CN109576350A - A kind of kit, method and the quality control method of DNA and RNA simultaneous quantitative - Google Patents
A kind of kit, method and the quality control method of DNA and RNA simultaneous quantitative Download PDFInfo
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- CN109576350A CN109576350A CN201910047187.3A CN201910047187A CN109576350A CN 109576350 A CN109576350 A CN 109576350A CN 201910047187 A CN201910047187 A CN 201910047187A CN 109576350 A CN109576350 A CN 109576350A
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Abstract
The present invention relates to a kind of kit, method and quality control methods to DNA in the sample containing nucleic acid and RNA simultaneous quantitative, belong to molecular biology field.In kit of the invention, nucleic acid includes house-keeping gene, and house-keeping gene includes First Intron and two exons adjacent with First Intron, and two exons are respectively First Exon and Second Exon；Kit includes amplimer, the first hybridization probe that can hybridize with First Intron and the second hybridization probe that can hybridize with First Exon or Second Exon that specific can be annealed to the nucleic acid sequence of First Exon and Second Exon, has different markers on the first hybridization probe and the second hybridization probe.The present invention is also by DNA compared with the measured value of RNA simultaneous quantitative carries out Quality Control with high-flux sequence detected value, provide double quality control methods of DNA Yu RNA quality, this method be suitable for quickly and easily assessment sample qualities if appropriate for carrying out operation and cost requires higher high-flux sequence genetic test.
Description
Technical field
The present invention relates to kit, method and the quality control methods of a kind of DNA and RNA simultaneous quantitative, belong to molecular biology
Field.
Background technique
Next generation's sequencing (NGS) technology revolutionized genomics field in past ten years.Each NGS operation is logical
Often generate parallel hundreds of thousands of thousands of million sequence informations to billions of a DNA profilings about each sequencing operation.Currently used for
The cost of human genome sequencing has reached 1000 dollars of benchmark.The low cost and high throughput of NGS technology enable people
Enough use nucleic acid sequencing as clinical tool.
However, expected cost needed for reaching NGS clinical application, speed, sensitivity for analysis and accuracy, however it remains
Many challenges.Clinical sample, such as biopsy sample and formalin fix paraffin embedding (FFPE) sample, provide only a small amount of
Starting material.NGS, which builds library, can be used DNA or RNA as starting template, be respectively suitable for the inspection of different gene mutation types
It surveys.Often due to storing for a long time in room temperature, nucleic acid therein especially RNA's nucleic acid in clinical sample largely degrades, and makes RNA mould
Version is built library and is become difficult.The clinically used treatment process fixed to sample progress formalin makes nucleic acid generate crosslinking or broken
It is bad, decline so that NGS builds library efficiency and increase testing result background.Therefore, it is taking considerable time and cost building NGS text
Content quickly and easily is carried out to DNA and RNA template before library and quality evaluation is necessary.
Summary of the invention
The purpose of the present invention is to provide a kind of kit, method and kit for DNA and RNA simultaneous quantitative and
The quality control method of DNA and RNA.
To achieve the above object, the technical scheme adopted by the invention is as follows:
In a first aspect, the present invention provides a kind of kit to DNA in the sample containing nucleic acid and RNA simultaneous quantitative,
Wherein, the nucleic acid includes house-keeping gene, and the house-keeping gene includes base number less than 300 First Intron and with described the
Two adjacent exons of one introne, two exons are respectively First Exon and Second Exon；The reagent
Box include can specificity be annealed to the First Exon and Second Exon nucleic acid sequence amplimer, can be with first
First hybridization probe of introne hybridization and the second hybridization probe that can hybridize with First Exon or Second Exon, it is described
Different markers is had on first hybridization probe and the second hybridization probe.
Preferably, the kit also includes PCR reaction solution and inverse transcription reaction liquid.Preferably, the PCR reaction solution packet
Include archaeal dna polymerase, deoxynucleotide, inorganic salts and pH buffer；The inverse transcription reaction liquid includes reverse transcriptase, deoxyribonucleoside
Acid, inorganic salts and pH buffer.
Preferably, the kit further includes random primer or the poly-dT polymerized nucleoside complementary with mRNA polyA tail
Sour primer.
Preferably, the different luminophore of emission wavelength is had on first hybridization probe and the second hybridization probe.
Preferably, the luminophore is FAM, ROX, HEX, CY5, TET or VIC.
