CN106033087B - The method system of built-in property standard curve detection substance molecular number - Google Patents

Abstract

The application discloses the method and apparatus that a kind of functional unit to biological sample carries out absolute quantitation detection.The application includes the calculating analysis of property standard curve and biological substance molecular number built in manufacture.The application is further to the application in biological detecting method, for example, in the application of biochip, PCR arrays, microarray and ELISA arrays.

Description

The method system of built-in property standard curve detection substance molecular number

Technical field

The present invention relates in biosensor detection system new design and calculation and analysis methods, so as to reach to sampleIn functional constituent molecule copy number carry out absolute quantitation detection.

Background technology

The success of the Human Genome Project has paved solid foundation to understand the function of each gene.However, we instituteThe next challenge faced is functional genomics research, because we lack strong and reliable expression analysis and method to commentEstimate apparent gene group (referring to bibliography 1).In gene expression research, many methods are developed to measure expression,As RT-PCR (referring to bibliography 2), DNA chip (referring to bibliography 3-5) and sequencing are used for (referring to bibliography 5-8)The expression of rna level measures, Western blotting (referring to bibliography 9), ELISA (referring to bibliography 10) and protein-chip(referring to bibliography 11) is analyzed for protein expression.But they cannot meet functional genomics research to expression pointThe needs of analysis (referring to bibliography 1,12,13).

Causing the ultimate impediment of transcriptional profile analysis difficulty, to come from the expression data of most experimental results be relatively fixedQuantized data (for example, disease is compared with normal) rather than Absolute quantification data (referring to bibliography 14-20).This be byBig data changes into a severe bottleneck of knowledge (BD2K), so, the current BD2K item numbers of National Institutes of HealthCall the standardization of data/metadata together (referring to bibliography 21).Therefore, we can be to expression there is an urgent need to an efficient methodSpectrum carries out absolute quantitation detection.

Polymerase chain reaction (PCR) is a kind of widely used biomedical technology, which usually leads in gene quantificationDomain is referred to as the golden standard for measuring gene expression dose.However, most of quantitative PCR analysis data belong to related data.ThoughRight digital pcr (dPCR) provides a kind of new approach to measure the copy number of specific nucleic acid (referring to bibliography 22), butSaturation easily occurs for dPCR, causes the failure of an experiment, and dPCR both the advantage of cost free efficiency nor high-throughput techniques (referring to ginsengExamine document 23).Therefore, PCR/dPCR cannot meet needs of the functional genomics research to express spectra quantitative analysis.

Sequencing technologies also have been used for gene expression abundance analysis.The method of early development is the gene table based on sequence labelUp to serial analysis (SAGE) (referring to bibliography 24,25) and large-scale parallel sequencing technology (MPSS) (referring to bibliography26).Nearest next-generation sequencing technologies make it possible that (RNA-seq) (referring to bibliography 6-8) is sequenced in RNA.But RNA is surveyedSequence has and the associated deviation of Transcript Length, it is impossible to all mRNA regions of uniform fold (referring to bibliography 27).RNA-seqBasic principle be that the expression of each RNA is weighed by the way that reading of the fragment map to the gene is sequenced(RPKM：Reads number in every million reads on map to every thousand bases of extron), it is expected RPKM can directly with it is a certainGene expression abundance is associated (referring to bibliography 28).Although RPKM is known as absolute gene expression data, and be used to turnThe foundation (referring to bibliography 29,30) of record group database, but it is in no way intended to reflect gene copy number.Because mRNA is richThe accurate quantification of degree is most important, and various statistical methods are used to the variance in simulation biocenose sample, it is intended to improve RNA-The overall fit of seq enumeration datas (referring to bibliography 31-33).The Encyclopedia of DNA Elements(ENCODE) Consortium (referring to bibliography 34), an international conjunction subsidized by American National human genome research instituteMake research project, also had been working hard attempt the copy number for finding out transcription from RPKM/FPKM information since 2003, but produce effectsLittle (referring to bibliography 35-37).Dr.Lior Pachter are pointed out in a comment on April 30th, 2014 (referring to ginsengExamine document 37), the work that best connection transcription copy number and RNA-seq are measured is Marinov et al. " from unicellular to thinIt transcribes in born of the same parents storehouse：The careful work made in the randomness of gene expression and RNA montages " (referring to a bibliography 38) text.The workMake to complete (spike-in methods are described in detail see microarray part) by using spike-in methods.However, this work is appointed so " veryIt is undesirable, because it depends on the quantitative accuracy of spike-in and is not deposited between spike-in RNA and endogenous RNAIn the hypothesis of systematic divergence.If these hypothesis are wrong, the mRNA numbers of each cell calculated just have systemError presence " (referring to bibliography 38).In fact these assume that really non-a hundred percent is correct (referring to bibliography 37,39),Therefore, sequencing technologies find the approach of definite gene copy number not yet.

Genetic chip is also known as DNA microarray, is first quick, high-throughput techniques (ginseng that gene expression quantifies fieldSee reference document 3), it is that research gene expression profile is most strong and one of most widely used instrument is (referring to bibliography 4,5,40-47), but genetic chip is also faced with identical challenge always：The data obtained is Relative quantification data rather than Absolute quantification numberAccording to, thus data normalization becomes impossible (referring to bibliography 14-20).The basic principle of expressing gene chip is to pass throughBiosensor detection platform based on affinity measures the signal strength of each RNA, judges its expression with this.It is sending outIn first microarray article of table, Schena et al. marks sample -1 and sample -2 respectively with color -1 and color -2, and inAdd in the exogenous mRNA of equivalent before mark in sample -1 and sample -2 respectively.To measure the gene expression of the two samplesThe change of pattern, sample signal is normalized with exogenous mRNA signals first for they, then compares two through returningOne changes the data of processing, and the multiple for obtaining gene expression changes (referring to bibliography 3).Double-colored hybridizing method can be cancelled special by targetDifference (referring to bibliography 48,49) caused by property and gene order, so as to substantially increase the essence of reliability and relative quantificationExactness is widely used (referring to bibliography 50-53).But the gene that double-colored hybridizing method can only be detected between paired sample is oppositeThe difference of expression quantity, the real strength of microarray data analysis carry out the gene expression pattern information of self-identifying different experiments.It comparesIn double-colored platform, Affymetrix companies have developed monochromatic platform, and alternative adds in various concentration known to (spike-in)Exogenous mRNA into test specimen (referring to bibliography 54).Sample signal is normalized with spike-in,So that different chip chambers can make the comparison of relative expression levels, this method is developed to assess genetic chip by further againMinimum detectable range measured frequency, i.e. chip sensitivity, can as the quality-controlling parameters hybridized every time, while be also adjusted for comeFrom the signal of low transcription (referring to bibliography 54-58)." the definitely expression of each transcription copy of Affymetrix platforms extractingIt is worth " regardless of whether the normalized through spike-in, so result can be straight between each experimental data of same probe group is usedIt connects and compares, therefore the mathematical algorithm of many complexity is referred to the data analysis of microarray, including linear model (referring to reference to text59) and Empirical Bayes offer (referring to bibliography 60).It is all these all to concentrate on using computational methods to ensure the base of designBecause array experiment is capable of detecting when the differential expression being truly present, because to identify that those have the gene of differential expression is one reallyIt is a to be involved in the problems, such as numerous inspections on a large scale, one or many test (ginsengs are carried out to each in tens thousand of a genesSee reference document 59-62).By monochromatic platform measuring and " absolute expression value " in mathematical model is used to refer to each microarrayGene copy number in the signal strength rather than biological significance of point (referring to bibliography 55,63,64).

Frigessi etc. proposes a TransCount based on Bayesian statistical model, can be from each microarray pointThe signal strength of probe calculates concentration, and the concentration of estimation seems than survey of the usually used expression ratio to transcript abundanceFixed more reliable (referring to bibliography 65).Same seminar further with TransCount methods, by a reference value comeEstimate the absolute concentration of transcription (referring to bibliography 66,67).However the discoveries such as Held, in terms of absolute concentration is determined,Spike-in methods are better than statistical model (referring to bibliography 68)；In addition, TransCount methods also do not have it is wide by the fieldGeneral use.Although it is well known that the normalized of microarray data is most important to the expression analysis in downstream, all normalizingsChange the method for processing, including spike-in, none success, reason is that they all rely on some hypothesis to make opposite normalizeProcessing (referring to bibliography 54-58,69).Quantify Microarray signals data it is exceedingly difficult come from given gene RNA amount andComplex relationship between signal strength caused by target-probe of hybridization.This relational dependence is in many factors, including mark sideMethod, hybridization conditions, gene order and target feature.Therefore, the method based on microarray is generally used for measuring the relative expression of RNA(for example, disease comparison is normal) (referring to bibliography 3,54-58,68-71).Yang et al. is every with a common reference substanceA gene provides the baseline of an expression measurement, so that the normalization of different experiments data and being compared to each other and being possibly realized (ginsengSee reference document 72), this method is further developed into again as generalized reference (universal reference, UR) RNA(referring to bibliography 73,74).But this method expends twice of cost, and to establish the UR of a real meaningIt is infeasible.Scientific research personnel pursues the standardization of microarray data with being continually striving to always, such as the quality control of microarray(MAQC) II projects of MAQC- that alliance advocates, it is intended to the bias source that assessment is each studied, it is therefore intended that understand in terms of predictionInfluence of the source and assessment endpoint signal intensity of variation in data (referring to bibliography 75).However, golden standard is stillIt is not within the foreseeable future.

