WO2016084848A1 - 小型rnaの発現量の補正方法及び装置 - Google Patents
小型rnaの発現量の補正方法及び装置 Download PDFInfo
- Publication number
- WO2016084848A1 WO2016084848A1 PCT/JP2015/083079 JP2015083079W WO2016084848A1 WO 2016084848 A1 WO2016084848 A1 WO 2016084848A1 JP 2015083079 W JP2015083079 W JP 2015083079W WO 2016084848 A1 WO2016084848 A1 WO 2016084848A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- value
- small rna
- standard substance
- expression level
- sample
- Prior art date
Links
Images
Classifications
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- 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
-
- 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
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
- G16B25/20—Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
-
- 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
-
- 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
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Definitions
- the present invention relates to a method for correcting an expression level for comparative analysis of the expression level of a target small RNA contained in a plurality of specimens, and an apparatus for correcting the expression level.
- Non-coding RNA is a general term for RNA that does not encode proteins, and is broadly divided into housekeeping RNA and regulatory RNA. There are ncRNAs of various lengths, and especially molecules with less than 200 bases are called small RNAs.
- RNAs include ribosomal RNA (rRNA), transport RNA (tRNA), small nuclear RNA (snRNA) involved in splicing, and small nuclear RNA (snoRNA) involved in rRNA modification. ing.
- rRNA ribosomal RNA
- tRNA transport RNA
- snRNA small nuclear RNA
- snoRNA small nuclear RNA
- RNA interference RNA interference
- NcRNA as a regulatory RNA has a chain length of approximately 20-25 bases, and its mechanism of action is translational suppression by microRNA (miRNA), cleavage of target mRNA by small interference RNA (siRNA) and target DNA It is broadly divided into gene silencing through heterochromatinization of regions.
- miRNA microRNA
- siRNA small interference RNA
- MiRNA is transcribed from genomic DNA as hairpin-like RNA (precursor). This precursor is cleaved by a dsRNA cleaving enzyme (Drosha, Dicer) having a specific enzyme RNase III cleavage activity, then changed to a double-stranded form, and then becomes a single strand.
- a dsRNA cleaving enzyme Rosha, Dicer
- RNase III cleavage activity One of the antisense strands is incorporated into a protein complex called RISC and is considered to be involved in mRNA translational suppression.
- miRNA has different aspects at each stage after transcription. Therefore, when miRNA is targeted (detection target), normally, such as a hairpin structure, a double-stranded structure, a single-stranded structure, etc.
- miRNA consists of RNA of 15 to 25 bases, and its existence has been confirmed in various organisms.
- miRNAs are present not only in cells but also in body fluids such as serum, plasma, urine, spinal fluid, which are specimens (biological samples) that do not contain cells, and their expression levels vary in various ways including cancer. It has been suggested to be a biomarker for various diseases. As of June 2014, more than 2500 miRNAs exist in humans (miRBase Release 20). When using a highly sensitive measurement system such as a DNA microarray, more than 1000 miRNAs are expressed in serum and plasma. Can be detected simultaneously. Therefore, biomarker search research for body fluids such as serum / plasma, urine, spinal fluid and the like has been carried out using the DNA microarray method.
- Patent Documents 4 to 6 present a standard substance which is a nucleic acid or a probe nucleic acid sequence and a design method for detecting the standard substance, and the accuracy in the amplification process and the detection process is shown. Although it is possible to evaluate the performance, it does not show that the error correction between the experiments including the step of extracting the nucleic acid from the specimen is actually performed.
- Patent Document 6 also shows the sequence of a standard substance that is a nucleic acid for correcting an error in the detection value of gene expression in a specimen. This sequence is used from the nucleic acid amplification step, and the amplification step It can only correct for errors between experiments.
- the methods described in Patent Documents 4 to 6 described above include measurement steps including amplification and detection of nucleic acid only when there is a sufficient amount of nucleic acid extracted from the specimen and the nucleic acid to be used can be accurately quantified.
- the target small RNA to be extracted is particularly useful when correcting measurement results between actual experiments, especially when handling a small amount of sample or when handling body fluid as a sample. Since the amount becomes very small and the amount of this small RNA cannot be measured with high accuracy, it is practically impossible to correct by such a method. For this reason, it is very important to correct errors between experiments including not only a small RNA detection process but also an extraction process from a specimen.
- the nucleic acid has the same base length as a small RNA. It has been considered to make corrections using standard substances. For example, a short RNA of about 20 bases, which is the same base length as miRNA as shown in Non-Patent Document 1, is used as a standard substance, and a certain amount of this is put into a sample and extracted, and each experiment is performed. A method for correcting an error in the extraction process of the target small RNA in the extraction process has been proposed.
- RNA with the same base length as small RNA is used as a standard substance, especially when using a body fluid as a specimen, the extraction efficiency of the standard substance from the specimen depends on the various conditions of the specimen and the various contaminants contained in it. Is not stable, and as a result, the measurement value is not stable, and the accuracy cannot be guaranteed, so that it cannot be used to correct the measurement result between experiments.
- the inventors of the present invention have found that in a method for correcting the expression level for comparative analysis of the expression level of a target small RNA contained in a plurality of specimens, the nucleic acid length is smaller than that of a small RNA for a plurality of specimens.
- Add a reference material which is a nucleic acid with a length of 200 bases or more, to the sample, extract the nucleic acid from the sample, measure the amount of each target small RNA expressed, and measure the abundance of the reference material. It has been found that by correcting using the measured value of the amount, the expression level between the samples can be corrected more accurately than in the past, and the following invention has been completed.
- An expression level correction method for comparatively analyzing the expression level of a target small RNA in a plurality of specimens An extraction step of adding a nucleic acid sample to each of the plurality of samples by adding at least one standard substance having a nucleic acid length of 200 bases or more and then extracting the nucleic acid from each sample; A measuring step of measuring the amount of target small RNA and standard substance present in each extracted nucleic acid sample, and obtaining the measurement value of the expression level of target small RNA and the amount of standard substance extracted for each specimen; A representative value acquisition step of acquiring a representative value from the measured value of the extraction amount of the standard substance for each specimen; Correct the target small RNA expression level for each sample by using the difference or ratio between the reference value arbitrarily set for the standard substance extraction amount and the representative standard value of each sample acquired in the representative value acquisition process.
- the at least one standard substance comprises at least one selected from standard substances that are nucleic acids having the nucleotide sequences shown in SEQ ID NOs: 1 to 5 and 15 to 17.
- the measurement step includes a probe for capturing a plurality of target small RNAs immobilized on a support, a probe for capturing at least one standard substance, and a nucleic acid sample labeled with a labeling substance.
- the representative value acquired in the representative value acquisition step is an average value or median value expressed by a logarithmic value calculated from a measured value of the extracted amount of at least one standard substance, (1) to The correction method according to any one of (8).
- the reference value is a fixed value arbitrarily determined with respect to the extraction amount of the standard substance, or a representative value of the standard substance extraction amount acquired for the first sample arbitrarily selected from the plurality of samples. The correction method according to any one of (1) to (9).
- a device for correcting the expression level in order to perform comparative analysis of the expression level of the target small RNA in a plurality of specimens Each sample measured using a nucleic acid sample obtained by adding at least one standard substance having a nucleic acid length of 200 bases or more to each sample and then extracting the nucleic acid from each sample
- Correction coefficient acquisition means for acquiring each; And a correction means for correcting the expression level of the target small RNA measured in each specimen using each correction coefficient acquired by the correction coefficient acquisition means.
- the representative value is an average value or a median value represented by a logarithmic value, which is calculated from a measured value of at least one standard substance extraction amount.
- the target small RNA is miRNA.
- the correction means includes (a) In the correction coefficient acquisition means, when acquiring a value obtained by subtracting the reference value from the representative value as a correction coefficient, subtracting the correction coefficient from the measured value of the expression level of the target small RNA, (b) In the correction coefficient acquisition means, when acquiring a value obtained by subtracting the representative value from the reference value as a correction coefficient, adding a correction coefficient to the measured value of the expression level of the target small RNA, (c) In the correction coefficient acquisition means, when the value obtained by dividing the representative value by the reference value is acquired as a correction coefficient, the measured value of the expression level of the target small RNA is divided by the correction coefficient, or (d) In the correction coefficient acquisition means, when acquiring a value obtained by dividing the reference value by the representative value as a correction coefficient, multiplying the measured value of the expression level of the target small RNA by a correction coefficient,
- the apparatus according to any one of (12) to (14), wherein the correction is performed by the following.
- the measurement values of the target small RNA expression level and standard substance extraction amount in a plurality of specimens stored in the storage means are a plurality of nucleic acid samples labeled with a labeling substance immobilized on a support.
- Hybridization is carried out by contacting with a probe for capturing a target small RNA and a probe for capturing at least one standard substance, and the expression level of each target small RNA and the extracted amount of the standard substance are measured as signal intensity.
- the device according to any one of (12) to (15), wherein (17) In order to correct the expression level for comparative analysis of the expression level of the target small RNA among a plurality of specimens, After adding at least one standard substance having a nucleic acid length of 200 bases or more to each sample, the nucleic acid sample obtained by extracting the nucleic acid from each sample is used in each nucleic acid sample.
- a correction coefficient acquisition step acquired as a coefficient; and a program for executing a correction step of correcting the expression level of the target small RNA measured for each specimen using each of the acquired correction coefficients.
- a correction coefficient acquisition unit that acquires each of the correction coefficient acquired by the correction coefficient acquisition unit, and corrects the expression level of the target small RNA measured in each sample to function as a correction unit.
- Program. (19) A computer-readable recording medium on which the program according to (17) or (18) is recorded. (20) A probe for capturing a plurality of small target RNAs and for capturing at least one standard substance selected from standard substances that are nucleic acids having the nucleotide sequences shown in SEQ ID NOs: 1 to 5 and 15 to 17
- a small RNA expression analysis chip comprising a support on which a probe is immobilized.
- the target small RNA expression level can be corrected more accurately than before when measuring the expression level of the small RNA extracted from the specimen and comparing the small RNA expression level between the specimens.
- comparative analysis of target small RNAs between samples can be performed more accurately.
- the standard substance in the present invention should be stably coexisted with the target small RNA-containing sample in the extraction process to the measurement process, and measure the amount of the standard substance present together with the target expression of the small RNA.
- the substance is used to obtain a reference for correcting the variation (measurement variation or error between measurements) of the measurement value of the expression level of the target small RNA between the measurements of a plurality of specimens. That is, it is possible to correct the measured value of the expression level of the target small RNA between the measurements of a plurality of specimens based on the abundance of the standard substance.
- RNA capture probe a probe for capturing a plurality of types of target small RNAs
- small RNA capture probes a probe for capturing a standard substance
- the result of detection with a microarray to which “standard substance capture probe” is fixed is schematically shown by a histogram of signal values.
- a probe for capturing a small RNA or a probe for capturing a standard substance is also collectively referred to as a “capture probe” or simply “probe”.
- FIG. 1A the result of analyzing each target small RNA extracted from the specimen A and the specimen B using a DNA microarray is shown by a histogram.
- a distribution (histogram) of measurement values obtained from a plurality of target small RNA capture probes mounted on the microarray and a representative value of measurement values obtained from a plurality of standard substance capture probes are shown.
- the histograms of small RNAs are greatly shifted. From this, it can be interpreted that there is a large difference in the expression level of small RNA between samples. On the other hand, it can be interpreted that there is a difference due to experimental error, particularly the nucleic acid extraction efficiency in the nucleic acid extraction step from the specimen. Which is correct cannot be judged from the histogram alone.
- the representative values of the measurement values obtained from the standard substance capture probe, which is a nucleic acid, shown in FIG. 1A are almost the same for sample A and sample B. That is, it can be determined that the sample A and the sample B are correctly subjected to the experiment and there is no experimental error. In this case, there is a large difference in the expression level of the small RNA between the specimens AB, and correction of the measurement value of the small RNA is not necessary for comparison between the specimens.
- FIG. 1B schematically shows the results of analyzing sample C and sample D using a DNA microarray. The histogram of the measured value obtained from the small RNA capture probe and the representative value of the measured value obtained from the standard substance capturing probe are shown.
- Specimen C and Specimen D have similar distributions of histograms of small RNA measurement values.
- the representative values of the measured values obtained from the standard substance capturing probe are greatly different between the sample C and the sample D. From this, it can be seen that an experimental error has occurred in the detection results of the sample C and the sample D for some reason. In such a case, it is necessary to appropriately correct the measurement value of the small RNA when comparing between the specimen CDs.
- FIG. 1C shows a histogram after correcting the measurement value of the target small RNA according to the present invention.
- a specific method of correction is as described later.
- the data of specimen C was corrected so that the measured values obtained from the standard substance capture probes of specimen C and specimen D would match.
- the representative values of the measurement values obtained from the standard substance capture probe are matched between the sample C and the sample D, and the histogram of the measurement values of the target small RNA capture probe corrected using the same correction coefficient is , It will shift greatly. In other words, there is a large difference in the expression level of small RNA even between specimen CDs.
- the expression level of the target small RNA is comparatively analyzed (measured) among a plurality of specimens.
- the number of specimens may be two, or three or more.
- the measurement between a plurality of samples mentioned here includes measurement of a plurality of different types of target small RNAs, measurement of each sample when measuring the same target small RNA a plurality of times, or a combination of both. .