Preferably, any one of following (a)~(e):
(a) nucleic acid includes genomic DNA or its segment；
(b) nucleic acid includes RNA；
(c) nucleic acid includes cDNA or its segment, it is preferable that the cDNA passes through reverse transcription total serum IgE, mRNA, miRNA
Or other non-coding RNAs obtain；
(d) nucleic acid includes genomic DNA and RNA；
(e) nucleic acid includes genomic DNA and cDNA.
Preferably, the samples sources are in blood sample, cell sample, tissue samples, food sample, environmental samples or life
Object sample.
Preferably, the kit further includes the operation instructions of kit.
Second aspect, the present invention provides a kind of using mentioned reagent box to DNA in the sample containing nucleic acid and RNA simultaneously
Quantitative method comprising following steps:
(1) house-keeping gene is chosen, reverse transcription is carried out by template of the nucleic acid, is converted into the RNA in nucleic acid
cDNA；
(2) using the product after step (1) reverse transcription as template, using the amplimer as primer, while described is added
One hybridization probe and the second hybridization probe carry out PCR amplification, obtain amplicon；
(3) signal caused by marker on the first hybridization probe and the second hybridization probe is detected, to the sample containing nucleic acid
DNA and RNA are quantified in this, quantitative method are as follows: record marker on the first hybridization probe and the second hybridization probe respectively
Corresponding Ct value passes through the quality of DNA and RNA in the absolute value and differential analysis sample of Ct value；Wherein, the Ct value is mark
Signal produced by note object reaches corresponding amplification cycles number when setting detection threshold value.
The third aspect, it is same to DNA in the sample containing nucleic acid and RNA using mentioned reagent box that the present invention provides another kinds
The method of Shi Dingliang comprising following steps:
(1) house-keeping gene is chosen, reverse transcription is carried out by template of the nucleic acid, is converted into the RNA in nucleic acid
cDNA；
(2) using the product after step (1) reverse transcription as template, using the amplimer as primer, while described is added
One hybridization probe and the second hybridization probe carry out PCR amplification, obtain amplicon；
(3) using the nucleic acid as template, using the amplimer as primer, while first hybridization probe and is added
Two hybridization probes carry out PCR amplification；
(4) respectively in detecting step (2) and step (3) on the first hybridization probe and the second hybridization probe produced by marker
Signal, DNA in the sample containing nucleic acid and RNA quantify, quantitative method are as follows: distinguish recording step (2) and step
(3) the corresponding Ct value of marker on the first hybridization probe and the second hybridization probe, passes through the first hybridization probe in step (3) in
And/or second marker on hybridization probe Ct value analysis sample in DNA quality, pass through the first hybridization probe in step (2)
The difference of the Ct value of marker analyses the quality of RNA in sample on the first hybridization probe in the Ct value of upper marker and step (3)；Its
In, the Ct value is that signal produced by marker reaches corresponding amplification cycles number when setting detection threshold value.
Fourth aspect, the present invention provides a kind of method for carrying out Quality Control to DNA in the sample containing nucleic acid and RNA,
The following steps are included:
A reference gene is chosen from nucleic acid, the reference gene includes First Intron of the base number less than 300, alkali
Radix greater than 300 intron 2, two exons adjacent with the First Intron and with the intron 2 phase
Two adjacent exons；Or two reference genes are chosen from nucleic acid, one of reference gene includes base number less than 300
First Intron and two exons adjacent with the First Intron, another reference gene include base number be greater than
300 intron 2 and two exons adjacent with the intron 2；Wherein, two adjacent with First Intron
Exon is respectively First Exon and Second Exon, and two exons adjacent with intron 2 are respectively aobvious outside third
Son and the 4th exon；
To contain the reference gene of First Intron as house-keeping gene, using any one of claim 10~13 side
Method is to DNA in the sample containing nucleic acid and RNA simultaneous quantitative；
One of third exon edge builds library primer for building library by the single-ended anchoring of the RNA of template of the nucleic acid；With
The DNA cloning product of the base of intron 2 and third exon junctional area sequencing reading metering reference gene, outside the 4th
The RNA amplification product of the sequencing reading metering reference gene of aobvious son, the RNA in the library NGS is judged with the ratio of two sequencing readings
Build library quality；
According to the result of DNA and RNA simultaneous quantitative and judge the library NGS RNA build library quality as a result, comprehensive analysis
The quality of DNA and RNA in sample.