In conclusion the absolute quantitation detection technique of exploitation gene expression is urgently required for current biomedical sector.

The content of the invention

Today big data to knowledge (BD2K) epoch, we there is an urgent need to gene expression copy number analysis on have oneA real breakthrough, the breakthrough particularly in high-throughput techniques (referring to bibliography 21).It is contemplated that one side of innovationJust the detection of existing gene expression relative quantification is transformed by effective method can carry out quantifying for absolute gene copy numberDetection, this target link closely National Institutes of Health BD2K projects advocate data/metadata standardization and biology doctorLearn the requirement of Data Integration.The method of the present invention is suitable for different biosensor detection system platforms, so as to utmostlyGround contributes to the needs that biological data is standardized and integrated, and valid data are provided for current biomedical large database concept.

Biosensor detection system is generally set by bio-identification part, bio-sensing part and biosensor readout meterStandby composition, using selectively interacting between some biomolecule and the characteristic with reference to (affinity), to identify molecule knotStructure and the further content of measurement analyte (referring to bibliography 76-78).Affinity type biosensor utilizes some biologiesMolecule (probe) selective binding and the principle of interaction detect the particular target analytes in biological sample (target).BelowIt is the example of some biosensor detection platforms.Based on immunoassay principle, using film as the antibody array of carrier, typically willDifferent antibodies (probe, function are capture) are fixed on the surface of the film by specifically binding to capture albumen (target).ELISA arraysIt is identical as the antibody array of carrier with using film, based on immunoassay principle, the difference is that antibody is fixed on the specific of porous plateKong Zhong.Genetic chip is the biosensor array of highly dense, be in great demand in genomics, proteomics, molecule diagnosis andThe fields such as systems biology.Round pcr falls within biosensor platform, is related to by expanding to measure specific DNA sequence dna.The basic task of biosensor platform is using probe-target specific binding complex, to generate detectable signal, signalWith target there are associated, so as to speculate the abundance of tested target, detection method can be mark or unmarked technology.

1) method of a kind of absolute quantity for measuring at least one measuring samples target molecule provided by the invention, including：It carriesIt is attached on the solid phase carrier to control sample affiliate with reference to even molecule 1 and at least there are one serial for a solid phase carrierOne of which in both one measuring samples probe molecule and at least one measuring samples target molecule, it is described at least one to be checkedOne of which and one series control sample knot in both sample probe molecule and at least one measuring samples target moleculeIt closes and matches somebody with somebody the different position that even molecule 1 is attached on solid phase carrier, one series control sample knot on each of which positionClosing has known and different copy number with even molecule 1；

2) solid phase carrier is combined at least one control sample with even molecule 2 and at least one measuring samples targetAnother contact in both molecule and at least one measuring samples probe molecule, wherein：One series control sampleIt is combined with reference to 1 specific recognition of even molecule and with reference at least one control sample with even molecule 2, direct or indirect real estateThe raw detectable control sample signal of a series, wherein the detectable control sample signal intensity in each position with it is oneSeries control sample combine it is related with known copy number of the even molecule 1 on different position, and on each position it is described extremelyFew control sample combines the no less than known control sample of the copy number with even molecule 2 and combines the copy number with even molecule 1；TogetherWhen：At least one measuring samples probe molecule specific recognition and with reference at least one measuring samples target molecule, directlyConnect or generate at least one detectable measuring samples signal indirectly, wherein each detectable measuring samples signal strength withCopy number of at least one measuring samples target molecule in the position is associated, and at least one measuring samples probeThe copy number of molecule is no less than the copy number of at least one measuring samples target molecule of the position；

3) the detectable control sample signal intensity of one series and at least one detectable measuring samples are detectedSignal strength；

4) according to one series sample signal intensity and control sample is controlled to combine with even molecule 1 in one systemKnown copy number on row different position makes the standard curve of the relation in relation to signal strength copy number；And

5) according to the standard curve, the measuring samples signal strength of at least one detection is converted into copy number,So that it is determined that the copy number of measuring samples target molecule.

One embodiment of the above method is that at least one control sample combines each molecule with even molecule 2Directly or indirectly by one or more first basic token unit marks and at least one measuring samples target molecule or extremelyAnother in few measuring samples probe molecule the two is directly or indirectly by one or more second basic token listsMeta-tag, wherein, one detectable control sample signal of series and at least one detectable measuring samples signal pointIt is not generated by the first basic token unit and the second basic token unit.

Another embodiment is each detectable control sample signal intensity divided by control sample combination at thisIt the first basic token unit number that each molecule with even molecule 2 directly or indirectly carries and described each detectable treatsSample product signal strength divided by this measuring samples target molecule or measuring samples probe molecule the direct or indirect band of each moleculeThe second basic token unit number having.

Another embodiment is that one series control sample is combined with even molecule 1, at least one control sample knotConjunction is selected from even molecule 2, at least one measuring samples target molecule and at least one measuring samples probe molecule：Molecule is selected from：(i) DNA, (ii) RNA, (iii) protein, (iv) peptide, (v) polysaccharide, (vi) chemical compound and (vii) antibody.

Another embodiment is that the solid phase carrier is selected from：(i) glass, (ii) plastics, (iii) silicon, (iv) metal,(v) nylon membrane, (vi) nitrocellulose filter, (vii) pvdf membrane, (viii) porous plate and (ix) microcosmic polystyrene bead.

Another embodiment is that the step 1) includes：One series control sample is provided to combine with even molecule1 and both at least one measuring samples probe molecule and at least one measuring samples target molecule in one of which；AndSample is controlled to combine with even molecule 1 and at least one measuring samples probe molecule and at least one one seriesOne kind in measuring samples target molecule the two is attached on solid phase carrier.

Another embodiment is that the step 1) includes：It is in situ on solid phase carrier directly to synthesize one seriesSample is controlled to combine with even molecule 1 and at least one measuring samples probe molecule and at least one measuring samples target moleculeOne kind in the two.Another embodiment is that the original position directly synthesis is photoetching synthesis, light guiding chemical synthesis or sprayInk printing.The fabricated in situ is photoetching synthesis, light guiding chemical synthesis or inkjet printing.

Another embodiment is that at least one control sample is combined with even molecule 2 by one or more first basesIn this indexing unit mark or both at least one measuring samples target molecule and at least one measuring samples probe moleculeIt is a kind of marked by one or more second basic token units, be one kind selected from following label to realize：(i) fluorescenceGroup, (ii) chemiluminescent agent, (iii) silver, (iv) affinity label, (v) photochemical agent, (vi) chromophore, (vii) enzyme and(viii) radio isotope.

Another embodiment is, the detectable control sample signal intensity of one series and at least one detectableMeasuring samples signal by microarray scanner, plate reader or Western blot scanners detect obtain.

Another embodiment is, the detectable control sample signal of one series and described at least one detectableMeasuring samples signal strength by unmarked technology detect obtain.

Another embodiment is to measure the kit of at least one measuring samples target absolute quantity, including：

With one series sample is controlled to combine with even molecule 1 and at least one measuring samples probe moleculeWith a kind of solid phase carrier in both at least one measuring samples target molecules；At least one control sample combination spouse pointSon 2；

Another kind in both at least one measuring samples probe molecule and at least one measuring samples target；With

Operation instructions.

Another embodiment is the kit, further comprises the first basic token unit and the second basic tokenUnit, wherein：The first basic token unit and the second basic token unit be respectively used to the solid phase carrier withAt least one control sample combines with even molecule 2 and at least one measuring samples target and at least one treats sampleBefore one of which contact in product probe molecule the two, at least one control sample combination spouse is directly or indirectly markedOne of which in molecule 2 and both at least one measuring samples target molecule and at least one measuring samples probe molecule；And the first basic token unit and the second basic token unit are respectively used to examine in the one series of detectionWhen the control sample signal of survey and at least one detectable sample signal, one detectable control of series is generatedSample signal processed and at least one detectable sample signal.

Another embodiment is, by computer software, according to the one series control sample signal detectedIntensity and the control sample are combined with known copy number of the even molecule 1 on the different position of one series, and making hasThe standard curve of OFF signal intensity and copy number relation, and according to the standard curve, by each position described at least oneThe measuring samples signal strength of a detection is converted into copy number, so that it is determined that the copy number of the measuring samples target molecule.

Another embodiment is the kit, wherein the solid phase carrier is selected from：(i) glass, (ii) plastics,(iii) silicon, (iv) metal, (v) nylon membrane, (vi) nitrocellulose filter, (vii) pvdf membrane, (viii) porous plate and (ix) are micro-See polystyrene bead.