- small RNA means RNA having a base length of less than 200 bases produced in vivo.
- ribosomal RNA 5S rRNA, 5.8S rRNA
- transfer RNA tRNA
- small nuclear ribonucleoprotein particle RNA snoRNA
- small nuclear RNA snRNA
- miRNA microRNA
- miRNA immature before processing
- stem-looped pre-miRNA and double-stranded miRNA / miRNA duplex but are not limited thereto.
- miRNA can be mentioned.
- the standard substance in the extraction process and the measurement process for comparative analysis of the expression level of the target small RNA, the standard substance is present in a certain content with respect to the target small RNA.
- the extraction step it is preferable that the standard substance which is a nucleic acid is extracted with the same extraction efficiency as that of the target small RNA.
- the standard substance used in the present invention is a nucleic acid.
- the nucleic acid length is 200 bases or more longer than the target small RNA, preferably 200 bases or more and 1200 bases or less, more preferably 500 bases or more and 1200 bases or less.
- single-stranded RNA when the nucleic acid length is 200 to 300 bases or more, tends to form a hydrogen bond in the strand and is physically stabilized, and various salts, lipids, proteins, etc. It is possible to maintain a more chemically stable state by associating with the.
- the nucleic acid length of the standard substance which is a nucleic acid
- the extraction efficiency from the sample and the measurement results vary greatly between experiments due to the extraction conditions, sample conditions, and the influence of contaminants contained in the sample.
- the standard substance preferably has the following properties (1) and (2).
- the GC content is in the range of 30 to 70%.
- Tm value is 10 °C or more and 95 °C or less.
- the GC content of (1) can be obtained from the abundance ratios of G and C in all bases of A, T, G and C in the base sequence of the standard substance used. Since the number of hydrogen bonds increases as the GC content increases, the structure and properties of the nucleic acid tend to be stable, but if it is too high, the sequence specificity at the time of measurement decreases. Therefore, the GC content of the standard material is preferably in the range of 30 to 70%, more preferably in the range of 40 to 60%.
- the Tm value of (2) can be calculated by using the nearest base pair (Nearest Neighbor) method (PNAS, 1998, 95: 1460-1465) based on the base sequence of the standard substance.
- PNAS Nearest Neighbor
- the higher the Tm value, the higher the structural stability of the nucleic acid, and the Tm value of the standard substance is preferably 10 ° C or higher and 95 ° C or lower, more preferably 30 ° C or higher and 95 ° C or lower, More preferably, it is 86 ° C or higher and 95 ° C or lower.
- a standard substance that does not cross-hybridize with a genetic transcript contained in a sample to be used in the present invention is preferable to select a standard substance that does not cross-hybridize with a genetic transcript contained in a sample to be used in the present invention.
- a homology search program select a nucleic acid that has a sequence homology of 50% or less for any gene transcript of the same species as the target small RNA recorded in a public database. can do.
- the homology search program is not particularly limited.
- public programs such as FASTA, BLAST, and Mega Blast can be applied.
- the public database is not particularly limited, and databases such as Genbank (NCBI), EMBL (EBI), Ensembl, and miRbase that store sequence information of gene transcripts can be used.
- the standard substance used in the present invention can be synthesized by applying an organic chemical synthesis method of nucleic acid, or using a vector in which the standard substance sequence is incorporated into a plasmid or the like in a host of a microorganism such as Escherichia coli. It can be prepared by incorporating a sequence that can be recognized by an RNA synthase such as T7 promoter into the upstream part of the substance sequence and applying a biological synthesis method such as a method of synthesis using an enzyme such as T7 polymerase.
- Nucleic acid standards that can be used as criteria for the validity evaluation and accuracy control of analyzers and analytical methods are known, and there are also commercially available products. Such known products are also used as standards in the present invention. Can be used.
- Standard materials can include not only single-stranded materials but also those in which double strands are formed together with complementary strands.
- a part of the base sequence of a naturally occurring nucleic acid may be included, or a base sequence that does not exist in nature may be included.
- the same base sequence may be a sequence that is repeated a plurality of times or randomly arranged a plurality of times, and a start codon or a termination codon may be included in a part of the sequence.
- primer sites such as poly A may be provided on both sides or one side of the sequence.
- the standard substance is preferably DNA or RNA, but nucleic acid derivatives such as artificial nucleic acids such as PNA and LNA can also be used.
- the nucleic acid derivative means a labeled derivative with a fluorophore, a modified nucleotide (for example, a nucleotide containing a group such as halogen, alkyl such as methyl, alkoxy such as methoxy, thio, or carboxymethyl, and base reconstruction, Means a derivative containing nucleotides subjected to saturation of double bonds, deamination, substitution of oxygen molecules with sulfur molecules, and the like.
- the terminal may be modified with various functional groups, and examples of such functional groups include a phosphate group, an amino group, and a thiol group.
- one type of standard substance may be used, or a plurality of types may be used.
- it may be used in a state of being associated with a protein or being encapsulated in a vesicle formed of lipids.
- the specimen that can be used in the method of the present invention is not particularly limited.
- various foods and drinks or diluted products thereof can be exemplified.
- the plurality of specimens to be compared and analyzed may be a plurality of specimens derived from different tissues, a plurality of specimens derived from the same tissue separated from different living bodies, or different sites (for example, A plurality of specimens derived from a lesioned part such as a tumor and a non-lesioned part) may be used.
- a certain amount of standard substance is added to a certain amount of specimen.
- the unit of quantity in this case is not particularly limited, and may be weight or volume.
- the unit for measuring a certain amount of standard substance solution may be any unit such as weight, volume, number of moles, and the like.
- various known methods such as an absorbance measurement method, an electrophoresis method, a column method, and a capillary electrophoresis method can be used.
- RNA Before adding nucleic acid, add a certain amount of standard substance to a certain amount of specimen.
- a standard substance after mixing a specimen with an extraction solution and inactivating a nucleolytic enzyme that may be present in the specimen with a guanidinium salt or the like, and before separating a solution containing RNA. It is more preferable to add.
- the standard substance may be added to the specimen in a solution state or in a dried solid state. However, in order to add an exact constant amount, it is preferably added in a solution state, from several ⁇ L to several hundreds. More preferably, it is added in an amount of ⁇ L.
- a pipette is usually used, but if it is less than ⁇ L, it is difficult to accurately measure it. If it is more than mL, the volume for the specimen increases and the composition of the extraction solution changes greatly. There is a concern that this may affect the extraction operation.
- a buffer solution such as water or PBS as the solvent.
- the amount of the standard substance to be added is added to the specimen so that the final concentration is comparable to that of the target small RNA contained in the specimen.
- the concentration of small RNA varies depending on the type of specimen, but when the specimen is a body fluid, the molar unit is z (zepto) mol / mL to p (pico) mol / mL, more preferably a ( Add the standard substance to the sample at a concentration of atto) mol / mL to f (femto) mol / mL.
- the method for measuring the concentration include an absorbance measurement method, a fluorescence method, an electrophoresis method, a column method, and a capillary electrophoresis method.
- the presence of the target small RNA and standard substance can be confirmed by measuring the extraction solution by absorbance measurement method, fluorescence method, electrophoresis method, column method, capillary electrophoresis method, etc. You may measure.
- a nucleic acid containing a target small RNA is extracted from each specimen in the presence of a standard substance (extraction process). Since the standard substance added to each specimen is also extracted together with the target small RNA, the nucleic acid sample obtained from each specimen in this extraction step includes the target small RNA and the standard substance.
- the extraction solution is preferably a solution containing 2 to 5 M guanidine and 40 to 60% phenol.
- an extraction solution that can effectively remove contaminants such as proteins
- a sample is homogenized in an extraction solution to form a homogenate, and an organic solvent for separating an aqueous solution containing RNA is added to the homogenate, followed by centrifugation.
- an organic solvent for separating an aqueous solution containing RNA is added to the homogenate, followed by centrifugation.
- the solution containing the extracted small RNA may be further purified by subjecting it to steps such as precipitation, chromatography, centrifugation, electrophoresis, and affinity separation.
- steps such as precipitation, chromatography, centrifugation, electrophoresis, and affinity separation.
- a step of adding a lower alcohol to a solution containing small RNA to precipitate small RNA and recovering the precipitated small RNA can be applied.
- a carrier capable of adsorbing RNA such as a silica membrane column, and then eluted from the carrier (column). You may apply the process to collect
- ⁇ Measurement process> the amounts of target small RNA and standard substance contained in the nucleic acid samples extracted from a plurality of specimens by the above extraction process are measured (measurement process).
- an amplification method such as a PCR method and a sequencing method
- a hybridization method such as a Northern hybridization method, a Southern hybridization method
- an array method it is preferable to perform the measurement by an array method using an array chip such as a microarray in which a probe that specifically binds to the target RNA or standard substance is immobilized on a support.
- an array chip including a support on which a target small RNA capture probe and a standard substance capture probe are aligned and immobilized can be preferably used.
- Capture probe or “probe for capturing” means a substance capable of binding directly or indirectly, preferably directly and selectively to a nucleic acid such as RNA to be captured, Examples include nucleic acids, proteins, saccharides and other antigenic compounds.
- a nucleic acid probe can be preferably used.
- the nucleic acid probe can be prepared using nucleic acid derivatives such as PNA (peptide nucleic acid) and LNA (Locked NucleicleAcid) in addition to DNA and RNA.
- the nucleic acid derivative means a labeled derivative with a fluorophore, a modified nucleotide (for example, a nucleotide containing a group such as halogen, alkyl such as methyl, alkoxy such as methoxy, thio, or carboxymethyl, and base reconstruction, Means a derivative containing nucleotides subjected to saturation of double bonds, deamination, substitution of oxygen molecules with sulfur molecules, and the like.
- a modified nucleotide for example, a nucleotide containing a group such as halogen, alkyl such as methyl, alkoxy such as methoxy, thio, or carboxymethyl
- base reconstruction Means a derivative containing nucleotides subjected to saturation of double bonds, deamination, substitution of oxygen molecules with sulfur molecules, and the like.
- the base length of the nucleic acid probe is preferably 20 bases or more from the viewpoint of ensuring hybridization stability. Usually, if the chain length is about 20 to 100 bases, the probe can sufficiently exhibit selective binding to the target RNA. Such an oligonucleic acid probe having a short chain length can be easily prepared by a known chemical synthesis method or the like.
- the nucleic acid probe has a base sequence that is completely complementary to the target nucleic acid (target small RNA or standard substance that is a nucleic acid). Any base sequence having a homology high enough to hybridize with can be used as a capture probe.
- stringency during hybridization is a function of temperature, salt concentration, probe chain length, GC content of the nucleotide sequence of the probe, and the concentration of the chaotropic agent in the hybridization buffer.
- stringent conditions for example, the conditions described in “Sambrook, J. et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York (1998)” be able to.
- Stringent temperature conditions are about 30 ° C or higher.
- Other conditions include the hybridization time, the concentration of the detergent (eg, SDS), the presence or absence of carrier DNA, and various stringencies can be set by combining these conditions.
- a person skilled in the art can appropriately determine conditions under which a nucleic acid probe prepared for detection of a target small RNA contained in a desired specimen or a standard substance to be used can appropriately function as a capture probe.
- Small RNA sequence information can be obtained from databases such as GenBank (http://www.ncbi.nlm.nih.gov/genbank/).
- the miRNA sequence information can be obtained from, for example, the miRbase website (http://www.mirbase.org/ftp.shtml).
- Small RNA capture nucleic acid probes can be designed based on sequence information available from these sites.
- the number of small RNA capture probes immobilized on the support is not particularly limited.
- the expression level of a small RNA may be measured using a fixed number of small RNA capture probes covering all known miRNAs whose sequences have been identified on a support.
- a number of small RNA capture probes immobilized on a support may be used.
- one or more specific small RNA capture probes related to a specific disease or biological condition may be used.
- the probe for capturing the standard substance may be any probe that can capture the standard substance used in a complementary manner.
- the homology with the base sequence of the standard substance is preferably 50% or more and preferably does not take a higher order structure. For example, it can be designed by the method described in JP 2011-239708 A .
- the same supports as those used in known microarrays and macroarrays can be used, and for example, slide glass, membranes, beads, and the like can be used.
- a support having a shape having a plurality of convex portions on the surface described in Japanese Patent No. 4244788 can also be used.
- the material of the support is not particularly limited, and examples thereof include inorganic materials such as glass, ceramic, and silicon; polymers such as polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polymethyl methacrylate, and silicone rubber.
- a method for immobilizing a capture probe on a support there are known a method of synthesizing oligo DNA on the surface of the support and a method of dropping oligo DNA synthesized in advance on the surface of the support and immobilizing it.
- the former method examples include the method of Ronald et al. (US Pat. No. 5,705,610), the method of Michel et al. (US Pat. No. 6,142,266), and the method of Francesco et al. (US Pat. No. 7037659).
- the support since an organic solvent is used during the DNA synthesis reaction, the support is preferably made of a material resistant to the organic solvent.
- DNA synthesis is controlled by irradiating light from the back surface of the support, and therefore the support is preferably made of a light-transmitting material.
- Examples of the latter method include the method of Hirota et al. (Patent No. 3922454) and the method using a spotter.
- Examples of the spot method include a pin method based on mechanical contact of a pin tip with a solid phase, an ink jet method utilizing the principle of an ink jet printer, and a capillary method using a capillary tube.