Compared with prior art, determine simultaneously the invention has the benefit that being used for DNA and RNA the present invention provides one kind
The method and kit of amount can analyze DNA and RNA using the kit and method simultaneously.Also, the present invention is also by DNA and RNA
The measured value of simultaneous quantitative provides double Quality Control sides of DNA Yu RNA quality compared with high-flux sequence detected value carries out Quality Control
Method, this method be suitable for quickly and easily assessment sample qualities if appropriate for carry out operation and cost require higher high throughput
Genetic test is sequenced.
Detailed description of the invention
Fig. 1 is the method schematic diagram that the present invention carries out Quality Control to DNA in the sample containing nucleic acid and RNA；
Fig. 2 be in the embodiment of the present invention 3 by the Ct difference of the exon probe of reverse transcription quantitative PCR and introne probe with
Reference gene RNA is sequenced to the result figure of DNA reading scale in NGS；
Fig. 3 is in the embodiment of the present invention 3 by the Ct of reverse transcription quantitative PCR and the exon probe of no reverse transcription quantitative PCR
Difference and NGS sequencing reference gene RNA read the result figure of scale to DNA；
Fig. 4 is that internal reference is sequenced in the ratio of the RNA and DNA concentration that use Qubit quantitative and NGS in the embodiment of the present invention 3
Result figure of the gene RNA to DNA reading scale；
Fig. 5 is that reference gene RNA is sequenced to DNA in the RNA concentration for using Qubit quantitative and NGS in the embodiment of the present invention 3
Read the result figure of scale.
Specific embodiment
In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology
The normally understood meaning of personnel institute.Also, molecular genetics used herein, nucleic acid chemistry and molecular biology relational language
It is widely used term and conventional steps in corresponding field with laboratory operation step.Meanwhile in order to better understand originally
Invention, is provided below the definition and explanation of relational language.
Term:
" nucleic acid " refers to the nucleotide polymer of any length, and including DNA and RNA.Nucleotide can be deoxidation core
Ribotide, ribonucleotide, modified nucleotide or base and/or its analog, or can be poly- by DNA or RNA
Synthase and mix any substrate in polymer.
The process for typically referring to generate two or more copies of expectation sequence " is expanded " as used herein.Amplification is anti-
The component answered can include but is not limited to, such as primer, polynucleotide template, polymerase, nucleotide, dNTP etc..
" PCR amplification " or " PCR amplification " refers to the specific fragment of geometric progression amplification target double-stranded DNA
Or the method for subsequence.PCR is well-known to those skilled in the art；See, e.g. United States Patent (USP) No.4,683,195 and 4,
683,202；" the PCR Protocols:A Guide to Methods " edited with Innis et al. nineteen ninety.PCR amplification causes
The quantity of target nucleotide sequences is in exponential increase.
" amplified production " or " amplicon " refers to the oligonucleotides generated by pcr amplification reaction, is particular target template core
The copy of a part of sour chain and/or its complementary series, corresponds to template nucleic acid sequence in nucleotide sequence and/or it is mutual
Complementary series.Amplified production can also comprising to primer have specificity sequence, and the sequence of the sequences flank target nucleic acid and/
Or its complementary series.Amplicon as described herein is usually double-stranded DNA, although its single chain can be referred to.
" house-keeping gene ", also known as housekeeping gene refer to that the genoid for being intended to stablize expression in all cells, product are
To necessary to maintenance radical cellular activities." reference gene " is equivalent to " house-keeping gene " in the present invention.Reference gene with
The expression of house-keeping gene relative abundance and stabilization, expression quantity in the derived tissues of Different categories of samples are not regulated and controled by biotic factor.
Term " exon " refers to the part being retained in mature mRNA, i.e. maturation mRNA corresponds to the portion in gene
Point.Introne is the part being cut away in mRNA process, is not present in mature mRNA.Exon and introne are all
It is for gene, the part of coding is exon, and not encoding is introne, and introne does not have hereditary effect.
" primer " is usually shorter single stranded polynucleotide, usually has free 3'-OH group, by with target sequence
Hybridize and be bound to target of interest, then promotes to polymerize with the polynucleotides of target-complementary.
" hybridization " and " annealing " used interchangeably herein refers to such reaction: wherein one or more polynucleotides are anti-
It should carry out stable compound with the hydrogen bond formed between the base by nucleotide groups.Hydrogen bond can pass through Watson-Crick
Base pairing, Hoogstein occur in conjunction with or by any other sequence-specific fashion.