The present invention also provides a kind of method for measuring at least one measuring samples nucleic acid molecule absolute quantity, including：

1) a porous plate solid phase carrier is provided, the different holes of the porous plate solid phase carrier are included to be had there are one seriesThe nucleic acid molecule of known and different copy numbers is as control sample form；

2) it is not controlled as one in measuring samples template to above-mentioned porous plate to be put at least one nucleic acid moleculeIn the hole that sample template occupies；

3) with a pair of primer that sample form complementation is controlled with one series and a pair with it is described at least one to be checkedThe primer of sample template complementation, by PCR (PCR) method expand it is one series control sample form andAt least one measuring samples template, wherein the copy number reflection control of the PCR product of one series control sample formA serial known copy number of the sample form processed before PCR reactions, wherein the PCR of at least one measuring samples templateThe copy number of product reflects at least one measuring samples mould of at least one measuring samples template before PCR reactionsThe copy number of plate；

4) with one respectively and it is one series control sample form PCR product specific bond combination coupled moleculePCR product is detected, wherein the combination coupled molecule has one or more basic token units, wherein the basic tokenUnit emits detectable signal；

5) at least one cycling of the detectable signal and the exponential phase to being located at PCR reactions are detected, makes related instituteThe standard curve of the signal strength and the relation of the known control sample form copy number of one series of control sample form is stated,Wherein described standard curve is obtained with regression analysis；

6) at least one measuring samples signal strength is converted into measuring samples template using the standard curveCopy number, so that it is determined that in the hole of place PCR reaction before the sample sample template to be checked copy number.

Measuring an embodiment of the method for at least one measuring samples nucleic acid molecule absolute quantity is, in the stepIt is rapid 4) in the combination coupled molecule be with the PCR product specific binding oligonucleotides.

Measuring another embodiment of the method for at least one measuring samples nucleic acid molecule absolute quantity is, the widowNucleotide is dual labelled probe molecule, and the double labelling includes being connected to the fluorogen of probe molecule 5' ends and is connected to probeThe quencher of molecule 3' ends.

Measuring the further embodiment of the method for at least one measuring samples nucleic acid molecule absolute quantity is, whereinThe combination coupled molecule of the step 4) is double chain nucleotide dyestuff, is specifically combined with the double stranded PCR products.

Measuring the further embodiment of the method for at least one measuring samples nucleic acid molecule absolute quantity is, according to instituteIt states the basic token unit described in step 4) and carries out data normalization processing, wherein each detectable control sample signalIntensity divided by the basic token unit number combined at this with each PCR product molecule.According to the step 4) instituteThe basic token unit stated carries out data normalization processing, wherein each detectable control sample signal intensity divided by shouldLocate the basic token unit number combined with each PCR product molecule；Each detectable sample letter to be checkedNumber intensity divided by the basic token unit number combined at this with each PCR product molecule.

Measuring the further embodiment of the method for at least one measuring samples nucleic acid molecule absolute quantity is, whereinThe signal strength of the standard curve of the step 6) and at least one measuring samples is taken from the same of the PCRXun Huan.Another object of the present invention is using a kind of side for the absolute quantity for measuring at least one measuring samples target moleculeThe data or use a kind of side for the absolute quantity for measuring at least one measuring samples nucleic acid molecule that method detectsThe database that the data that method detects are encoded.

The a kind of of the present invention detects the method for biomolecule number with built-in property standard curve, fundamentally solves life sectionActive demand of the Disciplinary Frontiers to absolute quantitation is learned, the availability of gene expression detection data can be increased substantially, so as toCost and promotion functions genomics research are reduced, and the data detected by this method can directly should in following clinicWith.

Key definition：Signal strength unit is each detectable unit on every copy molecule.

Description of the drawings

Fig. 1 is that spike-in methods use the schematic diagram in biochip technology.

Fig. 2 provides a method with spike-in in gene expression profile microarray technology to estimate the copy number of sampleExample.

Fig. 3 show hybridized with the single concentration of a spike-in exogenous rna with the probe points being serially diluted andIt is compared with traditional spike-in exogenous rnas being serially diluted, two methods can generate identical signal strength result.

Fig. 4 provides the schematic diagram that standard curve built in SC-spike-in is used in the microarray technology of end mark.

Fig. 5 show built in property standard curve how for quantitative PCR.

Fig. 6 shows the principle of sandwich immunoassay.

Fig. 7 show built in standard curve how for sandwich ELISA.

Fig. 8 show built in standard curve how for sandwich membrane antibody array.

Fig. 9 provides standard curve built in SC-spike-in in the schematic diagram of the microarray technology of polynucleotide labelling.

Figure 10 is to carry out linear regression analysis with the known copy number in control signal volume unit summation and control point/holeEquation and obtained hypothesis standard curve.

Figure 11 charts illustrate the composition of computer software.

Figure 12 is the equation of linear regression of prior art absolute quantitation PCR and the schematic diagram for assuming standard curve.

Specific embodiment

The invention discloses one kind biology point is detected with built-in property standard curve in biosensor detection system platformThe method of subnumber, the principle utilized are the combinations of bio-sensor system probe-target specificity, with reference in constant ratio knotIt closes, which can generate detectable signal, and there are associated with target for signal.Those skilled in the art can be used for reference in this paperHold, be suitably modified and be applied to different biosensor detection system platforms, it is accordingly required in particular to, it is noted that all similar replacesChange and change apparent to those skilled in the art, they are considered as being included in the present invention.

In order to which those skilled in the art is made to more fully understand technical scheme, below with different technology platformsThe present invention is made it is further disclose, and open signal volume unit this biosensor detection system innovation concept andApplication method of the signal strength unit in copy number standard curve is disclosed, is described in detail in combination with specific embodiment.This explanationIn all bibliography be incorporated herein by reference, degree for they entirety and be all purposes.Tool described belowBody embodiment is explanation of the invention rather than restriction.

Embodiment one:Built-in property standard curve is in the application of microarray

One microarray is a complicated chip lab, and common microarray is two-dimensional array, is fixed on solid carrierOn, solid phase carrier can be glass slide, nitrocellulose filter, silicon chip, plastic sheet, microballon or microtiter plate etc., can be to bigIt measures biological substance and carries out high flux screening.Microarray can be DNA chip, protein-chip, peptide chips, organization chip, cellChip, compound chip.MiRNA chips detect miRNA express spectras；Peptide chips can be used for protein-protein interactionDetailed analysis or preferred；Organization chip provides numerous histologic analysis；Cell chip provide it is a kind of to living cells in solid phase branchHold the approach that numerous researchs are made on object surface；Compound chip be by organic compound point sample in surface of solid phase carriers, for find withThe protein that specific compound combines provides instrument；DNA chip is most famous and currently in biology and medical research using mostWide microarray, applying includes gene expression profile, comparative genome hybridization, the chromatin imrnunoprecipitation on chip, SNP inspectionsIt surveys, alternative splicing detection, fusion gene microarray and tile array；The application of protein-chip includes protein expression profile,With identification protein-protein, protein-DNA, protein-RNA, protein-phospholipid and protein-small molecule it is mutualIt acts on (functional protein microarray).The probe-target hybridization formed is usually via detection by fluorogen, silver, chemiluminescence, parentThe relative abundance for measuring with target/probes of the marks such as photochemistry or radio isotope and determining analyte.SomeMarkless detection method has also been attempted for microarray technology, such as surface plasma resonance (SPR), carbon nanotubes, carbon nanocoilsThe cantilever of sensor and MEMS (MEMS).With unmarked technology, also it is expected to obtain 3-D effect.

Before more than ten years, Dudley etc. is pointed out, it is copy number preferably to express data, it will allow to test, in fact in differentTest between room, experimental system, data type and be compared, this be exactly functional genomics field it is highly desirable (referring to reference to textIt offers 79).But they can not accomplish to detect copy number, and method used is still similar to Yang, uses double-colored hybridization and markCommon oligonucleotides be used as with reference to object, measure sample with the ratio of sample signal intensity and oligonucleotides reference substance signal strengthProduct rna level.It again provides the method for an absolute expression value of measurement, but still can not measure copy number.Carter etc.People in order to control, using double-colored hybrid method, has carried out the trial of copy number measurement (referring to ginseng for the first time with spike-in exogenous rnasExamine document 80).However, this method is neither easy to operation, it also can not accurately measure, not adopted by colleague, also not sameIt is continuing in the article that the laboratory later stage delivers (referring to bibliography 70,71).Bissels et al. once with the UR of known quantity andDouble-colored hybrid method measures the copy number of RNA (referring to bibliography 74).However, UR will not cancel and mark related deviation,And cost and labor intensity all double.Therefore, microarray technology establishes a reliable measurement gene copy not yetSeveral methods.Convenience following for explanation and clear, selects one kind of microarray, RNA end mark microarrays, such as miRNAMicroarray for example come explain the principle of the present invention and how DNA microarray technology platform detection molecules copy number.

DNA microarray technology is attached to from Southern blot developments, DNA fragmentation on carrier, then with known dnaSequence hybridizes.There are DNA microarray thousands of microcosmic DNA spots to be attached to a surface of solid phase carriers.Each spot containsHave the specific dna sequence (be known as probe) of picomole quantity, can be a gene or a short-movie of other DNA, forThe cDNA or cRNA (being known as target) of sample hybridize under hybridization conditions, the nucleic acid of non-hybridized combination are then washed off, finally in micro- battle arrayProbe-target hybrid is scanned on column scan instrument, it is related to tested target abundance to obtain fluorescence intensity.The method of the prior art, oftenA experiment can only obtain relative quantification data, because can not possibly directly RNA be made by relying solely on measured array signal intensityAbsolute quantification analysis, this is because the hybridization efficiency of sequence dependent, difference RNA there is mark and each testingMark and the variation (referring to bibliography 40,73-75,79-84) of hybridization, this has resulted in being compared different experiments dataWith the difficulty of integration.So far, the requirement that only a small number of experimental methods can reach quantitative analysis and data compare,Spike-in attractes attention the most.The spike-in methods of the prior art are related to prints a different set of exogenous control in arrayManufacturing probe (is named as S- probes) herein, and handle RNA (S- targets) corresponding with different S- probes adds in before mark and hybridizationInto sample RNA (referring to bibliography 54,74,80-86), mark caused by this method cancels the variation of each experiment condition andHybridize deviation.Existing spike-in methods, by the way that the target amount added in is controlled to establish calculating reference value, that is to say, that S- targets existIt is known quantity in spike-in.