- post-treatment such as cross-linking by UV irradiation and surface blocking is performed as necessary.
- a functional group such as an amino group or an SH group is introduced at the end of the oligo DNA.
- the surface modification of the support is usually performed by treatment with a silane coupling agent having an amino group or the like.
- the capture probe may be detected by fixing it to a plurality of immobilization regions of the support for one kind of small RNA or standard substance.
- the same capture probe that captures one type of small RNA or standard substance may be immobilized at a plurality of locations on the support, or multiple types of capture probes may be attached to one type of small RNA or standard substance. If designed, multiple capture probes targeting the same small RNA or standard may be immobilized on the support.
- Hybridization with each probe immobilized on a support is performed by binding a labeling substance to a nucleic acid sample extracted from a sample to which at least one standard substance is added, preparing a nucleic acid sample labeled with the labeling substance, This is carried out by contacting the labeled nucleic acid sample with a probe.
- nucleic acid sample includes not only RNA extracted from a specimen but also cDNA and cRNA prepared from the RNA by a reverse transcription reaction.
- a labeled nucleic acid sample can be a target small RNA and standard substance in a nucleic acid sample that are directly or indirectly labeled with a labeling substance, and can also be a cDNA or RNA prepared from RNA in a nucleic acid sample.
- cRNA when the standard substance is RNA, target small RNA and cDNA or cRNA obtained by reverse transcription reaction from the standard substance are included
- a method of binding a labeling substance to a nucleic acid sample a method of binding a labeling substance to the 3 ′ end of the nucleic acid sample, a method of binding a labeling substance to the 5 ′ end, and a method of incorporating a nucleotide bound to the labeling substance into the nucleic acid.
- An enzymatic reaction can be used in the method of binding a labeling substance to the 3 ′ end and the method of binding a labeling substance to the 5 ′ end.
- T4-RNA Ligase, Terminal Deoxitidil Transferase, Poly A-polymerase, etc. can be used.
- any labeling method can be referred to the method described in “Shao-YaoYYing, edited by miRNA experiment protocol, Yodosha, 2008”.
- Various kits for binding a labeling substance directly or indirectly to the end of RNA are commercially available.
- miRCURY ⁇ miRNA ⁇ HyPower labeling kit (Exicon), NCode miRNA Labeling system (Life Technologies), FlashTag Biotin RNA Labeling Kit (Genisphere) Etc.
- Life Technologies' NCode miRNA el Labeling system adds a poly A tail to miRNA, then ligates the capture sequence to the 3 ′ end using cross-linked oligo dT, hybridizes it to the array, and then converts it to the capture sequence.
- RNA having a phosphorylated 3 'end is used as a standard substance, labeling can be performed by these methods.
- cDNA or cRNA incorporating the labeling substance is prepared, A method of hybridizing this with a probe on the array is also possible. This method can be employed when RNA is used as a standard substance.
- a plurality of specimens are used, but the same labeling substance may be used for all, or a plurality of different labeling substances may be used.
- labeling substances examples include various labeling substances that are also used in known microarray analysis. Specific examples include fluorescent dyes, phosphorescent dyes, enzymes, and radioisotopes, but are not limited thereto. Preferred are fluorescent dyes that can be easily measured and signals can be easily detected. Specifically, cyanine (cyanine 2), aminomethylcoumarin, fluorescein, indocarbocyanine (cyanine 3), cyanine 3.5, tetramethylrhodamine, rhodamine red, Texas red, indocarbocyanine (cyanine 5), cyanine 5.5, Although well-known fluorescent dyes, such as cyanine 7 and oyster, are mentioned, It is not limited to these.
- semiconductor fine particles having a light emitting property may be used as the labeling substance.
- semiconductor fine particles include cadmium selenium (CdSe), cadmium tellurium (CdTe), indium gallium phosphide (InGaP), silver indium zinc sulfide (AgInZnS), and the like.
- the sample-derived nucleic acid (target small RNA) and the nucleic acid sample containing the standard substance labeled as described above are brought into contact with the probe on the support and hybridized.
- This hybridization step can be performed in the same manner as in the past.
- the reaction temperature and time are appropriately selected according to the chain length of the nucleic acid to be hybridized. In the case of nucleic acid hybridization, it is usually about 30 ° C. to 70 ° C. for 1 minute to several tens of hours.
- Hybridization is performed, and after washing, the signal intensity from the labeling substance in each probe-immobilized region on the support is detected. The signal intensity is detected using an appropriate signal reader according to the type of labeling substance. When a fluorescent dye is used as a labeling substance, a fluorescence microscope or a fluorescence scanner may be used.
- Detected measurement value (signal value) is compared with ambient noise. Specifically, the measured value obtained from the probe-immobilized region is compared with the measured value obtained from other positions, and the case where the former numerical value is exceeded is detected (effective determination positive). .
- the background noise may be subtracted.
- the ambient noise can be subtracted from the detected signal value as background noise.
- the method described in “Microarray Data Statistical Analysis Protocol (Yodosha)” may be used.
- the measured values of the amount of target small RNA and standard substance present in each nucleic acid sample that is, the amount of target small RNA expressed in each sample and the amount of standard substance extracted are obtained as signal intensity.
- ⁇ Representative value acquisition process> for each specimen, a representative value is acquired from the measured value of the standard substance abundance in the extracted nucleic acid sample (representative value acquisition step).
- standard substance abundance in a nucleic acid sample is synonymous with the term “standard substance abundance extraction from a specimen”.
- standard substance abundance and “standard substance abundance” are used. Use the same meaning.
- the amount of standard substance present in the nucleic acid sample and the amount of standard substance extracted from the specimen may be simply referred to as “standard substance amount”. That is, in this specification, “the amount of the standard substance of the sample” means not the amount of the standard substance added to the sample but the amount of the standard substance present in the nucleic acid sample or the amount of standard substance extracted from the sample.
- the measurement value of the one kind of abundance can be a representative value, but when there are two or more kinds, a representative value may be obtained by various methods below.
- the average value means an average value calculated from measured values of the extracted amounts of a plurality of standard substances (for example, measured values of signal intensity obtained using a microarray).
- the median means a median obtained from measured values of extracted amounts of a plurality of standard substances (for example, measured values of signal intensity obtained using a microarray).
- average values or median values may be average values or median values expressed in logarithmic values.
- the “average value expressed in logarithmic value” is a logarithmic value obtained by converting a measured value of the extraction amount of a plurality of standard substances (for example, a measured value of signal intensity obtained using a microarray) into a logarithm with a base of 2. This means the average value obtained in.
- the “median value expressed in logarithmic value” is a logarithmic value obtained by converting a measured value of the extraction amount of a plurality of standard substances (for example, a measured value of signal intensity obtained using a microarray) into a logarithm with a base of 2.
- the same value can be obtained whether the logarithmic conversion of the measured value is performed first or later.
- the average value and the median value may be obtained by using all the measured values of a plurality of standard substances to be measured, or a part of measured values selected from the plurality of standard substances. It may be obtained by using. For example, it may be obtained using all measured values obtained with the standard substance capture probe mounted on the microarray, or a part of the standard substance capture probe (for example, the standard substance capture probe mounted on the microarray). If there are ten probes, five of them may be obtained. For example, it is possible to select only the standard substance capture probes that are positive for the effective determination in common for all the samples to be compared and to obtain the representative value of the standard substance. Further, outliers may be excluded from the measured value of the abundance of the standard substance before obtaining the representative value.
- a plurality of types of capture probes for one type of standard substance can be detected. May be obtained.
- an average value or median value calculated from a plurality of measured values for example, an average value or median value represented by logarithmic values may be used as a representative value. it can.
- the representative value may be obtained using all of the measured values, or may be obtained using a part of the measured values.
- each average value may be logarithmically converted, and an average value or a median value may be obtained among a plurality of standard substances using each logarithmic value.
- the average value or median value thus obtained is also included in the “average value or median value expressed in logarithmic values”.
- the CV value (coefficient of variation) of representative values in a plurality of specimens is 0.5 or less.
- the CV value of the measured value when a microarray is used is 0.5 or less.
- the CV value is 0.5 or more, the variation is large, and the extraction efficiency of the standard substance in the extraction process is not stable, and as a result, the accuracy of the corrected data is expected to decrease.
- correction coefficient acquisition step the correction used for correcting the expression level of the target small RNA using the representative value of the standard substance extraction amount of each specimen obtained in the representative value acquisition step and the reference value arbitrarily set for the standard substance extraction amount A coefficient is acquired (correction coefficient acquisition step).
- the correction coefficient may be obtained using the difference between the representative value and the reference value, or the correction coefficient may be obtained using the ratio between the representative value and the reference value.
- a correction coefficient is obtained using a ratio
- a correction coefficient is obtained using a difference. preferable.
- the correction coefficient acquisition step -1 is a method that uses the difference between the representative value of the standard substance extraction amount and the reference value. In this step, the following 1-1. Reference specimen acquisition method, or 1-2. A fixed numerical correction method may be applied.
- Reference Sample Acquisition Method One sample (first sample) is arbitrarily selected from a plurality of samples to be analyzed, and this is set as a “reference sample”. The remaining one or more samples (second and subsequent samples) are “corrected samples”.
- the term “second and subsequent samples” includes the second sample. For example, if there are two samples to be compared, only the second sample is corrected, and if there are three samples to be compared, the sample to be corrected is the second sample. And a third specimen.
- the “standard value” is the representative value of the standard substance amount of the standard sample.
- a difference between the reference value and the representative value of the standard substance amount of each of the second and subsequent samples (sample to be corrected) is used as a correction coefficient for each of the second and subsequent samples. As many correction coefficients as the number of specimens to be corrected are acquired.
- the correction coefficient is obtained by Expression 1 or Expression 1 ′.
- c 1-1 (Representative value of standard substance amount of standard sample (standard value)) -(Representative value of standard substance amount of sample to be corrected)
- c 1-1 ' (Representative value of the standard substance amount of the corrected sample) -(Representative value of standard substance amount of standard sample (standard value)) ... Equation 1 '
- the correction coefficient for the corrected sample is expressed by Equation 2 or Equation 2 ′. Can be obtained.
- n is the total number of standard substance capture probe immobilization regions on the support;
- Aj is the measured signal value from the j-th (1 ⁇ j ⁇ n) standard substance capture probe immobilization region in the reference sample,
- Xj is a signal measurement value from the j-th (1 ⁇ j ⁇ n) standard substance capture probe immobilization region in the second specimen, It is.
- n is equal to the number of standard substances targeted by the standard substance capture probe on the support.
- the total number n' of the standard substance capture probe immobilization regions that are positive in the validity determination in common for all the samples to be compared can be used instead of n.
- the correction coefficient is obtained by Expression 3 or Expression 3 ′.
- r 1-2 (Fixed value (reference value))-(Representative value of the standard substance amount of the corrected sample) ...
- r 1-2 ' (Representative value of standard substance amount of sample to be corrected) ⁇ (Fixed value (reference value)) ...
- the correction coefficient for the corrected sample is expressed by Equation 4 or Equation 4 ′. Can be obtained.
- ⁇ is the reference value (fixed value)
- n is the total number of standard substance capture probe immobilization regions on the support
- Yj is a signal measurement value from the j-th (1 ⁇ j ⁇ n) standard substance capture probe immobilization region in the specimen, It is.
- n is equal to the number of standard substances targeted by the standard substance capture probe on the support.
- the total number n' of the standard substance capture probe immobilization regions that are positive in the validity determination in common for all the samples to be compared can be used instead of n.
- the fixed numerical value used as the reference value in the fixed numerical value correction method may be any numerical value (except 0) as long as the same numerical value is used consistently for all samples in at least one comparative analysis. . If the same expression measurement system is used and the same numerical value is always used as a fixed numerical value, comparative analysis can be performed even between samples whose expression levels are measured on different days. For example, since the amount of each standard substance added to the specimen is the same for all specimens, the fixed numerical value may be determined based on the amount of standard substance added. However, since the magnitude of the signal value detected by the system used in the measurement process can vary, the fixed numerical value can be freely selected according to the system used.
- the correction coefficient acquisition step-2 is a method that uses the ratio between the representative value of the standard substance amount and the reference value. In this step, the following 2-1. Reference specimen acquisition method, or 2-2. A fixed numerical correction method may be applied.
- Reference Sample Acquisition Method One sample (first sample) is arbitrarily selected from a plurality of samples to be analyzed, and this is set as a “reference sample”. The remaining second and subsequent samples are “corrected samples”.
- the reference value of the reference substance amount of the reference sample is set as the “reference value”, and the ratio between the reference value and the representative value of the reference substance amount of each of the second and subsequent samples (corrected samples) is Used as a correction coefficient for each of the second and subsequent samples. As many correction coefficients as the number of specimens to be corrected are acquired.
- the correction coefficient is obtained by Expression 5 or Expression 5 ′.
- c 2-1 (Representative value of standard substance amount of standard sample (standard value)) / (Representative value of standard substance amount of sample to be corrected)
- c 2-1 ' (Representative value of the standard substance amount of the corrected sample) / (Representative value of standard substance amount of standard sample (standard value)) ...
- the correction coefficient for the second specimen is expressed by Equation 6 or Equation 6 ′. Can be obtained.
- n is the total number of standard substance capture probe immobilization regions on the support;
- Aj is the measured signal value from the j-th (1 ⁇ j ⁇ n) standard substance capture probe immobilization region in the reference sample,
- Xj is a signal measurement value from the j-th (1 ⁇ j ⁇ n) standard substance capture probe immobilization region in the second specimen, It is.
