount of human-derived RNA to be used for the analysis between each biological sample based on the amount of human-derived RNA thus quantified;
The method includes: The method according to claim 1, wherein a primer pair consisting of a primer having the base sequence of SEQ ID NO: 2 and a primer having the base sequence of SEQ ID NO: 3, a primer pair consisting of a primer having the base sequence of SEQ ID NO: 4 and a primer having the base sequence of SEQ ID NO: 5, or a primer pair consisting of a primer having the base sequence of SEQ ID NO: 6 and a primer having the base sequence of SEQ ID NO: 7 is used in the nucleic acid amplification reaction targeting the human 18S rRNA gene. The method according to claim 1 or 2, wherein a primer pair consisting of a primer having the base sequence of SEQ ID NO: 4 and a primer having the base sequence of SEQ ID NO: 5 is used in the nucleic acid amplification reaction targeting the human 18S rRNA gene. The method according to any one of claims 1 to 3, wherein the nucleic acid amplification reaction is carried out by RT-PCR. The method according to any one of claims 1 to 4, wherein the nucleic acid amplification reaction is carried out by a one-step RT-PCR method. The method according to any one of claims 1 to 5, wherein the quantitative range of the amount of human-derived RNA is 100 ag/μL to 100 ng/μL. The method according to any one of claims 1 to 6, wherein the amount of human-derived RNA to be subjected to the analysis is adjusted to a concentration range of 10 fg/μL to 50 pg/μL.