Fig. 1 is that spike-in methods use the schematic diagram in biochip technology.Have in target pond 100 (i.e. miRNA) pre-The exogenous miRNA 101 and 102 (S- targets) of spike-in for first adding in and marking together are respectively with being fixed on glass slide 200Specific probe 201 and 202 (S- probes) is combined by hybridizing.The exogenous miRNA 101 and 102 of spike-in must beDifferent abundance, the probe 201 being fixed on glass slide and 202 have identical abundance.Scanning process detects markFluorogen, the scanning signal intensity in 201 and 202 positions represent the quantity of S- targets 101 and 102 respectively.This method is wideQuality control applied to microarray and relative quantitative assay generally.

Carter et al. proves that spike-in methods can be used to carry out endogenous rna expression the measurement (ginseng of absolute quantitationSee reference document 80).They are by the use of 7 different saccharomycete mRNA sequences as exogenous control parameter (S- targets).The base surveyedBecause of the total serum IgE sample that express spectra is triplicate mice embryonic, placenta, ES cells and TS cells, by yeast sequences (S- targets)With in different known copy number spike-in samples RNA.Then to each micro-array chip signal strength of these S- targetsIt is average normalized to numerical value carry out linear regression analysis.Dotted line is the tropic for the matter that is averaged in a organized way in Fig. 4, C_hmi=(I_i- a)/b equations be used for counterplot calculate endogenous RNA transcript estimation copy number, wherein, C_hmiIt is that microarray is miscellaneous at probe iThe estimation copy number of the endogenous transcript of friendship, I_iIt is the logarithm normalized value of signal strength at probe i, a and b are then cutting for S- targetsAway from and slope.

Fig. 2 provides a method using spike-in sample to estimate in gene expression profile microarray technologyThe example of copy number.This work is by Carter et al. completions, and Fig. 2 is in original text Zhang Zhongwei Fig. 2 b.7 saccharomycete S- target copy numbersLinear regression and the RNA sample of mice embryonic, placenta, ES cells and TS cells average die signal strength show it is aobviousThe signal strength of work and the correlation of copy number, r²Matter is 0.99.

If however, make accurate normalized to each independent experiment using the method for Carter,The S- target levels of spike-in necessary completely the same (referring to bibliography 56,77,84-86) in all samples, and this pointIt is often extremely challenging to experimental implementation, it is seldom used in gene copy number analysis.Existing spike-in methods it is anotherA shortcoming is that a S- target sequence can only generate a signal strength data on an array, and linear regression can only use multiple S-Target sequence could be completed.So all endogenous RNA to be normalized it is not sound feasible with such standard curveBorder, because it cannot reflect the linear dynamic range of the sequence with different characteristics, therefore, these S- targets cannot generation with reference to matterThe characteristic of all RNA of table.Meanwhile existing spike-in methods further drawback is, it cannot reflect that different genes sequence is carriedMark metering difference.So, it is necessary first to be the existing spike-in methods of reform, will be by each independent controlThe amount of spike-in, reform into can be equal between all samples the control reference quantity for having unified standard.In order to realizeThis target, the best way are to be standardized S- probes by manufacturing process, and the S- probes of standardization pass through a pair in turnOne probe-target molecule combination ratio controls the amount of S- targets.

The feasibility of above-mentioned imagination controls the quantity of exogenous spike-in can be by Yauk with fixed known probeEt al. experimental data in the article delivered in 2006 carry out indirect proof (referring to bibliography 87).It is more suitable in order to find oneIn the quality assurance and/or normalized parameter of making toxicogenomics microarray, compared to commercially available product, they are micro- battle arrayAnother method of the control design case of row.Different from previously described common spike-in, they test such a possibilityProperty, with the single spike-in concentration of single exogenous rna sequence, but the complementary probe with being attached on array is multiple and differentDilution spot is hybridized.Using arabidopsis ' chlorophyll synthase gene, they have made reciprocal two kinds of dilution experiments, withIt determines during bimolecular hybridization reaction, the cRNA that the concentration and probe concentration in which kind of degree on chip can be simulated in liquid phase is denseThe variation of degree.Chlorophyll synthase cRNA is by in single concentration (5ng) spike-in to mouse RNA (5 μ g), and and arrayThe upper diluted concentration and probe concentration of difference (0.000015 to 100 μM) is hybridized；On the contrary, the spike-in references being serially dilutedCRNA is added in one by one in mouse RNA solution to be hybridized from different array chips.The result is shown in Fig. 3 (artworks 5), show with oneThe single concentration of spike-in exogenous rnas is hybridized with the probe points being serially diluted and is serially diluted with traditionalSpike-in exogenous rnas are compared, and two methods can generate identical signal strength result (referring to bibliography 87).However,The method of Yauk cannot be used for making absolute quantitation, because not having to can be used for the parameter for carrying out absolute quantification analysis in text.Test oneThe purpose of the reversed serial reference of kind, they so represent in article：" design and placement location of this series allow to miscellaneousQA (quality assurance) problem handed over and frequently encountered in printing is checked.It can be with LOWESS in addition, we demonstrating the seriesIt normalizes and is used in combination, compared with LOWESS normalization is used alone, can improving the verification and measurement ratio of gene differential expression, (what is improved is sensitiveDegree and predictability) " (referring to bibliography 87).Obviously, the main target of Yauk etc. is to meet the need of chip quality guaranteeIt asks；Secondly, merge when with LOWESS normalization in use, this design can improve the recall rate of gene differential expression.Yauk'sExperiment, which is appointed, so to be rested on relative quantification, and the method for relative quantification depends on UR and Bicolor-code.

Although they have abandoned the method (referring to bibliography 88,89) to Yauk in the article delivered later, but if IExamined closely from brand-new angle, with reference to establish the method for absolute quantitation in microarray technology, must find can be same with sampleStep mark and hybridization, and the reference substance of all samples characteristic can be represented, and such a series of requirement is easily standardized, weAdvantage with regard to that can find and using Reverse Turning Control：1) control reference quantity can be completed by manufacturing process, be easily able to standardize；2)A plurality of standard curve can be developed on same array chip, can preferably represent the feature of all samples.For the foregoing reasons, insteadIt will be used in below to reform current relative quantification by property standard curve built in manufacture, so as to reach to diluted principleThe purpose of exploitation microarray detection gene copy number.

For the ease of being distinguished with the spike-in of the prior art, to establish general absolute copy number in the applicationThe spike-in of new generation developed for the purpose of standard curve is named as the SC-spike-in (spike- of standard curve hereinin).It selects with sequence of the laboratory sample target without crisscrossing as SC-spike-in, by the probe with SC-spike-in complementations(SC- probes) is printed to a series of different copy numbers on same an array, and the span of SC- probe copy numbers covers from closeBackground is to the signal strength of saturation.The SC-spike-in biomolecule (SC- targets) of hybridization reaction is added in, copy number should be greater thanTotal copy number of SC- probes on chip array.Fig. 4 provides standard curve built in SC-spike-in in end mark microarray skillThe schematic diagram of art.There is the SC-spike- for the single saturation capacity of tool for being previously added and marking together in target pond 300 (i.e. miRNA)In miRNA 301 (SC- targets) are combined by hybridizing with a series of specific probe point of copy numbers, such as are fixed on slidePoint 401 and 402 (SC- probes) on 400.Point 401 and 402 has the identical base sequence complementary with SC- targets 301, but copiesNumber is different.The fluorogen of mark, scanning signal intensity generation respectively of 401 and 402 positions of point can be detected in scanning processTable SC- targets 301 the point quantity, quantity be equal to known copy number of the SC- probes at 401 and 402.To SC- target signalsLinear regression analysis is carried out with SC- probes to make the standard curve in relation to signal strength and copy number.Endogenous miRNA targetsCopy number will be drawn according to the formula to calculating obtained from the regression analysis of standard curve.Fig. 4 explains that 1 SC- target can generateOne standard curve.Using different SC- targets and SC- probes can in same an array the built-in a plurality of mark for representing different characteristicDirectrix curve.

In protein-chip, it can be that antibody, antigen, nucleic acid are fitted to arrange the capture molecule being fixed on surface of solid phase carriersLigand, affine body or full length protein.Technical principle using antibody as capture molecule refers to the explanation in ELISA parts.The principle of its SC-spike-in method is same as above, but with the capture molecule of serial copy number arrangement, (SC- is visited on solid phase carrierPin) it is antibody, antigen, aptamer, affine body or full length protein.