- n is equal to the number of standard substances targeted by the standard substance capture probe on the support.
- the total number n' of the standard substance capturing probe immobilization regions that are positive in the determination of validity in common for all the samples to be compared can be used instead of n.
- the correction coefficient is obtained by Expression 7 or Expression 7 ′.
- r 2-2 (Fixed value (reference value)) / (Representative value of the standard substance amount of the corrected sample) ...
- Formula 7 r 2-2 ' (Representative value of standard substance amount of sample to be corrected) / (Fixed value (reference value)) ...
- the correction coefficient for the corrected sample is expressed by Equation 8 or Equation 8 ′. Can be obtained.
- ⁇ is a fixed value
- n is the total number of standard substance capture probe immobilization regions on the support
- Yj is a signal measurement value from the j-th (1 ⁇ j ⁇ n) standard substance capture probe immobilization region in the specimen, It is.
- n is equal to the number of standard substances targeted by the standard substance capture probe on the support.
- the total number n' of the standard substance capturing probe immobilization regions that are positive in the validity determination in common for all the samples to be compared can be used instead of n.
- the correction step-1 is a method for correcting the expression level of the target small RNA using the correction coefficient obtained in the correction coefficient acquisition step-1.
- the correction factor-1 is added to the expression level of the target small RNA, or the expression Correction is performed by subtracting the correction coefficient from the quantity.
- this step there are two correction methods respectively corresponding to the reference specimen acquisition method and the fixed numerical value correction method in the correction coefficient acquisition step-1.
- Reference specimen acquisition method The correction of the expression level of the target small RNA in the second and subsequent specimens is performed using the correction coefficients for the second and subsequent specimens, respectively.
- the correction coefficient (c2 1-1 or c2 1-1 ′) for the second sample is used, and for the third sample
- the correction coefficient (c3 1-1 or c3 1-1 ′) for the third specimen is used.
- the correction coefficient that is, in the case of Equation 1 above, each of the second and subsequent specimens
- the target small RNA expression level is corrected for each of the second and subsequent samples.
- the corrected expression level Ei of the i-th target small RNA in a certain “corrected sample” can be obtained by the following equation 9.
- Wi is a signal measurement value from the i-th small RNA capture probe immobilization region.
- the correction coefficient that is, in the case of the above formula 1 ′
- the target small RNA expression level is corrected for each of the second and subsequent samples by subtracting the correction coefficient from the measurement value of each target small RNA expression level in the second and subsequent samples or the logarithm of the measured value. Is done.
- the corrected expression level Ei of the i-th target small RNA in a certain “corrected sample” can be obtained by the following equation 9 ′.
- Wi Wi
- c2 is added to the measurement value of each target small RNA expression level in the second sample or the logarithm of the measurement value, or What is necessary is just to subtract c2 '.
- the difference between the representative value of the first sample as the reference sample and the reference value is naturally 0, but because of the program configuration, 0 is added to or subtracted from the target small RNA expression level of the first sample. Can be carried out.
- the target small RNA expression level is corrected using a correction coefficient obtained by the difference between the representative value and the fixed numerical value (reference value). That is, when the target small RNA expression level is corrected for a certain sample, the correction coefficient (r 1-2 or r 1-2 ′) for that sample is used.
- the correction coefficient that is, in the case of Equation 3
- the measured value of the expression level of each target small RNA in the specimen or the pair of the measured values By adding the correction coefficient to the numerical value, the expression level of the target small RNA for each specimen is corrected.
- the corrected expression level Ei of the i-th target small RNA in a certain “corrected specimen” can be obtained by the following equation 10.
- Wi is a signal measurement value from the i-th small RNA capture probe immobilization region.
- the corrected expression level Ei of the i-th target small RNA in a certain “corrected sample” can be obtained by the following equation 10 ′.
- Wi Wi is the same as that in Equation 10 above.
- the correction step-2 is a method for correcting the expression level of the target small RNA using the correction coefficient obtained in the correction coefficient acquisition step-2, and the expression level of the target small RNA is divided by the correction coefficient or the expression Correction is performed by multiplying the quantity by a correction factor.
- this process there are two correction methods respectively corresponding to the reference specimen acquisition method and the fixed numerical value correction method in the correction coefficient acquisition step-2.
- Reference specimen acquisition method The correction of the expression level of the target small RNA in the second and subsequent specimens is performed using the correction coefficients for the second and subsequent specimens, respectively. That is, when correcting the expression level of the target small RNA for the second sample, the correction coefficient (c2 2-1 or c2 2-1 ′) for the second sample is used, and the third sample is corrected. When correcting the target small RNA expression level, the correction coefficient (c3 2-1 or c3 2-1 ′) for the third specimen is used.
- the correction coefficient when using a ratio in which the representative value of the standard substance amount of the second and subsequent specimens to be corrected is the denominator and the representative value of the standard substance quantity of the reference specimen is the numerator, that is, in the case of the above formula 5, By correcting the measurement value of each target small RNA expression level in the second and subsequent samples or the logarithm of the measurement value by the correction coefficient, the expression level of the target small RNA for each of the second and subsequent samples is corrected.
- the corrected expression level Ei of the i-th target small RNA in a certain “corrected sample” can be obtained by the following equation 11.
- Wi is a signal measurement value from the i-th small RNA capture probe immobilization region.
- the correction coefficient when using a ratio in which the representative value of the standard substance amount of the reference specimen is the denominator and the representative value of the standard substance quantity of the second and subsequent specimens is the numerator, that is, In this case, by dividing the measured value of each target small RNA expression level in the second and subsequent samples or the logarithm of the measured value by the correction coefficient, the expression level of the target small RNA for each of the second and subsequent samples is calculated. Correction is performed.
- the corrected expression level Ei of the i-th target small RNA in a certain “corrected sample” can be obtained by the following equation 11 ′.
- Wi Wi
- c2 2-1 is divided by the measurement value of each target small RNA expression level in the second sample or the logarithm of the measurement value. Or c2 2-1 ′.
- the value of the expression level Ei of the corrected target small RNA finally obtained is the same in the procedures of Formula 5 and Formula 11 and in the procedures of Formula 5 ′ and Formula 11 ′.
- the ratio between the representative value of the first sample as the reference sample and the fixed value (reference value) in the present method is naturally 1, but the target small RNA expression level of the first sample is 1 because of the program configuration. Or a calculation of dividing the target small RNA expression level by 1 may be performed.
- the target small RNA expression level is corrected using a correction coefficient obtained by a ratio with a fixed numerical value (reference value). That is, when the target small RNA expression level is corrected for a certain sample, the correction coefficient (r 2-2 or r 2-2 ′) for that sample is used.
- the corrected expression level Ei of the i-th target small RNA in a certain “corrected sample” can be obtained by the following equation 12.
- Wi is a signal measurement value from the i-th small RNA capture probe immobilization region.
- each target small RNA expression in the specimen is corrected by dividing the measured value of the quantity or the logarithm of the measured value by the correction coefficient.
- the corrected expression level Ei of the i-th target small RNA in a certain “corrected sample” can be obtained by the following equation 12 ′.
- Wi Wi
- the corrected target small RNA expression level is used to compare the target small RNA expression level among multiple samples.
- the target small RNA expression level of the first sample as the reference sample has not been corrected, and therefore, for example, between the first sample and the second sample
- the comparison is a comparison between the target small RNA expression level of the uncorrected first sample and the corrected target small RNA expression level of the second sample, but at least one of the samples to be compared One is always a corrected sample. Therefore, in the case of “compare the target small RNA expression level among multiple body fluid samples based on the corrected target small RNA expression level”, between the uncorrected reference sample and other corrected samples, Contrasting embodiments are also included.
- the comparative analysis process itself can be performed in the same manner as the conventional method.
- a scatter plot of expression level data called a scatter plot may be created.
- two scatter plots for example, between the first specimen and the second specimen
- a scatter plot between the first specimen and the third specimen and if necessary, between the remaining two specimens (in the case of the above example, further the first scatter plot).
- a scatter plot that is comparatively analyzed between the second specimen and the third specimen may be created.
- a comparative analysis of four or more specimens can be performed in the same manner.
- the corrected target small RNA expression level is used to calculate the difference in target small RNA expression level between any one sample and the remaining other samples, and the logarithmic change rate (fold) -change) may represent the comparative analysis results.
- the expression level of the target small RNA in the reference sample in the case of the reference sample acquisition method
- the corrected expression level of the target small RNA in the first sample in the case of the fixed numerical correction method
- the difference from the corrected target small RNA expression level may be calculated.
- the present invention is not limited to calculating the difference between the first sample and the other sample, and the difference between any one of the second and subsequent samples and the other sample is calculated. It is good.
- the apparatus of the present invention is an apparatus that corrects the expression level in order to compare and analyze the expression level of the target small RNA.
- the device About each sample measured using a nucleic acid sample obtained by adding nucleic acid to each sample after adding at least one standard substance having a nucleic acid length of 200 bases or more to each sample.
- Storage means for storing measured values of target small RNA expression level and standard substance extraction level of Representative value acquisition means for acquiring a representative value, preferably a representative value represented by a logarithmic value, from the measured value of the standard substance extraction amount for each specimen; The difference or ratio between the reference value arbitrarily set for the extraction amount of the standard substance and the representative value of each sample acquired by the representative value acquisition means is used as a correction factor for the target small RNA expression level for each sample.
- Correction coefficient acquisition means for acquiring each; Correction means for correcting the expression level of the target small RNA measured in each specimen using each correction coefficient acquired by the correction coefficient acquisition means.
- the reference value is a representative value of the standard substance amount of the arbitrarily selected first specimen (reference specimen), and the expression level of the target small RNA measured in the second and subsequent specimens is corrected. Is done. That is, in this aspect, the device of the present invention About each sample measured using a nucleic acid sample obtained by adding nucleic acid to each sample after adding at least one standard substance having a nucleic acid length of 200 bases or more to each sample.
- Storage means for storing measured values of target small RNA expression level and standard substance extraction level of Representative value acquisition means for acquiring a representative value, preferably a representative value represented by a logarithmic value, from the measured value of the standard substance extraction amount for each specimen;
- the arbitrarily selected first sample is used as a reference sample
- the representative value of the standard substance extraction amount of the reference sample is used as a reference value
- the difference or ratio between the reference value and the representative values of the remaining second and subsequent samples is determined.
- Correction coefficient acquisition means for acquiring correction coefficients for the second and subsequent samples, respectively; Correction means for correcting the expression level of the target small RNA measured in the second and subsequent specimens using the correction coefficients for the second and subsequent specimens acquired by the correction coefficient acquisition means.
- the reference value is a fixed numerical value arbitrarily determined with respect to the standard substance extraction amount, and the target small RNA expression level is corrected for all the samples including the first sample. That is, in this aspect, the device of the present invention About each sample measured using a nucleic acid sample obtained by adding nucleic acid to each sample after adding at least one standard substance having a nucleic acid length of 200 bases or more to each sample.
- Storage means for storing measured values of target small RNA expression level and standard substance extraction level of Representative value acquisition means for acquiring a representative value, preferably a representative value represented by a logarithmic value, from the measured value of the standard substance extraction amount for each specimen;
- a correction coefficient acquisition means that uses a fixed numerical value as a reference value, and acquires a difference between the reference value and the representative value of each specimen as a correction coefficient for the specimen;
- Correction means for correcting the expression level of the target small RNA measured in each specimen using the correction coefficient for each specimen obtained by the correction coefficient obtaining means.
- the small RNA expression level comparison / analysis apparatus including the correction device further includes an output means for outputting a result of comparing the target small RNA expression level between at least two samples based on the corrected target small RNA expression level. May be included.
- FIG. 2 is a block diagram showing an outline of the configuration of the analysis apparatus provided with the correction device.
- the analysis apparatus 10 includes an input unit 110, a display unit 120, an output unit 130, a storage unit 140, a control unit 150, a conversion unit 160, and an analysis unit 170.
- FIG. 3 shows an example of a flowchart of the target small RNA expression level correction process according to the present invention.
- the input unit 110 is a means for inputting information related to the operation of the analysis apparatus 10.
- Conventionally known input means such as a keyboard can be preferably used.
- the data of the small RNA expression amount and the standard substance extraction amount obtained by the hybridization assay are read by a reading means such as a scanner different from the apparatus of the present invention and converted into numerical data, for example. Thereafter, the numerical data may be input to the analysis device 10 from the input unit 110.
- reading means such as a scanner may be included in the analysis device 10 of the present invention (not shown).
- the expression amount and extraction amount data input from the input unit 110 or the expression amount and extraction amount data read and digitized by the reading unit incorporated in the analysis apparatus 10 is stored in the storage unit 140.
- the storage unit 140 serves as a storage unit that stores, for each of the plurality of specimens, the measured values of the expression amount of the plurality of target small RNAs and the extraction amount of at least one standard substance measured simultaneously.
- Measured value data of the small RNA expression level and the standard substance extraction amount of each specimen stored in the storage unit 140 is converted into a logarithm such as 2 by the conversion unit 160 in some cases.
- the analysis unit 170 acquires a representative value of the measured value of the standard substance extraction amount for each specimen.
- the representative value is, for example, an average value or a median value of at least one kind of standard substance measurement value (even if only one kind of standard substance is used for correction, In the case where there are a plurality of probe-immobilized regions on the array, the representative value can be an average value or a median value), or a measurement value of a specific type of standard substance.
- the analysis unit 170 calculates the difference or ratio between the reference value and the representative value of the standard substance amount of each sample for each sample, and acquires the correction coefficient. Details of the correction coefficient acquisition are as described in ⁇ Correction coefficient acquisition step> of the correction method.
- the correction coefficient 0 (when calculating the difference) or correction coefficient 1 (when calculating the ratio) is also applied to the first sample that is set as the reference sample due to the program configuration. ).
- a person who operates the apparatus 10 may designate any one sample from the input unit 110.
- the apparatus 10 may automatically select a reference value, or may select one sample as a reference sample.
- a sample in which data is input from the input unit 110 and data is first stored in the storage unit 140 can be selected as a reference sample by the apparatus 10.
- the step of selecting or inputting the reference specimen is positioned after the representative value acquisition step (S-3) in FIG. 3 for convenience, but is not limited to this, and is an earlier step, for example, when storing data. May be executed.
- a fixed numerical value designated in advance may be registered in the conversion unit 160 or the like as a reference value.
- the analysis unit 170 corrects the measured target small RNA expression level data using the correction coefficient for each specimen.
- the details of the correction operation are as described in ⁇ Correction process> of the correction method.
- the correction coefficient 0 when calculating the difference
- correction is also applied to the target small RNA expression level data of the first sample set as the reference sample due to the program configuration.
- a correction operation using the coefficient 1 when calculating the ratio may be executed.
- the analysis unit 170 performs comparison and statistical analysis of the target small RNA expression level of each specimen.
- the result of the comparison and statistical analysis is output to the display unit 120 by the output unit 130 and displayed.
- a comparison result or a statistical analysis result can be output to an output device such as a printer, a recording medium, or the like.
- the output unit 130 can be configured to output the comparison analysis result and the statistical analysis result to an external storage device such as a database existing outside the device via a network.
- the storage unit 140 stores measured values of the expression levels of a plurality of small RNAs and the extraction amounts of a plurality of standard substances, and also appropriately stores intermediate analysis results generated in the above steps.
- control unit 150 The various operations described above of the apparatus 10 are controlled by the control unit 150. Specifically, as indicated by the broken-line arrows in FIG. Control information is output from the control unit 150, and each unit operates in cooperation with the control information, and the entire apparatus 10 operates.
- the present invention also provides a program for causing a computer to function as the correction device or analysis device described above.
- the program is a program for causing a computer to function as each of the above-described means (that is, storage means, representative value acquisition means, correction coefficient acquisition means, correction means, and output means in the analysis apparatus). It is.
- the present invention provides a program for causing a computer to execute each step of the above-described correction method of the present invention or a comparative analysis method including the correction method.
- the correction method includes the measurement process, the representative value acquisition process, the correction coefficient acquisition process, and the correction process described above.
- the corrected target small RNA expression level is further used between a plurality of samples.
- a comparative analysis step of comparing the target small RNA expression level may be included.
- These programs are programs for causing a computer to correct the expression level of the target small RNA using data of the measurement value of the standard substance extraction amount measured simultaneously with the expression level of the target small RNA by a microarray or the like.
- the present invention provides a computer-readable recording medium on which any one of the above programs is recorded.
- the “recording medium” can be any “portable physical medium” (non-transitory recording medium) such as a flexible disk, magneto-optical disk, ROM, EPROM, EEPROM, CD-ROM, MO, DVD, and the like.
- it may be a “communication medium” that holds a program in a short period of time, such as a communication line or a carrier wave when transmitting a program via a network, represented by a LAN, WAN, or the Internet.
- Program is a data processing method described in an arbitrary language or description method, and may be in any form such as source code or binary code.
- the “program” is not necessarily limited to a single configuration, but is distributed in the form of a plurality of modules and libraries, or in cooperation with a separate program represented by an OS (Operating System). Including those that achieve the function. Note that a well-known configuration and procedure can be used for a specific configuration for reading a recording medium, a reading procedure, an installation procedure after reading, and the like in each device described in the embodiment.
- An array chip comprising a support on which a plurality of target small RNA capture probes and at least one, preferably a plurality of standard substance capture probes, that can be preferably used in the present invention, is immobilized as a chip for small RNA expression analysis Can be provided.
- Preferred conditions for the chip are as described in the measurement process of the present invention.
- RNA500- A SEQ ID NO: 1
- RNA500-B SEQ ID NO: 2
- RNA500-C SEQ ID NO: 3
- B SEQ ID NO: 5
- RNA1000-NMIJ CRM 6204-a a certified reference material consisting of B (SEQ ID NO: 5) (all of which are RNAs with a nucleic acid length of about 1000 bases)
- hsncs_071028 SEQ ID NO: 15
- hsncs_404161 SEQ ID NO:
- cel-miR-39 As a reference material for a comparative example, which is a nucleic acid having a nucleic acid length of less than 200 bases, cel-miR-39 (SEQ ID NO: 6), which is a non-human miRNA having a nucleic acid length of 20 bases shown in Non-Patent Document 1. , Cel-miR-54 (SEQ ID NO: 7), ath-mir-159a (SEQ ID NO: 8), and cel-miR-238 (SEQ ID NO: 9) were selected. Each miRNA was synthesized as an RNA having a phosphate group modified at the 5 ′ end by Eurofin Genomics.
- RNAs with a nucleic acid length of 60 bases sold by Eurofin Genomics H2NC000001 (SEQ ID NO: 10), H2NC000002 (SEQ ID NO: 11), H2NC000003 (SEQ ID NO: 12), H2NC000005 (SEQ ID NO: 13) and H2NC000006 (SEQ ID NO: 14) were used.
- Table 1 shows the physical properties of each reference material. For all the standard substances used, it was confirmed that the sequence homology was 50% or less with respect to all human gene transcripts recorded in the public database BLAST.
- Target small RNAs 2555 human miRNAs obtained from miRBase Release 19 were selected, and DNAs having their complementary sequences were used as target small RNA capture probes (http://www.sanger.ac.uk/Software/ Rfam / mirna / index.shtml).
- DNA having a complementary sequence to the sequences of RNA500-A, RNA500-B, RNA500-C, RNA1000-A, RNA1000-B, hsncs_071028, hsncs_404161, and hsncs_647981 was used.
- cel-miR-39 22 bases
- cel-miR-54 24 bases
- ath-mir-159a 21 bases
- cel-miR obtained from miRBase DNA having a complementary sequence of -238 23 bases
- DNA having complementary sequences of the above-mentioned five kinds of RNA H2NC000001, H2NC000002, H2NC000003, H2NC000005, H2NC000006 was used.
- RNA capture probe and the capture probe for the standard nucleic acid substance synthetic DNA having an amino group introduced at the 3 ′ end (combined synthesis by Eurofin Genomics) was used.
- miRNA target small RNA
- capture probes introduced with an amino group at the 3 ′ end and all 17 standard substance capture probes were combined with the “3D-Gene” (registered trademark) substrate (3000 pillars) manufactured by Toray Industries, Inc.
- the DNA microarray was prepared by immobilizing on the convex portions of the substrate.
- the standard substance capture probes were fixed at 8 points each. The following experiment was performed using this DNA microarray.
- RNA500-A and RNA500-B are standard substances for the above-mentioned examples.
- RNA500-C, RNA1000-A, RNA1000-B, hsncs_071028, hsncs_404161 and hsncs_647981 were each added at 10 fmol, and in the comparative example, cel-miR-39, which is the above-mentioned standard for comparative examples, Add 10 fmol each of cel-miR-54, ath-mir-159a, cel-miR-238, H2NC000001, H2NC000002, H2NC000003, H2NC000005 and H2NC000006 and collect the upper aqueous layer by centrifugation Then, RNA was purified using miRNeasy mini kit (Qiagen).
- the obtained target small RNA (miRNA) was labeled using 3D-Gene miRNA labeling kit (Toray).
- the labeled miRNA was hybridized and washed according to the standard protocol of 3D-Gene miRNA chip (Toray Industries, Inc.).
- the reacted DNA microarray detected a fluorescent signal using a microarray scanner (Toray Industries, Inc.). The scanner settings were 100% laser output and 42% photomultiplier voltage setting.
- Example 1 In accordance with the above procedure, add the standard materials for examples shown in SEQ ID NOs: 1 to 5 (all 5 types) or the standard materials for examples shown in SEQ ID NOs: 15 to 17 (all 3 types) to 300 ⁇ L of commercially available serum samples Then, a nucleic acid extraction process and a measurement process of target small RNA (miRNA) expression level and standard substance abundance using a DNA microarray were performed. The above process was repeated 10 times in order to compare the effect of correcting the fluctuation (variation) in the measured value of the expression level between the measurement experiments.
- miRNA target small RNA
- the correction of the expression level of each miRNA was performed as follows. First, the abundance of each standard substance in each experiment was obtained as an average value of eight measured values on the DNA microarray, and this was calculated as a representative value. Next, the representative value of the standard substance in the first experiment was used as a reference value, and the ratio to the representative value in the second to tenth experiments was obtained as a correction coefficient. Using this correction coefficient, the measurement value of the miRNA expression level in the second to tenth experiments was corrected. As a result, after correction, the median CV value between the 10 measurement experiments of the expression level of each miRNA was about 0.32 (SEQ ID NO: 1) and 0.40 (sequence) for all about 1000 detected miRNAs, respectively. No. 2), 0.40 (SEQ ID NO: 3), 0.38 (SEQ ID NO: 4), 0.30 (SEQ ID NO: 5), 0.41 (SEQ ID NO: 15), 0.36 (SEQ ID NO: 16), and 0.39 (SEQ ID NO: 17).
- the median CV value of the expression levels of about 1000 types of detected miRNAs before correction was 0.45.
- the median CV value of the expression level of each miRNA after correction is 1.07 (SEQ ID NO: 6), 1.14 (SEQ ID NO: 7), 0.98 (SEQ ID NO: 8), 0.99 (SEQ ID NO: 9), 0.94 (SEQ ID NO: 10), 0.95 (SEQ ID NO: 11), 0.84 (SEQ ID NO: 12), 0.82 (SEQ ID NO: 13), and 0.88 (SEQ ID NO: 14).
- Example 1 the median CV value of the detection value of the target small RNA after correction was smaller than 0.45 before correction, whereas in Comparative Example 1, it was larger than 0.45 before correction. That is, in the correction of variation (variation) in the amount of expression between the experiments in an experiment in which the same specimen was used for multiple times from the extraction of the nucleic acid to the DNA microarray experiment under the same conditions, compared to Comparative Example 1, It was shown that the accuracy of correction is improved by performing correction using the standard substance used in Example 1.
- Example 2 Using three types of sera A to C collected from three humans, using the standard substances for examples shown in SEQ ID NOs: 1 to 5 as standard substances, the extraction date of nucleic acid and the experimenter for each serum 2 times each (1st and 2nd) under different conditions, adding the standard substance to the serum specimen and extracting the nucleic acid, and measuring the target small RNA expression level and standard substance abundance by DNA microarray The process was performed.
- the target small RNA about 1000 kinds of detected miRNAs were used as in Example 1.
- the abundance of each standard substance obtained in the measurement process was calculated as an average value of eight measurement values on the DNA microarray, and this was converted into a logarithmic value with a base of 2.
- an average value of logarithmic conversion values of the obtained five kinds of standard substances was calculated and used as a representative value.
- the first and second representative values were calculated for each serum AC. Table 2 shows logarithmic conversion values and average values (representative values) of each standard substance.
- the measurement value of miRNA expression level in each serum was converted into a logarithm with a base of 2, and correction was performed by subtracting the correction coefficient in each serum from the second logarithmic conversion value of each serum. .
- the measurement value of the miRNA expression level in the second experiment was corrected.
- Example 3 Experiments similar to those in Example 2 were performed using the standard materials for examples shown in SEQ ID NOs: 15 to 17 as standard materials.
- Example 4 shows the results of calculating the regression line of the measured value of the quantity.
- the horizontal axis represents the first expression (reference sample, no correction), and the vertical axis represents the second expression (corrected specimen, with correction).
- the scatter plot (serum A) was shown in FIG. 4 ((A): correction result of Example 2 and (B): correction result of Comparative Example 2).