Embodiment two：Built-in property standard curve is in the application of PCR arrays

Round pcr is used for the DNA fragmentation for expanding single or multiple copies, and copy number is crossed over up to several orders of magnitude.The DNA being amplified is known as template, and the template of PCR reactions falls within target.Quantifying PCR method allows to the tested sequence in sampleThe amount of (i.e. template) is estimated that this technology is usually used in quantitative measurement of gene expression level.Quantitative PCR is one and generally acknowledgesDNA quantitative tools, it detects the accumulation matter of the DNA product after each cycle during PCR amplification.This technology is crowdIt is well known, thus omit the datail description of the technology herein, only refer to that (they belong to probe, and function is for the pair of primers of PCRFind the target with amplifying specific) determine amplicon sequence-specific and length.There are two types of quantitative PCR detecting methods：BaseIn the dyestuff of double-stranded DNA or based on probe (this probe in detecting PCR product abundance).SYBR Green I are that double-stranded DNA combinesOne in dyestuff, it is attached on the ditch of double-stranded DNA, with reference to SYBR Green I its fluorescence increase over one hundred times.Therefore,The increment that can monitor the DNA of amplification by making fluoremetry at the end of the extension step of each PCR cycle, so as to be specialThe identification of product and quantitatively one fabulous instrument of offer.Based on the quantitative PCR of probe dependent on sequence-specific to detectDesired PCR product, it utilizes the target-specific probe of fluorescent marker, such as TaqMan probe, and gained fluorescence signal allows to PCRThe accumulation of the product of exponential phase quantitative determines (referring to bibliography 90-95).

Fig. 5 show built in property standard curve how for quantitative PCR.DNA profiling will be controlled in advance with a series of copy numbersIt is placed in the different holes of porous plate, such as the hole 601 and 602 on 96 orifice plates 600.With real-time quantitative PCR instrument (such asQuantStudioTM real-time PCR systems) signal that detects carries out linear regression analysis to Control architecture, formulate signal strengthWith the standard curve of copy number.The copy number of sample target will be obtained according to the formula to calculating obtained from the regression analysis of standard curveGo out.Since each reaction is isolated in a hole, Control architecture sequence can be exogenous or endogenous.

Embodiment three：Built-in property standard curve is in the application of ELISA arrays and membrane array

The principle of enzyme linked immunosorbent assay (ELISA) (ELISA) is that the survey of one " analyte " is determined with antibody and color changeExamination.ELISA detections are related to the antigen that at least one antibody is directed to specificity.Sample with unknown quantity antigen passes through non-specificProperty (through being adsorbed to carrier surface) or specifically (through another antibody capture, which is also specifically directed to same primary antibodyOriginal forms double-antibody sandwich elisa) it is fixed on solid phase carrier (being typically polystyrene micropore plate).When antigen is fixedAfterwards, detection antibody is added in, compound is formed with antigen.Antibody can be covalently attached or detect with enzyme again by second by detecting antibodyAntibody test, secondary antibody are connected by biological coupling with enzyme.Between per step, commonly use mild detergent and remove any non-spyThe protein or antibody that the opposite sex combines.After completing washing, the substrate of enzyme is added in generate visible signal, which represents in sampleAmount of antigen.ELISA detections are carried out in liquid, so reactant needs to be maintained in reacting hole.The species of ELISA methodIncluding：Determined antigen (is fixed in the hole of microwell plate) by Salmonella, and (double-antibody sandwich elisa is shown in figure to indirect ELISA6), competitive ELISA (such as two kinds of antibody competitions are combined with same antigen, and signal strength is caused to change) or detect simultaneously notSame antibody and antigen.In addition to classical enzyme mark detection, it is also possible to other detection methods of Western blotting.

The principle of membrane array is similar to Western blotting, but it can be antigen or antibody to be fixed in advance on film.Therefore, instituteThe detection method having suitable for Western blotting can be used in membrane array use, such as colorimetric detection, chemiluminescence detection, radioactivity inspectionIt surveys and fluorescence is examined

It surveys.Since membrane array belongs to protein immunoblotting, immune response principle is identical with ELISA.

Fig. 6 shows the principle of sandwich immunoassay.(1) capture antibody is fixed on plate film and coats；(2) sample is added in, is ownedAntigen with capture antibody combined；(3) detection antibody and antigen binding are added in；(4) enzyme-linked secondary antibody and detection antibody are added inWith reference to；(5) substrate is added in, detectable signal is become by enzymatic conversion.

Fig. 7 show built in standard curve how for sandwich ELISA.Capture antibody as control is by advance with a series ofKnown copy number is fixed in the different holes of porous plate, the hole 801 and 802 on such as 96 orifice plates 800, to generate standard curve；The antibody that sample is tested antigen is fixed on saturation capacity in other holes of porous plate in advance；Determined antigen is added in per hole.To readingSignal in the above-mentioned control hole that plate device detects carries out linear regression analysis, to formulate the standard of signal strength and copy number songLine.The copy number of sample target will be drawn according to the formula to calculating obtained from the regression analysis of standard curve.Due to each reactionIt is isolated in a hole, control capture antibody can be exogenous or endogenous.

Fig. 8 show built in standard curve how for sandwich membrane antibody array.As control capture antibody by advance withSerial copy number is fixed on the different position on film, such as the point 1001 and 1002 on nitrocellulose filter 1000, is marked to generateDirectrix curve；Sample is tested the different position that the antibody of antigen is fixed in advance with saturation capacity on film；By the control of single saturation capacityAfter antigen and sample processed mixing with antibody hybridization.The detection of chemiluminescence signal is identical with the method for Western blotting.To detectingAbove-mentioned control signal carry out linear regression analysis, to formulate the standard curve of signal strength and copy number.The copy of sample targetNumber will be drawn according to the formula to calculating obtained from the regression analysis of standard curve.Capture antibody and tested antigen as controlIt is combined without intersecting, but can be endogenous or exogenous.

Embodiment four：The calculating of various labeling methods and signal strength unit

Although SC-spike-in can solve standardization parameter by a series of SC- probes of copy numbers built in industrializationThe problem of, different RNA features can be represented by built-in a plurality of standard curve, but only can't by SC-spike-inThe difference of signal strength that may be present between reflection different molecular.Therefore, it is also desirable to it solves between each sample and control moleculeIt can be compared in accurate identical level.The target will pass through the concept for introducing signal strength unit and following computational methodsTo realize.Another signal strength for being characterized in be detected of the application is converted into signal strength unit (IU).Signal strengthUnit is each detectable unit on every copy molecule.The concept for introducing signal strength unit be in order to ensure sample andIt can be carried out between SC-spike-in, between sample and sample room and SC-spike-in and SC-spike-in in equal levelCompare.The purpose of signal strength unit is to allow mathematical computations (regression analysis of standard curve and the calculating of samples copy number) canAccurately to be carried out in same standard.

A) when using labelling method, signal strength unit is emitted from each basic token unit on every copy moleculeSignal.Basic token unit can be a molecule that can emit/re-emit detectable signal, such as fluorogen is excited through lightAnd re-emit light；Basic token unit can be an energy metabolism substrate so as to generate the molecule of detectable signal, such as enzymeIt cracks chemiluminescent agent and shines or convert a substrate into detectable color；Basic token unit can be a connection quiltIt surveys molecule and causes the molecule of detectable signal molecule, such as the biotin that can be covalently attached with tested protein, biotin is againIt is identified by the antibiotin conjugate of gold-coupling, the latter causes detectable silver-colored set point.Name three kinds of different mark/dyesColor method explains how measured signal strength to be converted into signal strength unit, and wherein SC-spike-in is used as markingAccurate control, sample is interested detection sample：

1) labeling method one：Each tested molecule is marked by a basic token unit, in miRNA chips as shown in Figure 4MiRNA end marks, the fluorescence labeling probe in quantitative PCR shown in Fig. 5.Or biomolecule is each tested by identical quantityBasic token unit marks, and is covalently attached in sandwich ELISA or membrane antibody array as shown in 6 figures, Fig. 7 and Fig. 8 with secondary antibodyEnzyme number.Each sample molecule and control molecule can generate the signal of identical given value.

In labeling method one, each biology copy number generates identical signal strength.Therefore, if CN_S=CN_C, thenINTENSITY_S=INTENSITY_C。

2) labeling method two：Each tested molecule is marked by basic token unit in varying numbers, mRNA tables as shown in Figure 9The cRNA that Cy3-CTP is marked up in spectrum chip.The nucleotide each marked on control molecule and sample molecule can produceThe signal of raw identical given value.

In labeling method two, different signal strengths can be generated per copy number sample molecule, the control molecule per copy numberAlso different signal strengths can be generated, the signal strength per copy number sample molecule can be different from control molecule.Divide per copy numberThe signal strength of son depends on being incorporated into the total quantity of the basic token unit of the biomolecule.

3) labeling method three：Basic token unit is the dye molecule combined with double chain nucleotide, such as SYBR Green I,PicoGreen, EvaGreen, Hoechst 33258, BEBO, QuantiFluor, AccuClear or AccuBlue.Dyestuff withThe double chain nucleotide of quantitative PCR product or other detection platforms shown in Fig. 5 combines.The A-T or C- that each pair hybridizes in double-stranded DNAG can be combined with same amount of basic token unit, therefore, the letter of identical given value can be generated per a pair of A-T/C-GNumber.

In labeling method three, different signal strengths can be generated per copy number sample molecule, the control molecule per copy numberAlso different signal strengths can be generated, the signal strength per copy number sample molecule can be different from control molecule.Per copy numberSignal strength depends on the sequence length (nucleotide number) of duplex molecule, and longer, signal strength is stronger.