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Computational Biology (AREA)
- Evolutionary Biology (AREA)
- Medical Informatics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Theoretical Computer Science (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Pathology (AREA)
- Medicinal Chemistry (AREA)
- Sustainable Development (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
前記複数の各検体に、核酸長が200塩基以上の核酸である少なくとも1種の標準物質を加えた後、各検体から核酸を抽出して核酸試料を得る抽出工程;
抽出された各核酸試料中に存在する標的小型RNA及び標準物質の量をそれぞれ測定し、各検体について標的小型RNAの発現量及び標準物質の抽出量の測定値を得る、測定工程;
各検体について、標準物質の抽出量の測定値から代表値を取得する、代表値取得工程;
標準物質の抽出量に関して任意に設定された基準値と、代表値取得工程で取得された各検体の標準物質の代表値との差又は比を、各検体のための標的小型RNA発現量の補正係数として取得する、補正係数取得工程;
取得された各補正係数をそれぞれ用いて、各検体について測定された標的小型RNAの発現量を補正する補正工程;
を含む、補正方法。
(2) 標準物質の核酸長が200塩基以上1200塩基以下である(1)に記載の補正方法。
(3) 少なくとも1種の標準物質が、配列番号1~5及び15~17に示す塩基配列の核酸である標準物質から選択される少なくとも1種を含む、(2)に記載の補正方法。
(4) 2種類以上の標準物質を用いる、(1)ないし(3)のいずれか1項に記載の補正方法。
(5) 検体が体液由来検体である(1)ないし(4)のいずれか1項に記載の補正方法。
(6) 標的小型RNAがmiRNAである(1)ないし(5)のいずれか1項に記載の補正方法。
(7) 前記抽出工程における核酸試料の抽出が、フェノール・クロロホルム法により行なわれる、(1)ないし(6)のいずれか1項に記載の補正方法。
(8) 前記測定工程が、標識物質で標識された核酸試料を、支持体上に固定化された複数の標的小型RNAを捕捉するためのプローブ及び少なくとも1種の標準物質を捕捉するためのプローブと接触させてハイブリダイゼーションを行ない、各標的小型RNAの発現量及び標準物質の抽出量をシグナル強度測定値として得ることを含む、(1)ないし(7)のいずれか1項に記載の補正方法。
(9) 前記代表値取得工程で取得される代表値が、少なくとも1種の標準物質抽出量の測定値から算出された、対数値で表された平均値又は中央値である、(1)ないし(8)のいずれか1項に記載の補正方法。
(10) 前記基準値は、標準物質の抽出量に関して任意に定められた固定数値、又は、前記複数の検体から任意に選択された第1の検体について取得された標準物質抽出量の代表値である、(1)ないし(9)のいずれか1項に記載の補正方法。
(11) 前記補正工程では、
(a)前記補正係数取得工程において、前記代表値から前記基準値を減算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値から補正係数を減算すること、
(b)前記補正係数取得工程において、前記基準値から前記代表値を減算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値に補正係数を加算すること、
(c)前記補正係数取得工程において、前記代表値を前記基準値で除算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値を補正係数で除算すること、又は
(d)前記補正係数取得工程において、前記基準値を前記代表値で除算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値に補正係数を乗算すること、
により前記補正を行なう、(1)ないし(10)のいずれか1項に記載の方法。
(12) 複数の検体における標的小型RNAの発現量を比較解析するために発現量を補正する装置であって、
複数の各検体に、核酸長が200塩基以上の核酸である少なくとも1種の標準物質を加えた後、各検体から核酸を抽出することにより得られた核酸試料を用いて測定された、各検体についての標的小型RNA発現量及び標準物質抽出量の測定値を記憶する記憶手段と;
各検体について、標準物質抽出量の測定値から代表値を取得する、代表値取得手段と;
標準物質の抽出量に関して任意に設定された基準値と、前記代表値取得手段によって取得された各検体の代表値との差又は比を、各検体のための標的小型RNA発現量の補正係数としてそれぞれ取得する、補正係数取得手段と;
前記補正係数取得手段によって取得された各補正係数を用いて、当該各検体において測定された標的小型RNAの発現量の補正を行なう、補正手段と
を含む、前記装置。
(13) 前記代表値が、少なくとも1種の標準物質抽出量の測定値から算出された、対数値で表された平均値又は中央値である、(12)に記載の装置。
(14) 標的小型RNAがmiRNAである(12)又は(13)に記載の装置。
(15) 前記補正手段は、
(a)前記補正係数取得手段において、前記代表値から前記基準値を減算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値から補正係数を減算すること、
(b)前記補正係数取得手段において、前記基準値から前記代表値を減算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値に補正係数を加算すること、
(c)前記補正係数取得手段において、前記代表値を前記基準値で除算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値を補正係数で除算すること、又は
(d)前記補正係数取得手段において、前記基準値を前記代表値で除算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値に補正係数を乗算すること、
により前記補正を行う、(12)ないし(14)のいずれか1項に記載の装置。
(16) 前記記憶手段に記憶される、複数の検体における標的小型RNAの発現量及び標準物質抽出量の測定値は、標識物質で標識された核酸試料を、支持体上に固定化された複数の標的小型RNAを捕捉するためのプローブ及び少なくとも1種の標準物質を捕捉するためのプローブと接触させてハイブリダイゼーションを行ない、各標的小型RNAの発現量及び標準物質の抽出量をシグナル強度測定値としてそれぞれ測定した値である、(12)ないし(15)のいずれか1項に記載の装置。
(17) 複数の検体間で標的小型RNAの発現量を比較解析するための発現量の補正をするために、1又は複数のコンピューターに、
複数の各検体に、核酸長が200塩基以上の核酸である少なくとも1種の標準物質を加えた後、各検体から核酸を抽出することにより得られた核酸試料を用いて、各核酸試料中に存在する標的小型RNA及び標準物質の量をそれぞれ測定し、各検体について標的小型RNAの発現量及び標準物質の抽出量の測定値を得る、測定工程;
各検体について、標準物質の抽出量の測定値から代表値を取得する、代表値取得工程;
標準物質の抽出量に関して任意に設定された基準値と、代表値取得工程で取得された各検体の標準物質の代表値との差又は比を、各検体のための標的小型RNA発現量の補正係数として取得する、補正係数取得工程;及び
取得された各補正係数をそれぞれ用いて、各検体について測定された標的小型RNAの発現量を補正する補正工程
を実行させるためのプログラム。
(18) 複数の検体間で標的小型RNAの発現量を比較解析するための発現量の補正をするために、1又は複数のコンピューターを、
複数の各検体に、核酸長が200塩基以上の核酸である少なくとも1種の標準物質を加えた後、各検体から核酸を抽出することにより得られた核酸試料を用いて測定された、各検体についての標的小型RNA発現量及び標準物質抽出量の測定値を記憶する記憶手段;
各検体について、標準物質抽出量の測定値から代表値を取得する、代表値取得手段;
標準物質の抽出量に関して任意に設定された基準値と、前記代表値取得手段によって取得された各検体の代表値との差又は比を、各検体のための標的小型RNA発現量の補正係数としてそれぞれ取得する、補正係数取得手段;及び
前記補正係数取得手段によって取得された各補正係数を用いて、当該各検体において測定された標的小型RNAの発現量の補正を行なう、補正手段
として機能させるためのプログラム。
(19) (17)又は(18)に記載のプログラムを記録した、コンピューター読み取り可能な記録媒体。
(20) 複数の標的小型RNAを捕捉するためのプローブと、配列番号1~5及び15~17に示す塩基配列の核酸である標準物質から選択される少なくとも1種の標準物質を捕捉するためのプローブとが固定化された支持体を含む、小型RNA発現解析用チップ。
本発明では、標的小型RNAの発現量を比較解析するための抽出工程及び測定工程において、標的小型RNAに対して標準物質が一定の含量で存在する。特に、抽出工程において、核酸である標準物質が標的小型RNAと同様の抽出効率で抽出されることが好ましい。
(1)GC含率が30~70%の範囲であること。
(2)Tm値が10℃以上95℃以下であること。
本発明の方法で用いることができる検体としては、特に限定されないが、例えば、血液、血清、血漿、尿、便、髄液、唾液、ぬぐい液、脳脊髄液、汗、涙液、精液、リンパ液、関節液などの各種組織液又は体液、各種組織、細胞の凍結検体やパラフィン包埋検体(FFPE)又はその切片、細胞や組織を培養した培養液など、生体から分離された検体、すなわち生体試料が挙げられる。また、各種飲食物又はその希釈物等を挙げることができる。一定量の検体に一定量の標準物質を添加して用いるので、特に体液であることが好適である。また、比較解析する複数の検体は、異なる組織に由来する複数の検体でもよいし、異なる生体から分離された同一の組織に由来する複数の検体でもよく、また、同一組織内の異なる部位(例えば、腫瘍等の病変部と非病変部)に由来する複数の検体でもよい。
本発明の方法では、検体の一定量に対して標準物質を一定量加える。この場合の量の単位については、特に限定されるものではなく、重さであっても、容積であってもよい。また、標準核酸の溶液を一定量加える際に、標準物質の溶液の一定量を測定する際の単位は、重量、容量、モル数など、どんな単位でもよい。標準物質の量を測定する方法としては、吸光度測定法、電気泳動法、カラム法、キャピラリー電気泳動法など既知の各種方法をとることができる。
本発明では、標準物質の共存下、各検体から標的小型RNAを含む核酸の抽出処理が行われる(抽出工程)。各検体に添加された標準物質も標的小型RNAと共に抽出されるので、この抽出工程で各検体から得られる核酸試料には、標的小型RNA及び標準物質が含まれる。
(a)50容量%超過のフェノール、
(b)溶液の総量に対して3~10容量%の多価アルコール、
(c)溶液の総量に対して0.5~2.0M濃度のグアニジニウム塩、
(d)溶液の総量に対して0.1~0.5M濃度のチオシアン酸塩、及び
(e)溶液のpHを4~6に維持するための緩衝剤、
を含む抽出溶液を使用することが好ましい。また、核酸が抽出されやすいように、抽出溶液に各種塩を添加してもよい。
本発明の方法では、以上の抽出工程により複数の検体からそれぞれ抽出された核酸試料に含まれる標的小型RNA及び標準物質の量を測定する(測定工程)。
本発明の方法では、次いで、各検体について、抽出された核酸試料中の標準物質存在量の測定値から、代表値を取得する(代表値取得工程)。なお、核酸試料中の標準物質存在量という語と、検体からの標準物質抽出量という語は同義であり、本明細書においては、「標準物質存在量」と「標準物質抽出量」という語を同じ意味で用いる。核酸試料中の標準物質存在量、及び検体からの標準物質抽出量を、単に「標準物質量」ということがある。すなわち、本明細書において、「検体の標準物質量」といった場合、検体中に添加した標準物質量ではなく、核酸試料中の標準物質存在量又は検体からの標準物質抽出量を意味している。
次いで、代表値取得工程で得られた各検体の標準物質抽出量の代表値と、標準物質抽出量に関して任意に設定された基準値とを用いて、標的小型RNAの発現量の補正に用いる補正係数を取得する(補正係数取得工程)。この補正係数取得工程では、代表値と基準値との差を利用して補正係数を求めてもよいし、代表値と基準値との比を利用して補正係数を求めてもよい。特に限定されないが、対数変換していない代表値を用いる場合には比を利用して補正係数を求め、対数で表された代表値を用いる場合には差を利用して補正係数を求めることが好ましい。以下、これら2通りの工程について説明する。
補正係数取得工程-1は、標準物質抽出量の代表値と基準値との差を利用する方法である。本工程には、下記の1-1.基準検体取得法、又は1-2.固定数値補正法を適用し得る。
解析対象とする複数の検体の中から任意に1検体(第1の検体)を選択し、これを「基準検体」とする。残りの1又はそれ以上の検体(第2以降の検体)が、「補正される検体」となる。
c1-1=(基準検体の標準物質量の代表値(基準値))
-(補正される検体の標準物質量の代表値) ・・・式1
c1-1’=(補正される検体の標準物質量の代表値)
-(基準検体の標準物質量の代表値(基準値)) ・・・式1’
nは、支持体上の標準物質捕捉プローブ固定化領域の総数、
Ajは、基準検体における、j番目(1≦j≦n)の標準物質捕捉プローブ固定化領域からのシグナル測定値、
Xjは、第2の検体における、j番目(1≦j≦n)の標準物質捕捉プローブ固定化領域からのシグナル測定値、
である。
標準物質量の代表値について、すべての検体において一定の数値をとるものとあらかじめ仮定する方法である。すなわち、固定した数値を「基準値」とし、この固定数値と各検体の標準物質量の代表値との差を得て、この差を補正係数として利用する。当該方法では、1-1.に示した「基準検体」は存在しないので、比較解析対象となる複数の検体の全てが「補正される検体」となり、比較解析対象となる検体の数だけ補正係数が取得されることになる。
r1-2=(固定数値(基準値))-(補正される検体の標準物質量の代表値)
・・・式3
r1-2'=(補正される検体の標準物質量の代表値)-(固定数値(基準値))
・・・式3’
αは基準値(固定数値)、
nは、支持体上の標準物質捕捉プローブ固定化領域の総数、
Yjは、検体における、j番目(1≦j≦n)の標準物質捕捉プローブ固定化領域からのシグナル測定値、
である。
補正係数取得工程-2は、標準物質量の代表値と基準値との比を利用する方法である。本工程には、下記の2-1.基準検体取得法、又は2-2.固定数値補正法を適用し得る。
解析対象とする複数の検体の中から任意に1検体(第1の検体)を選択し、これを「基準検体」とする。残りの第2以降の検体が、「補正される検体」となる。
c2-1=(基準検体の標準物質量の代表値(基準値))
/(補正される検体の標準物質量の代表値) ・・・式5
c2-1'=(補正される検体の標準物質量の代表値)
/(基準検体の標準物質量の代表値(基準値)) ・・・式5’
nは、支持体上の標準物質捕捉プローブ固定化領域の総数、
Ajは、基準検体における、j番目(1≦j≦n)の標準物質捕捉プローブ固定化領域からのシグナル測定値、
Xjは、第2の検体における、j番目(1≦j≦n)の標準物質捕捉プローブ固定化領域からのシグナル測定値、
である。
標準物質量の代表値について、すべての検体において一定の数値をとるものとあらかじめ仮定する方法である。すなわち、固定した数値を「基準値」とし、この固定数値と各検体の標準物質量の代表値との比を得て、この比を補正係数として利用する。当該方法では、2-1.に示した「基準検体」は存在しないので、比較解析対象となる複数の検体の全てが「補正される検体」となり、比較解析対象となる検体の数だけ補正係数が取得されることになる。
r2-2=(固定数値(基準値))/(補正される検体の標準物質量の代表値)
・・・式7
r2-2'=(補正される検体の標準物質量の代表値)/(固定数値(基準値))
・・・式7’
αは固定数値、
nは、支持体上の標準物質捕捉プローブ固定化領域の総数、
Yjは、検体における、j番目(1≦j≦n)の標準物質捕捉プローブ固定化領域からのシグナル測定値、
である。
次いで、補正係数取得工程-1又は補正係数取得工程-2で得られた補正係数を用いて、補正工程-1又は補正工程-2の方法を利用して、補正される検体における標的小型RNAの発現量の補正を行う。
補正工程-1は、補正係数取得工程-1で得られた補正係数を用いて標的小型RNAの発現量の補正を行なう方法であり、標的小型RNAの発現量に補正係数を加算、又は該発現量から補正係数を減算することによって補正を行なう。本工程には、補正係数取得工程-1の基準検体取得法と固定数値補正法にそれぞれ対応した2通りの補正方法がある。
第2以降の検体における標的小型RNAの発現量の補正は、第2以降の検体のための補正係数をそれぞれ用いて実施する。つまり、第2の検体について標的小型RNAの発現量を補正する場合には、第2の検体のための補正係数(c21-1又はc21-1’)を使用し、第3の検体について標的miRNA発現量を補正する場合には、第3の検体のための補正係数(c31-1又はc31-1’)を使用する。
標的小型RNA発現量の補正は、代表値と固定数値(基準値)との差によって得られた補正係数をそれぞれ用いて実施する。つまり、ある検体について標的小型RNA発現量を補正する場合には、その検体のための補正係数(r1-2又はr1-2’)を使用する。
補正工程-2は、補正係数取得工程-2で得られた補正係数を用いて標的小型RNAの発現量の補正を行なう方法であり、標的小型RNAの発現量を補正係数で除算、又は該発現量に補正係数を乗算することによって補正を行なう。本工程にも、補正係数取得工程-2の基準検体取得法と固定数値補正法にそれぞれ対応した2通りの補正方法がある。
第2以降の検体における標的小型RNAの発現量の補正は、第2以降の検体のための補正係数をそれぞれ用いて実施する。つまり、第2の検体について標的小型RNAの発現量を補正する場合には、第2の検体のための補正係数(c22-1又はc22-1’)を使用し、第3の検体について標的小型RNA発現量を補正する場合には、第3の検体のための補正係数(c32-1又はc32-1’)を使用する。
標的小型RNA発現量の補正は、固定数値(基準値)との比によって得られた補正係数をそれぞれ用いて実施する。つまり、ある検体について標的小型RNA発現量を補正する場合には、その検体のための補正係数(r2-2又はr2-2’)を使用する。
補正済みの標的小型RNA発現量を使用して、複数の検体間で標的小型RNA発現量を対比する。基準検体取得法により補正が実施される場合には、基準検体とした第1の検体の標的小型RNA発現量は補正を受けていないため、例えば第1の検体と第2の検体との間の対比は、補正されていない第1の検体の標的小型RNA発現量と、補正済みの第2の検体の標的小型RNA発現量との間での対比となるが、対比する検体のうちの少なくとも1つは必ず補正された検体となる。従って、「補正済みの標的小型RNA発現量により、複数の体液検体間で標的小型RNA発現量を対比する」といった場合には、補正されていない基準検体と補正された他の検体との間で対比する態様も包含される。
複数の各検体に、核酸長が200塩基以上の核酸である少なくとも1種の標準物質を加えた後、各検体から核酸を抽出して得られた核酸試料を用いて測定された、各検体についての標的小型RNA発現量及び標準物質抽出量の測定値を記憶する記憶手段と;
各検体について、標準物質抽出量の測定値から、代表値、好ましくは対数値で表された代表値を取得する、代表値取得手段と;
標準物質の抽出量に関して任意に設定された基準値と、前記代表値取得手段によって取得された各検体の代表値との差又は比を、各検体のための標的小型RNA発現量の補正係数としてそれぞれ取得する、補正係数取得手段と;
前記補正係数取得手段によって取得された各補正係数を用いて、当該各検体において測定された標的小型RNAの発現量の補正を行なう、補正手段と
を含む。
複数の各検体に、核酸長が200塩基以上の核酸である少なくとも1種の標準物質を加えた後、各検体から核酸を抽出して得られた核酸試料を用いて測定された、各検体についての標的小型RNA発現量及び標準物質抽出量の測定値を記憶する記憶手段と;
各検体について、標準物質抽出量の測定値から、代表値、好ましくは対数値で表された代表値を取得する、代表値取得手段と;
任意に選択された第1の検体を基準検体とし、基準検体の標準物質抽出量の代表値を基準値として、当該基準値と残りの第2以降の検体の代表値との差又は比を、当該第2以降の検体のための補正係数としてそれぞれ取得する、補正係数取得手段と;
前記補正係数取得手段によって取得された第2以降の検体のための補正係数をそれぞれ用いて、第2以降の検体において測定された標的小型RNAの発現量の補正を行なう、補正手段と
を含む。
複数の各検体に、核酸長が200塩基以上の核酸である少なくとも1種の標準物質を加えた後、各検体から核酸を抽出して得られた核酸試料を用いて測定された、各検体についての標的小型RNA発現量及び標準物質抽出量の測定値を記憶する記憶手段と;
各検体について、標準物質抽出量の測定値から、代表値、好ましくは対数値で表された代表値を取得する、代表値取得手段と;
固定数値を基準値として、当該基準値と各検体の代表値との差は比を、当該検体のための補正係数としてそれぞれ取得する、補正係数取得手段と;
前記補正係数取得手段によって取得された各検体のための補正係数をそれぞれ用いて、各検体において測定された標的小型RNAの発現量の補正を行なう、補正手段と
を含む。
核酸長が200塩基以上の核酸である実施例用の標準物質として、独立行政法人産業技術総合研究所から定量解析用リボ核酸(RNA)水溶液として購入できる、5種類のRNA水溶液:試料名RNA500-A(配列番号1)、RNA500-B(配列番号2)、RNA500-C(配列番号3)(以上、いずれも核酸長約500塩基のRNA)、RNA1000-A(配列番号4)、及びRNA1000-B(配列番号5)(以上、いずれも核酸長約1000塩基のRNA)からなる認証標準物質 NMIJ CRM 6204-a、並びに本願発明者らが設計しユーロフィンジェノミクス社にて委託合成した3種類のRNA、hsncs_071028(配列番号15)、hsncs_404161(配列番号16)、hsncs_647981(配列番号17)(以上、いずれも核酸長約200塩基のRNA)のRNAを使用した。
標的小型RNAとして、miRBaseリリース19から入手した2555種のヒトmiRNAを選択し、その相補配列を有するDNAを標的小型RNA捕捉プローブとして用いた(http://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml)。
(DNAマイクロアレイの作製)
3'末端にアミノ基を導入した上記2555種の標的小型RNA(miRNA)捕捉プローブ及び全17種の標準物質捕捉プローブを、東レ株式会社製の「3D-Gene」(登録商標)基板(3000柱基板)の凸部に固定化し、DNAマイクロアレイを作製した。なお、標準物質捕捉プローブは各8点で固定を行った。このDNAマイクロアレイを用いて以下の実験を行なった。
血清検体300μLに対し、RNA抽出試薬である3D-Gene RNA extraction reagent from liquid sample(東レ)を900μL混合し、実施例においては、上記した実施例用の標準物質であるRNA500-A、RNA500-B、RNA500-C、RNA1000-A、RNA1000-B、hsncs_071028、hsncs_404161及びhsncs_647981のうちの少なくとも1種を各10fmol添加し、比較例においては、上記した比較例用標準物質であるcel-miR-39、cel-miR-54、ath-mir-159a、cel-miR-238、H2NC000001、H2NC000002、H2NC000003、H2NC000005及びH2NC000006のうちの少なくとも1種を各10fmol添加し、遠心分離操作により、上層の水層を回収し、miRNeasy mini kit(キアゲン社)を用いてRNAを精製した。
得られた標的小型RNA(miRNA)を、3D-Gene miRNA labeling kit(東レ)を用いて標識した。標識したmiRNAは、3D-Gene miRNA chip(東レ社)の標準プロトコールに従い、ハイブリダイゼーションと洗浄を行った。反応済みのDNAマイクロアレイは、マイクロアレイスキャナ(東レ社)を用いて蛍光シグナルを検出した。スキャナーの設定は、レーザー出力100%、フォトマルチプライヤーの電圧設定を42%にした。
上記の手順に従って、配列番号1~5で示される実施例用標準物質(5種全て)又は配列番号15~17で示される実施例用標準物質(3種全て)を市販の血清検体300μLに添加し、核酸の抽出工程、及びDNAマイクロアレイによる標的小型RNA(miRNA)の発現量及び標準物質の存在量の測定工程を実施した。以上の工程は、その測定実験間での発現量の測定値の変動(ばらつき)を補正する効果を比較するために、10回繰り返して行った。
標準物質として、配列番号1~5で示される実施例用標準物質の代わりに、配列番号6~14で示される比較例用の標準物質9種を使用して、実施例1と同様の実験を行った。
3人のヒトから採取した血清A~Cの3種類を用い、標準物質として配列番号1~5で示される実施例用標準物質を使用し、各血清に対して核酸の抽出日と実験者が異なる条件で各2回(1回目、2回目)ずつ、上記の手順で血清検体への標準物質の添加と核酸の抽出工程ならびにDNAマイクロアレイによる標的小型RNAの発現量及び標準物質の存在量の測定工程を行った。標的小型RNA(miRNA)としては、実施例1と同じく、検出された約1000種類のmiRNAを用いた。
以上の操作により、2回目の実験のmiRNAの発現量の測定値の補正が実施された。
標準物質として配列番号15~17で示される実施例用標準物質を使用して、実施例2と同様の実験を実施した。
標準物質として配列番号6~9で示される比較例用標準物質を使用して、実施例2と同様の実験を実施した。
標準物質として配列番号10~14で示される比較例用標準物質を使用して、実施例2と同様の実験を実施した。
110 入力部
120 表示部
130 出力部
140 記憶部
150 制御部
160 変換部
170 解析部
Claims (20)
- 複数の検体における標的小型RNAの発現量を比較解析するための発現量の補正方法であって、
前記複数の各検体に、核酸長が200塩基以上の核酸である少なくとも1種の標準物質を加えた後、各検体から核酸を抽出して核酸試料を得る抽出工程;
抽出された各核酸試料中に存在する標的小型RNA及び標準物質の量をそれぞれ測定し、各検体について標的小型RNAの発現量及び標準物質の抽出量の測定値を得る、測定工程;
各検体について、標準物質の抽出量の測定値から代表値を取得する、代表値取得工程;
標準物質の抽出量に関して任意に設定された基準値と、代表値取得工程で取得された各検体の標準物質の代表値との差又は比を、各検体のための標的小型RNA発現量の補正係数として取得する、補正係数取得工程;
取得された各補正係数をそれぞれ用いて、各検体について測定された標的小型RNAの発現量を補正する補正工程;
を含む、補正方法。 - 標準物質の核酸長が200塩基以上1200塩基以下である請求項1に記載の補正方法。
- 少なくとも1種の標準物質が、配列番号1~5及び15~17に示す塩基配列の核酸である標準物質から選択される少なくとも1種を含む、請求項2記載の補正方法。
- 2種類以上の標準物質を用いる、請求項1ないし3のいずれか1項に記載の補正方法。
- 検体が体液由来検体である請求項1ないし4のいずれか1項に記載の補正方法。
- 標的小型RNAがmiRNAである請求項1ないし5のいずれか1項に記載の補正方法。
- 前記抽出工程における核酸試料の抽出が、フェノール・クロロホルム法により行なわれる、請求項1ないし6のいずれか1項に記載の補正方法。
- 前記測定工程が、標識物質で標識された核酸試料を、支持体上に固定化された複数の標的小型RNAを捕捉するためのプローブ及び少なくとも1種の標準物質を捕捉するためのプローブと接触させてハイブリダイゼーションを行ない、各標的小型RNAの発現量及び標準物質の抽出量をシグナル強度測定値として得ることを含む、請求項1ないし7のいずれか1項に記載の補正方法。
- 前記代表値取得工程で取得される代表値が、少なくとも1種の標準物質抽出量の測定値から算出された、対数値で表された平均値又は中央値である、請求項1ないし8のいずれか1項に記載の補正方法。
- 前記基準値は、標準物質の抽出量に関して任意に定められた固定数値、又は、前記複数の検体から任意に選択された第1の検体について取得された標準物質抽出量の代表値である、請求項1ないし9のいずれか1項に記載の補正方法。
- 前記補正工程では、
(a)前記補正係数取得工程において、前記代表値から前記基準値を減算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値から補正係数を減算すること、
(b)前記補正係数取得工程において、前記基準値から前記代表値を減算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値に補正係数を加算すること、
(c)前記補正係数取得工程において、前記代表値を前記基準値で除算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値を補正係数で除算すること、又は
(d)前記補正係数取得工程において、前記基準値を前記代表値で除算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値に補正係数を乗算すること、
により前記補正を行なう、請求項1ないし10のいずれか1項に記載の方法。 - 複数の検体における標的小型RNAの発現量を比較解析するために発現量を補正する装置であって、
複数の各検体に、核酸長が200塩基以上の核酸である少なくとも1種の標準物質を加えた後、各検体から核酸を抽出することにより得られた核酸試料を用いて測定された、各検体についての標的小型RNA発現量及び標準物質抽出量の測定値を記憶する記憶手段と;
各検体について、標準物質抽出量の測定値から代表値を取得する、代表値取得手段と;
標準物質の抽出量に関して任意に設定された基準値と、前記代表値取得手段によって取得された各検体の代表値との差又は比を、各検体のための標的小型RNA発現量の補正係数としてそれぞれ取得する、補正係数取得手段と;
前記補正係数取得手段によって取得された各補正係数を用いて、当該各検体において測定された標的小型RNAの発現量の補正を行なう、補正手段と
を含む、前記装置。 - 前記代表値が、少なくとも1種の標準物質抽出量の測定値から算出された、対数値で表された平均値又は中央値である、請求項12に記載の装置。
- 標的小型RNAがmiRNAである請求項12又は13に記載の装置。
- 前記補正手段は、
(a)前記補正係数取得手段において、前記代表値から前記基準値を減算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値から補正係数を減算すること、
(b)前記補正係数取得手段において、前記基準値から前記代表値を減算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値に補正係数を加算すること、
(c)前記補正係数取得手段において、前記代表値を前記基準値で除算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値を補正係数で除算すること、又は
(d)前記補正係数取得手段において、前記基準値を前記代表値で除算した値を補正係数として取得する場合、標的小型RNAの発現量の測定値に補正係数を乗算すること、
により前記補正を行う、請求項12ないし14のいずれか1項に記載の装置。 - 前記記憶手段に記憶される、複数の検体における標的小型RNAの発現量及び標準物質抽出量の測定値は、標識物質で標識された核酸試料を、支持体上に固定化された複数の標的小型RNAを捕捉するためのプローブ及び少なくとも1種の標準物質を捕捉するためのプローブと接触させてハイブリダイゼーションを行ない、各標的小型RNAの発現量及び標準物質の抽出量をシグナル強度測定値としてそれぞれ測定した値である、請求項12ないし15のいずれか1項に記載の装置。
- 複数の検体間で標的小型RNAの発現量を比較解析するための発現量の補正をするために、1又は複数のコンピューターに、
複数の各検体に、核酸長が200塩基以上の核酸である少なくとも1種の標準物質を加えた後、各検体から核酸を抽出することにより得られた核酸試料を用いて、各核酸試料中に存在する標的小型RNA及び標準物質の量をそれぞれ測定し、各検体について標的小型RNAの発現量及び標準物質の抽出量の測定値を得る、測定工程;
各検体について、標準物質の抽出量の測定値から代表値を取得する、代表値取得工程;
標準物質の抽出量に関して任意に設定された基準値と、代表値取得工程で取得された各検体の標準物質の代表値との差又は比を、各検体のための標的小型RNA発現量の補正係数として取得する、補正係数取得工程;及び