B) when using unmarked technology, signal strength unit is from emitting per basic component units on every copy moleculeSignal.For example, amino acid is the basic component units of protein, nucleotide is the basic component units of DNA.Unmarked biologySensor technology is to be a ripe analysis tool maying be used in terms of kinetic energy, affinity and concentration analysis, in high passThe application of amount is also ready for ready (referring to bibliography 96,97).Unmarked technology includes, but not limited to surface plasmaScreening, transmitted light imaging based on resonance (SPR), nuclear magnetic resonance (NMR), high-throughput mass spectrum, resonant wave guide tube sheet, isothermal dropDetermine calorimetry, the detection method based on cell impedance and other bio-physical methods.In the experiment of optical biosensor, withThe accumulation of analyte on the surface, the variation of the refractive index of buffer of the Systems for optical inspection measurement near sensor surface(referring to bibliography 96-99).By measuring reflectivity, SPR can be adsorbed with detection molecules, such as polymer, DNA or protein,Aptamer-protein-interacting etc..When there is different biopolymerization article patterns on surface, using appropriate optics and imaging sensor(i.e. video camera), the technology are expanded to as surface plasma resonance image-forming (SPRI).Molecular weight based on absorption, SPRIThe high contrast of image is provided, such as (wherein the combination of antibody-polypeptide is specifically composed by stereogram in antibody-polypeptide microarrayDetermine) (referring to bibliography 100,101) and aptamer-arrays of immobilized protein (referring to bibliography 102).

The calculation formula of unmarked signal strength unit is identical with above-mentioned labelling method three, but NN_CAnd NN_SCan be respectivelyThe number of other basic structural units of the number of the nucleotide of composition control and sample, the number of amino acid or tested molecule.Its formula is can to generate identical known value signal respectively based on each nucleotide/amino acid/other basic structural unitsIt is assumed that so as to meet the premise that signal strength unit is equal between sample and SC-spike-in.If future science finds thisAssuming that will not be accurate, then formula should be drunk according to new scientific discovery and change.

Embodiment five：The different mathematical formulaes of various labeling methods

Figure 10 is the equation that linear regression analysis is carried out using the known copy number in control signal intensity and control point/holeWith obtained hypothesis standard curve.This example application simple linear regression, generates formula Y=a+bX, and wherein X is log₁₀Copy number (the CN of conversion_C), Y is the point/hole log₁₀Summation (the SIU of the signal strength unit of conversion_C), b is the slope of straight line,A is intercept.Therefore, the copy number of sample can be calculated by following formula：X=10^(Y–a)/b, wherein X is the copy number (CN of sample_S),Y is X samples in the point/hole log₁₀Summation (the SIU of the signal strength unit of conversion_S)。

Because SIU=IU x CN,:In labelling method one, work as CN_S=CN_CWhen, SIU_S=INTENSITY_S=SIU_C=INTENSITY_C；In labelling method two, work as CN_S=CN_CWhen, SIU_S≤INTENSITY_S,SIU_C≤INTENSITY_C, SIUS=/≠SIUC, depending on whether NLS and NLC is equal；In labelling method three, as CNS=CNC, SIUS<INTENSITY_S,SIU_C<INTENSITY_C,SIU_S=/≠ SIU_C, depending on NN_SAnd NN_CIt is whether equal.

Each array/plate film can built-in one or more standard curve, control molecule can be a kind of molecule or it is a variety of notSame molecule.

Embodiment six：Computer software related to the present invention and kit

Another embodiment of the invention is the computer software for performing the present invention.Figure 11 charts illustrate that computer is softThe composition of part.Software may include the normalization of ambient noise, and array is in the normalizing of the hybridization of different position (such as each lattice)Change, the normalization of dye strength, the Logarithm conversion of signal strength and copy number, the pattern of the signal strength obtained according to measurement comeCalculate IU_C/SIU_CAnd IU_S/SIU_S, according to IU_C/SIU_CAnd CN_CStandard curve is formulated, math equation is established according to standard curveFormula finally provides copy number/absolute magnitude of each tested molecule.It is expanded if being related to not wait between built-in control and detection sampleThere are when, software also bear group signal strength carry out reasonable regulations task.Software can be used real with biologyIt tests on the computer that equipment is connected or the biological experimental data transmitted is analyzed on the remote computer.

Another embodiment of the invention is to provide the kit and/or SC- needed for property standard curve built in foundationSpike-in RNA, DNA, peptide/protein etc..Kit and/or SC-spike-in generally include container, label and SC- targets/Or SC- probes.The kit can further comprise it is other from business and the required material of user's position, including for ease ofBuilt-in standard curve is made to achieve the purpose that absolute quantitation and is designed element or equipment, buffer, enzyme, dNTPs, diluent,Filter, other analogs and operation instructions.

The present invention is illustrated further with reference to specific embodiment, it is therefore intended that explain rather than limit.SimultaneouslyAlso please understand, the following examples are mainly focused on the application of the present invention rather than in the biosensor detection side of each maturationThe details of method.

Embodiment 1:Built-in standard curve is in the application of the microarray of labeling method one.Below with mankind miRNA chips andFig. 4 explains the copy number that the biological sample of end mark how is detected using built-in standard curve as an example.

Exogenous miRNA is selected to make standard curve：With the mature sequence dme-miR-2a-1-5p of drosophila miRNA(miRBase accession number MIMAT0020780) makes the built-in standard curve of mankind's miRNA chips.It closesInto, purifying and quantitative dme-miR-2a-1-5p sequences, to prepare as SC- targets 301.

Prepare the standard curve of probe：Synthesis, purifying and quantitative and the complementation of dme-miR-2a-1-5p sequences antisense are fewNucleotide, and add additional " G " residue at 5' ends, to prepare as SC- probes.Then by SC- probes chip point sampleInstrument point enables the oligonucleotides to be fixed thereon, SC- in pretreated standard 1 " X3 " microscope slide surfaceA series of copy number scopes of probe are 10³To 10¹⁰, per the triplicate point sample of number of copies.Fig. 4 midpoints 401 and 402 are SC-The example of probe difference copy number point, is printed on the probe points of mankind miRNA on identical slide.

·SC-spike-in:Drosophila miRNA dme-miR-2a-1-5p, i.e. SC- targets 301 are copied with S- probes on slide2 times several of amounts is added into the total serum IgE sample of 100ng people, that is to say, that the copy number of SC- targets 301 is 2X 3X (10³+10⁴+10⁵+10⁶+10⁷+10⁸+10⁹+10¹⁰)。

Mark：The 3' ends of miRNA are marked with flower cyanines 3-pCp.

Hybridization：The miRNA of mark is added in the hybridization chamber equipped with microarray slide.

Washing：Microarray slide after cleaning hybridization with cleaning solution.

Scanning：The microarray slide after hybridization and cleaning is scanned using microarray scanner.

Crossing pattern：The data obtained using software processing microarray scanning will be carried by the information that probe capturesIt takes out and is transformed into signal strength associated with probe.The step also includes background normalization and the logarithm of signal strength turnsChange etc..

Mathematical equation：Based on obtained log₁₀(INTENSITY_C) and log₁₀(CN_C) information it is linear to carry outRegression analysis.Y=0.6161+0.569X is that a series of control signal intensity after being normalized in Fig. 6 and copy number are returnedReturn and equation is assumed caused by analysis.Namely to SC- targets 301 in the signal strength of point 401,402 etc. and SC- probesCopy number 401,402 etc. carries out assuming equation caused by regression analysis.

Determine the copy number of sample people miRNA：Samples copy number is by formula X=10^{(Y-0.6161)/0.569}Reckoning obtains.Therefore, if the hypothesis normalized signal intensity matter positioned at the Si people miRNA of point 400i is 3, there is (3-0.6161)/0.569=4.18963.In the total serum IgE sample of 100ng people, copy numbers of the Si miRNA in point 400i is X=10^4.18963=15475 copyShellfish.

Embodiment 2:Built-in standard curve is in the application of the microarray of labeling method two.Human gene express spectra core is used belowPiece and Fig. 9 explain the copy number that the biological sample for participating in mark how is detected using built-in standard curve as an example.

Exogenous mRNA is selected to make standard curve：With arabidopsis peroxisome small heat shock proteinMRNA (the GenBank accession number of Hsp15.7:DQ403190) the built-in standard curve of mankind's expression profiles of gene chip is made.The gene mRNA has 414 bases, including 112 " G " residues.By the Plasmid DNA (ERCC) containing DQ403190 sequencesA single linear chain is cut into, then using Ambion companiesT7 kits carry out in-vitro transcriptionAbove-mentioned mRNA (referring to bibliography 103).The DQ403190mRNA obtained is purified and quantified, to prepare as SC-Target 1101.

Prepare the standard curve of probe：One section of DQ403190 fragment sequence for having 60 nucleotide is selected to be visited as controlPin, and synthesize, purify and quantify to prepare as SC- probes.Then it is SC- probes is pretreated with chip point sample instrument pointStandard 1 " X3 " microscope slide surface, the oligonucleotides is enable to be fixed thereon, a series of copies of SC- probesNumber scope is 10³To 10¹⁰, per the triplicate point sample of number of copies.Fig. 9 midpoints 1201 are SC- probe difference copy numbers with 1202The example of point, is printed on the probe points of human mRNA on identical slide.