取得された各補正係数をそれぞれ用いて、各検体について測定された標的小型RNAの発現量を補正する補正工程
を実行させるためのプログラム。 - 複数の検体間で標的小型RNAの発現量を比較解析するための発現量の補正をするために、1又は複数のコンピューターを、
複数の各検体に、核酸長が200塩基以上の核酸である少なくとも1種の標準物質を加えた後、各検体から核酸を抽出することにより得られた核酸試料を用いて測定された、各検体についての標的小型RNA発現量及び標準物質抽出量の測定値を記憶する記憶手段;
各検体について、標準物質抽出量の測定値から代表値を取得する、代表値取得手段;
標準物質の抽出量に関して任意に設定された基準値と、前記代表値取得手段によって取得された各検体の代表値との差又は比を、各検体のための標的小型RNA発現量の補正係数としてそれぞれ取得する、補正係数取得手段;及び
前記補正係数取得手段によって取得された各補正係数を用いて、当該各検体において測定された標的小型RNAの発現量の補正を行なう、補正手段
として機能させるためのプログラム。 - 請求項17又は18に記載のプログラムを記録した、コンピューター読み取り可能な記録媒体。
- 複数の標的小型RNAを捕捉するためのプローブと、配列番号1~5及び15~17に示す塩基配列の核酸である標準物質から選択される少なくとも1種の標準物質を捕捉するためのプローブとが固定化された支持体を含む、小型RNA発現解析用チップ。
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15/529,165 US10622093B2 (en) | 2014-11-26 | 2015-11-25 | Method and device for correcting level of expression of small RNA |
BR112017009009-0A BR112017009009A2 (ja) | 2014-11-26 | 2015-11-25 | A correcting method and a device of the amount of revelation of small RNA |
EP15863331.3A EP3225689B1 (en) | 2014-11-26 | 2015-11-25 | Method and device for correcting level of expression of small rna |
CN201580062576.3A CN107109397B (zh) | 2014-11-26 | 2015-11-25 | 小型rna的表达量的修正方法和装置 |
KR1020177010333A KR102380453B1 (ko) | 2014-11-26 | 2015-11-25 | 소형 rna의 발현량의 보정 방법 및 장치 |
JP2015558051A JP6705171B2 (ja) | 2014-11-26 | 2015-11-25 | 小型rnaの発現量の補正方法及び装置 |
CA2974433A CA2974433C (en) | 2014-11-26 | 2015-11-25 | Method and device for correcting level of expression of small rna |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014238451 | 2014-11-26 | ||
JP2014-238451 | 2014-11-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2016084848A1 true WO2016084848A1 (ja) | 2016-06-02 |
Family
ID=56074400
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2015/083079 WO2016084848A1 (ja) | 2014-11-26 | 2015-11-25 | 小型rnaの発現量の補正方法及び装置 |
Country Status (8)
Country | Link |
---|---|
US (1) | US10622093B2 (ja) |
EP (1) | EP3225689B1 (ja) |
JP (1) | JP6705171B2 (ja) |
KR (1) | KR102380453B1 (ja) |
CN (1) | CN107109397B (ja) |
BR (1) | BR112017009009A2 (ja) |
CA (1) | CA2974433C (ja) |
WO (1) | WO2016084848A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2020519256A (ja) * | 2017-01-30 | 2020-07-02 | ジーエムアイ−グレガー−メンデル−インスティテュート フォー モレキュラー プランツェンバイオロジー ゲゼルシャフト ミット ベシュレンクテル ハフツング | 配列データの正規化のための新規のspike inオリゴヌクレオチド |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116515976B (zh) * | 2023-06-16 | 2023-10-31 | 上海精翰生物科技有限公司 | 一种转录组测序的校正方法及其试剂盒 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007075095A (ja) * | 2005-04-11 | 2007-03-29 | Mitsubishi Rayon Co Ltd | miRNA搭載マイクロアレイ |
JP2007097429A (ja) * | 2005-09-30 | 2007-04-19 | Mitsubishi Rayon Co Ltd | non−codingRNAの検出データ補正方法 |
JP2010119331A (ja) * | 2008-11-19 | 2010-06-03 | National Institute Of Advanced Industrial Science & Technology | 核酸標準物質 |
JP2010519893A (ja) * | 2007-03-02 | 2010-06-10 | エスアイアールエス‐ラブ ゲーエムベーハー | 遺伝子発現分析データの正規化のための参照遺伝子 |
WO2011145614A1 (ja) * | 2010-05-17 | 2011-11-24 | 独立行政法人産業技術総合研究所 | 核酸標準物質検出用プローブの設計方法、核酸標準物質検出用プローブ及び当該核酸標準物質検出用プローブを有する核酸検出系 |
JP2014007995A (ja) * | 2012-06-29 | 2014-01-20 | Toray Ind Inc | 小型rna発現量の比較解析方法 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5229895B2 (ja) | 1972-03-31 | 1977-08-04 | ||
EP3118334A1 (en) * | 2009-11-04 | 2017-01-18 | DiamiR, LLC | Methods of using small rna from bodily fluids for diagnosis and monitoring of neurodegenerative diseases |
US10600503B2 (en) | 2011-08-04 | 2020-03-24 | Georgetown University | Systems medicine platform for personalized oncology |
-
2015
- 2015-11-25 CN CN201580062576.3A patent/CN107109397B/zh active Active
- 2015-11-25 CA CA2974433A patent/CA2974433C/en active Active
- 2015-11-25 KR KR1020177010333A patent/KR102380453B1/ko active IP Right Grant
- 2015-11-25 EP EP15863331.3A patent/EP3225689B1/en active Active
- 2015-11-25 BR BR112017009009-0A patent/BR112017009009A2/ja not_active IP Right Cessation
- 2015-11-25 JP JP2015558051A patent/JP6705171B2/ja active Active
- 2015-11-25 US US15/529,165 patent/US10622093B2/en active Active
- 2015-11-25 WO PCT/JP2015/083079 patent/WO2016084848A1/ja active Application Filing
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007075095A (ja) * | 2005-04-11 | 2007-03-29 | Mitsubishi Rayon Co Ltd | miRNA搭載マイクロアレイ |
JP2007097429A (ja) * | 2005-09-30 | 2007-04-19 | Mitsubishi Rayon Co Ltd | non−codingRNAの検出データ補正方法 |
JP2010519893A (ja) * | 2007-03-02 | 2010-06-10 | エスアイアールエス‐ラブ ゲーエムベーハー | 遺伝子発現分析データの正規化のための参照遺伝子 |
JP2010119331A (ja) * | 2008-11-19 | 2010-06-03 | National Institute Of Advanced Industrial Science & Technology | 核酸標準物質 |
WO2011145614A1 (ja) * | 2010-05-17 | 2011-11-24 | 独立行政法人産業技術総合研究所 | 核酸標準物質検出用プローブの設計方法、核酸標準物質検出用プローブ及び当該核酸標準物質検出用プローブを有する核酸検出系 |
JP2014007995A (ja) * | 2012-06-29 | 2014-01-20 | Toray Ind Inc | 小型rna発現量の比較解析方法 |
Non-Patent Citations (1)
Title |
---|
See also references of EP3225689A4 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2020519256A (ja) * | 2017-01-30 | 2020-07-02 | ジーエムアイ−グレガー−メンデル−インスティテュート フォー モレキュラー プランツェンバイオロジー ゲゼルシャフト ミット ベシュレンクテル ハフツング | 配列データの正規化のための新規のspike inオリゴヌクレオチド |
JP7044270B2 (ja) | 2017-01-30 | 2022-03-30 | ジーエムアイ-グレガー-メンデル-インスティテュート フォー モレキュラー プランツェンバイオロジー ゲゼルシャフト ミット ベシュレンクテル ハフツング | 配列データの正規化のための新規のspike inオリゴヌクレオチド |
Also Published As
Publication number | Publication date |
---|---|
CN107109397A (zh) | 2017-08-29 |
BR112017009009A2 (ja) | 2018-01-30 |
JP6705171B2 (ja) | 2020-06-03 |
KR20170081169A (ko) | 2017-07-11 |
KR102380453B1 (ko) | 2022-03-31 |
EP3225689A1 (en) | 2017-10-04 |
CA2974433A1 (en) | 2016-06-02 |
CA2974433C (en) | 2023-03-07 |
US20170351809A1 (en) | 2017-12-07 |
US10622093B2 (en) | 2020-04-14 |
EP3225689B1 (en) | 2020-07-29 |
JPWO2016084848A1 (ja) | 2017-10-12 |
CN107109397B (zh) | 2020-11-27 |
EP3225689A4 (en) | 2018-06-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
de Planell-Saguer et al. | Detection methods for microRNAs in clinic practice | |
US11408027B2 (en) | Method of evaluating quality of miRNA derived from body fluid | |
JP7363478B2 (ja) | 体液検体の品質評価方法 | |
KR102350747B1 (ko) | miRNA 발현량의 비교 해석 방법 및 장치 | |
JP6705171B2 (ja) | 小型rnaの発現量の補正方法及び装置 | |
CN110628914A (zh) | 与乳腺癌相关的lncRNA标志物及其检测引物和应用 | |
JP5998674B2 (ja) | 小型rna発現量の比較解析方法 | |
US20140099633A1 (en) | DETECTION OF miRNA USING CAPILLARY ELECTROPHORESIS WITH LASER-INDUCED FLUORESCENCE DETECTION | |
WO2021132231A1 (ja) | 血清検体の品質評価方法 | |
JP2021153487A (ja) | 血液検体からのmiRNAを含む血清検体の調製方法 | |
JP2010094075A (ja) | 小型rnaを検出する方法 | |
Lin et al. | MicroRNA Detection Methods for Mammalian Cell Lines and Their Applications in Therapeutic Protein Production |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
ENP | Entry into the national phase |
Ref document number: 2015558051 Country of ref document: JP Kind code of ref document: A |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 15863331 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 20177010333 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15529165 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REG | Reference to national code |
Ref country code: BR Ref legal event code: B01A Ref document number: 112017009009 Country of ref document: BR |
|
REEP | Request for entry into the european phase |
Ref document number: 2015863331 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2974433 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 112017009009 Country of ref document: BR Kind code of ref document: A2 Effective date: 20170428 |