·SC-spike-in:Hsp15.7mRNA, i.e. SC- targets 1101, with 2 times of amount of SC- probes copy number on slideIt is added into the total serum IgE sample of 1000ng people, that is to say, that the copy number of SC- targets 1101 is 2X 3X (10³+10⁴+10⁵+10⁶+10⁷+10⁸+10⁹+10¹⁰)。

Mark：The total serum IgE and SC- targets 1101 of employment carry out the first chain cDNA synthesis, are then carried out with the first chain cDNAThe synthesis of second chain cDNA.In-vitro transcription is carried out as template and simultaneously cyanines 3-CTP to be spent to mark, with the second chain cDNA so as to markThe transcript cRNA of note is antisense RNA, and possessed mark Cy3-C numbers are equal with " G " residue number of original mRNA, for example,The SC- targets 1101 of mark have 112 Cy3-C residues.

Hybridization, washing and scanning：With above-described embodiment 1.

Crossing pattern：The data obtained using software processing microarray scanning will be carried by the information that probe capturesIt takes out and is transformed into signal strength associated with probe.The step is also including background normalization etc..

The conversion of signal strength unit summation (SIU)：Signal strength after background normalization is converted into SIU.ForSC- targets, since SC- targets 1101 have " C " residue of 112 marks, just there is SIU for the SIU at point 1201₁₂₀₁=INTENSITY₁₂₀₁/112.This is a crucial switch process, and each gene has oneself specific conversion formula, basisIt is the gene sequence information obtained from GenBank.For example, mRNA (the GenBank accession number of human interleukin-12 (IL2)：NM_000586) there is the base of 822bp, wherein 85 " G " residues, the point 1200j of its probe location in fig.9, therefore,SIU_1200j=ITENSITY_1200j/85。

Logarithmic transformation：Log is made to SIU and CN₁₀Conversion.

Mathematical equation：Based on obtained log₁₀(SIU_C) and log₁₀(CN_C) information carry out linear regression pointAnalysis.Y=0.6161+0.569X is that a series of control signal volume unit summations after being normalized in Fig. 9 and copy number are carried outEquation is assumed caused by regression analysis.1201,1202 etc. signal strength and SC- namely are being put to SC- targets 1101Probe carries out assuming equation caused by regression analysis in 1201,1202 etc. copy number.

Determine the copy number of sample people mRNA：Samples copy number is by formula X=10^{(Y-0.6161)/0.569}Reckoning obtains.Therefore, if S positioned at point 1200j_1200jIt is 3 that the hypothesis of people mRNA, which normalizes transformed signal strength unit summation, then has(3-0.6161)/0.569=4.18963.In the total serum IgE sample of 1000ng people, copy numbers of the Sj mRNA in point 1200j is X=10^4.18963=15475 copies.

Embodiment 3:Built-in standard curve is in the application of the array of labeling method three.Reverse transcription quantitative PCR array is used belowWith Fig. 5 as an example come explain how using built-in standard curve come detect double chain nucleotide mark biological sample copyNumber.

MRNA sequence is selected to make standard curve：MRNA (the GenBank accession number of employment β actins (ACTB)：NM_001101) the built-in standard curve of PCR arrays is made.The mRNA of this gene has 1852 bp.Being contained by amplification shouldThe cloned plasmids of gene produce the cDNA of ACBT, then the ACBTcDNA for being inserted into plasmid cuts into single linear chain, andIt purifies and quantitative to prepare as Control architecture to use.

Prepare the standard curve of Control architecture：The ACBT cDNA of purifying are put into the hole of microwell plate 600, control mouldA series of copy number scopes of plate are 10²To 10⁸, each number of copies puts two holes.Fig. 5 mesoporous 601 and 602 is different copiesThe example of numerical control molding plate hole, with sample on same microwell plate.

Reverse transcription (RT)：Few (dT) primer is used respectively, using the total serum IgE of 50ng sample people and 50ng as controlMouse total serum IgE, carry out the first chain cDNA synthesis.

PCR amplification：Use double-stranded DNA dyestuff SYBR Green I and the real-time PCR method of this field standard.People RNART be added to sample well, the RT of mouse RNA is added to control/standard curve hole.

Sketch traditional method for carrying out absolute quantitation to the Initial abundance of amplicon with standard curve, it is therefore intended thatIt helps to illustrate the difference between the present invention and existing real-time PCR absolute quantitations technology：1) in conventional method, as standard curveControl architecture is the sequence identical with sample gene, so as to which each sample gene needs a standard curve, this isIt is very burdensome.2) in conventional method, the serial copy number of standard curve is separately completed by each experiment, and therefore, standardization is notFeasible.3) in conventional method, the linear regression of standard curve is based on threshold cycle number (Ct) and copy number, referring in Figure 12It is assumed that conventional linear return (referring to bibliography 104,105).

In embodiments of the present invention, it is (that is, unlimited when the fluorescence intensity that obtains from each PCR cycle is used in combinationIn a threshold cycle number Ct) and signal strength unit concept, can be different PCR cycles make standard curve respectively and selectSelecting property it is selected to：1) needs of standard curve number, a standard curve are reduced, the copy number available for multiple genes is surveyedIt is fixed；2) accuracy of gene copy number measure is greatly increased.The mathematical computations of the present invention are described in detail below.

PCR product length：The forward and reverse primer of ACBT genes be selected at 1045 for forward primer beginning and1108 be reverse primer termination, so, the PCR product length of ACBT is 64 base-pairs.Select human angiotensin II byBody variant 1 gene (AT1, GenBank accession number：AY221090) example as sample gene, AT1 have1080bp, it is that forward primer beginning and 501 is reverse primer termination that forward and reverse primer, which is selected at 434, so,The PCR product length of AT1 is 68 base-pairs.

The conversion of signal strength unit summation (SIU)：Signal strength after background normalization is converted into SIU.Therefore,When without considering fluorescence intensity difference between A-T and C-G, control the SIU of ACBT has SIU at hole 601₆₀₁=INTENSITY₆₀₁The SIU of/64, sample AT1 have SIU at the 600k of hole_600k=INTENSITY_600k/68.This is one crucialSwitch process, each gene have oneself specific conversion formula, and basis is determined by position of the pair of primers on geneFixed amplicon size.Above-mentioned conversion is all carried out to the data of all periods in the exponential amplification phase.

Logarithmic transformation：Log10 conversions are made to SIU and CN.

Mathematical equation：Use log₁₀(SIU_C) and log₁₀(CN_C) information carry out linear regression analysis.Selection is theThe data of the ACBT measured at 20 PCR cycles a series of, Y=0.6161+0.569X are that the control after being normalized in Fig. 5 is believedNumber volume unit summation and copy number carry out assuming equation caused by regression analysis.Namely to ACBT in hole 601,602Etc. log₁₀(SIU_C) and log₁₀(CN_C) carry out assuming equation caused by regression analysis.

Determine the copy number of sample people mRNA：Selection calculates the regression equation of sample gene copy number first,The selection is determined by the Ct of gene.For example, if the Ct values of AT1 are 20, its copy number is by formula X=10^{(Y–0.6161)/0.569}It calculates and obtains.Therefore, as the S expanded by primer in hole 600k_AT1The hypothesis of people mRNA normalizes transformed signal strength unitSummation is 3 at Xun Huan 20, then has (3-0.6161)/0.569=4.18963.The total serum IgE sample of the people of institute adding hole 600kIn, the copy number of AT1mRNA is 10^4.18963=15475 copies.

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Claims (25)

1. a kind of method for the absolute quantity for measuring at least one measuring samples target molecule, including：

1) solid phase carrier is provided, it is attached on the solid phase carrier to be combined there are one series control sample with even molecule 1 and at leastOne of which in both one measuring samples probe molecule and at least one measuring samples target molecule, it is described at least one to be checkedOne of which and one series control sample knot in both sample probe molecule and at least one measuring samples target moleculeIt closes and matches somebody with somebody the different position that even molecule 1 is attached on solid phase carrier, one series control sample knot on each of which positionClosing has known and different copy number with even molecule 1；

2) solid phase carrier is combined at least one control sample with even molecule 2 and at least one measuring samples target moleculeWith another contact in both at least one measuring samples probe molecules, wherein：

One series control sample is combined with reference to 1 specific recognition of even molecule and with reference at least one control sampleWith even molecule 2, the detectable control sample signal of a series is directly or indirectly generated, wherein the detectable control in each positionThis signal strength of sample preparation is combined with one series control sample with known copy number phase of the even molecule 1 on different positionIt closes, and at least one control sample on each position combines the no less than known control sample of the copy number with even molecule 2The copy number of even molecule 1 is matched somebody with somebody in this combination；Meanwhile

At least one measuring samples probe molecule specific recognition and with reference at least one measuring samples target molecule, directlyConnect or generate at least one detectable measuring samples signal indirectly, wherein each detectable measuring samples signal strength withCopy number of at least one measuring samples target molecule in the position is associated, and at least one measuring samples probeThe copy number of molecule is no less than the copy number of at least one measuring samples target molecule of the position；

3) the detectable control sample signal intensity of a series and at least one detectable measuring samples signal strength are detected；

4) combined according to the detectable control sample signal intensity of one series and the control sample with even molecule 1 oneKnown copy number on a series different position makes the standard curve of the relation in relation to signal strength and copy number；And

5) according to the standard curve, at least one detectable measuring samples signal strength is converted into copy number, fromAnd determine the copy number of measuring samples target molecule.

2. a kind of method of absolute quantity for measuring at least one measuring samples target molecule according to claim 1, specialSign is, at least one control sample combines each molecule with even molecule 2 directly or indirectly by one or more theOne basic token unit marks and both at least one measuring samples target molecule and at least one measuring samples probe moleculeIn another directly or indirectly marked by one or more second basic token units, wherein, one series canThe control sample signal of detection and at least one detectable measuring samples signal respectively by the first basic token unit andSecond basic token unit generates.

3. a kind of method of absolute quantity for measuring at least one measuring samples target molecule according to claim 2, specialSign is, each detectable to control sample signal intensity divided by control sample straight with reference to each molecule with even molecule 2 at thisConnect or the first basic token unit number for carrying indirectly and each detectable measuring samples signal strength divided by this at it is to be checkedThe second base that each molecule of another in both sample target molecule and measuring samples probe molecule directly or indirectly carriesThe number of this indexing unit.

4. a kind of method of absolute quantity for measuring at least one measuring samples target molecule according to claim 1, specialSign is that one series control sample, which is combined, to be combined with even molecule 1, at least one control sample with even molecule 2, at least oneA measuring samples target molecule and at least one measuring samples probe molecule are selected from：(i) DNA, (ii) RNA, (iii) protein,(iv) peptide, (v) polysaccharide, (vi) chemical compound and (vii) antibody.

5. a kind of method of absolute quantity for measuring at least one measuring samples target molecule according to claim 1, specialSign is that the solid phase carrier is selected from：(i) glass, (ii) plastics, (iii) silicon, (iv) metal, (v) nylon membrane, (vi) nitric acidCellulose membrane, (vii) pvdf membrane and (viii) porous plate.

6. a kind of method of absolute quantity for measuring at least one measuring samples target molecule according to claim 5, specialSign is that the solid phase carrier is microcosmic polystyrene bead.

7. a kind of method of absolute quantity for measuring at least one measuring samples target molecule according to claim 1, specialSign is that the step 1) includes：

● it is in situ on solid phase carrier directly to synthesize one series control sample with reference to even molecule 1 and described at least oneOne kind in both a measuring samples probe molecule and at least one measuring samples target molecule；Or

● pre-synthesis one series control sample is combined and is visited with even molecule 1 and at least one measuring samplesOne kind in both pin molecule and at least one measuring samples target molecule, is attached on solid phase carrier.

8. a kind of method of absolute quantity for measuring at least one measuring samples target molecule according to claim 7, specialSign is that the original position directly synthesis is photoetching synthesis, light guiding chemical synthesis or inkjet printing.

9. a kind of method of absolute quantity for measuring at least one measuring samples target molecule according to claim 2, specialSign is that the first basic token unit and the second basic token unit are one kind selected from following label：(i) it is glimmeringLight blob, (ii) chemiluminescent agent, (iii) silver, (iv) affinity label, (v) photochemical agent, (vi) chromophore, (vii) enzyme,(viii) radio isotope.

10. a kind of method of absolute quantity for measuring at least one measuring samples target molecule according to claim 1, specialSign is that one detectable control sample signal of series and at least one detectable measuring samples signal are by micro-Array Scanner, plate reader or the detection of Western blot scanners obtain.

11. a kind of method of absolute quantity for measuring at least one measuring samples target molecule according to claim 1, specialSign is that one detectable control sample signal of series and at least one detectable measuring samples signal are by nothingLabelling technique detection obtains.

12. a kind of method of absolute quantity for measuring at least one measuring samples target molecule according to claim 1, specialSign is that wherein step 4) is combined and matched somebody with somebody according to the detectable control sample signal intensity of one series and the control sampleKnown copy number of the even molecule 1 on a serial different position makes the standard of the relation in relation to signal strength and copy numberAt least one detectable measuring samples signal strength is converted into copying by curve and step 5) according to the standard curveShellfish number, so that it is determined that the copy number of measuring samples target molecule, is completed by computer software.

13. a kind of method according to claim 11 measures the reagent of the absolute quantity of at least one measuring samples target moleculeBox, including：

● with one series control sample combine with even molecule 1 and at least one measuring samples probe molecule andThe solid phase carrier of one of which at least one measuring samples target molecule the two；

● at least one control sample is combined with even molecule 2；

● another in both at least one measuring samples probe molecule and at least one measuring samples target molecule；With

● operation instructions.

14. kit according to claim 13, which is characterized in that the solid phase carrier is selected from：(i) glass, (ii) modelingMaterial, (iii) silicon, (iv) metal, (v) nylon membrane, (vi) nitrocellulose filter, (vii) pvdf membrane and (viii) porous plate.

15. kit according to claim 14, which is characterized in that the solid phase carrier is microcosmic polystyrene bead.

16. a kind of method for the absolute quantity for measuring at least one measuring samples nucleic acid molecule, including：

1) a porous plate solid phase carrier is provided, the different holes of the porous plate solid phase carrier are included have there are one series it is knownAnd the nucleic acid molecule of different copy numbers is as control sample form；

2) be put at least one nucleic acid molecule as one in measuring samples template to the porous plate solid phase carrier not byIn the hole that control sample form occupies；

3) respectively with a pair of primer with the control sample form complementation and a pair of drawing with measuring samples template complementationObject expands the control sample form and the measuring samples template, wherein described by PCR (PCR) methodControl the control sample of the copy number reflection control sample form of the PCR product of sample form before PCR reactionsThe known copy number of template, wherein the copy number of the PCR product of the measuring samples template reflects that the measuring samples template existsThe copy number of the measuring samples template before PCR reactions；

4) specifically bound respectively with the PCR product of PCR product and the measuring samples template with the control sample formCombination coupled molecule detect PCR product, wherein the combination coupled molecule has one or more basic token units,Described in basic token unit transmitting detectable signal；

5) at least one cycling of the detectable signal and the exponential phase to being located at the PCR reactions are detected, makes related instituteThe standard curve of the signal strength and the relation of the known control sample form copy number of a series of control sample form is stated, whereinThe standard curve is obtained with regression analysis；

6) at least one measuring samples signal strength is converted into the copy number of measuring samples template using the standard curve, fromAnd the copy number of the measuring samples template before the PCR reactions where determining in hole.

17. a kind of side of absolute quantity for measuring at least one measuring samples nucleic acid molecule according to claim 16Method, which is characterized in that the combination coupled molecule in the step 4) is the few core with PCR product specific bindingThuja acid.

18. a kind of side of absolute quantity for measuring at least one measuring samples nucleic acid molecule according to claim 17Method, which is characterized in that the oligonucleotides is dual labelled probe molecule, and the double labelling includes being connected to 5 ' end of probe moleculeFluorogen and be connected to the quencher of 3 ' end of probe molecule.

19. a kind of side of absolute quantity for measuring at least one measuring samples nucleic acid molecule according to claim 16Method, which is characterized in that in the combination coupled molecule of the step 4) be double chain nucleotide dyestuff, specifically with double-strandPCR product combines.

20. a kind of side of absolute quantity for measuring at least one measuring samples nucleic acid molecule according to claim 16Method, which is characterized in that data normalization processing is carried out according to the basic token unit of the step 4), including：

● each detectable control sample signal intensity divided by the basic token combined at this with each PCR product moleculeUnit number；

● each detectable sample signal intensity to be checked divided by the basic token combined at this with each PCR product moleculeUnit number.

21. a kind of side of absolute quantity for measuring at least one measuring samples nucleic acid molecule according to claim 16Method, which is characterized in that be taken from the standard curve of the step 6) and at least one measuring samples signal strengthSame the cycling of the PCR reactions.

22. a kind of side of absolute quantity for measuring at least one measuring samples nucleic acid molecule according to claim 16Method, which is characterized in that wherein described step 1) includes：

● it is in situ in the different holes on porous plate solid phase carrier directly to synthesize one series with known and different copiesSeveral nucleic acid molecules is as control sample form；Or

● there is the nucleic acid molecule of known and different copy numbers to be attached to porous plate pre-synthesis one series and consolidateAs control sample form in different holes on phase carrier.

23. a kind of side of absolute quantity for measuring at least one measuring samples nucleic acid molecule according to claim 16Method, which is characterized in that wherein described step 5) is made known to signal strength and a series in relation to the control sample formThe standard curve and the step 6) for controlling the relation of sample form copy number will be at least one to be checked using the standard curveSample signal intensity-conversion into measuring samples template copy number, so that it is determined that in the hole of place PCR reaction before it is described to be checkedThe copy number of sample template, is completed by computer software.

24. a kind of method according to claim 11 measures the absolute quantity of at least one measuring samples nucleic acid moleculeKit, including：

● there is the nucleic acid molecule of known and different copy numbers as the porous of control sample form with one seriesPlate solid phase carrier；

● a pair of primer that sample form complementation is controlled with a series；With

● operation instructions.

25. a kind of a kind of method for the absolute quantity for measuring at least one measuring samples target molecule according to claim 1,Or a kind of method of absolute quantity for measuring at least one measuring samples nucleic acid molecule according to claim 16 is surveyedData and the database that encodes.