As used herein, term " specificity annealing " refers to that nucleic acid hybridizes with the nucleic acid of complementary series.As used herein, core
The a part of of acid molecule can specifically hybridize with the complementary series on another nucleic acid molecules.That is, nucleic acid sequence
Whole length is not necessarily required to hybridize with a part of such sequence with " with another molecular specificity hybridizing ", for example,
The end 5' of molecule may exist one section of non-hybridized nucleotide, and one section of sequence of the end 3' of same molecule and another molecule
Specifically hybridize.
" hybridization probe " is a bit of Single-stranded DNA fragments (more than ten arrive several hundred a bases), for detecting the core being complementary
Acid sequence.Band is possible to detected signaling molecule on the nucleic acid molecules of hybridization probe, such as isotope or the nucleic acid of fluorescent marker
Molecule.When by probe and sample hybridization, probe and the nucleic acid being complementary (DNA or RNA) sequence are closely coupled by hydrogen bond.
In the present invention, " the first hybridization probe ", " the second hybridization probe ", " First Exon ", " Second Exon ",
" first ", " second " in " third exon ", " the 4th exon ", " First Intron ", " intron 2 " etc. etc. are only
In order to be distinguished to hybridization probe, exon, introne etc., and not its quantity is defined.
" reverse transcription " is to pass through reverse transcriptase, the process of synthetic DNA using RNA as template.It is one kind of DNA biosynthesis
Particular form.Reverse transcription is that during carrying out genetic engineering, the process of DNA hereditary information is gone out using RNA as template extraction.
" random primer " refers to when specific mRNA containing the sequence for terminating reverse transcriptase due to being difficult to copy its overall length
When sequence, this non-specific primer of random hexamer can be used to copy full length mRNA.
" reaction system " is the combination of component (such as one or more polypeptides, nucleic acid and/or primer), in suitable item
It is specifically reacted under part, such as primer extension reaction or pcr amplification reaction.
" library " refers to the set of nucleic acid sequence.
Term " determination ", " detection ", " measurement ", " evaluation ", " assessment ", " assaying " and " analysis " is interchangeably herein
Using to refer to any type of measurement, and including a certain element presence or absence of determination.These terms include quantitatively determine and/
Or qualitative determination.
It should be appreciated that the aspect and embodiment of invention as described herein include " Consists of " and/or " substantially by ...
Composition " aspect and embodiment.
As it is used herein, unless otherwise specified, singular "one", "an" and "the" draw including plural number
With.
As understood by those skilled in the art, the value or parameter being mentioned above " about " include that (and description) is related to this
The embodiment of value or parameter itself.For example, the description as described in " about X " includes the description to " X ".
In the case where providing numberical range, it should be understood that each median between the upper and lower bound of the range, with
And any other specified value in the range or median are included in the scope of the present disclosure.Include in the range of defined
In the case where the upper limit or lower limit, the range of any of boundary included by those is excluded, is also included in the disclosure.
Unless otherwise stated, the present invention be carried out using standardization program described in following bibliography, such as
(USA New York Cold SpringHarbor goes out " Molecular Cloning:A Laboratory Manual (the 3rd edition) " of Sambrook et al.
Version society, 2001)；With (USA New York " Basic Methods in Molecular Biology " of Davis et al.
Elsevier Science publishing company, nineteen ninety-five).
In order to investigate the quality of DNA and RNA in the sample containing nucleic acid, to improve the efficiency that subsequent high pass measures sequence,
In one embodiment of the present of invention, the present invention provides a kind of reagents to DNA in the sample containing nucleic acid and RNA simultaneous quantitative
Box, wherein the nucleic acid include house-keeping gene, the house-keeping gene include First Intron of the base number less than 300 and with institute
Two adjacent exons of First Intron are stated, two exons are respectively First Exon and Second Exon；It is described
Kit include can specificity be annealed to the First Exon and Second Exon nucleic acid sequence amplimer, can be with
First hybridization probe of First Intron hybridization and the second hybridization probe that can hybridize with First Exon or Second Exon,
Different markers is had on first hybridization probe and the second hybridization probe.
In mentioned reagent box of the invention, the length of First Intron is shorter, length is shorter refer to its with it is adjacent
The amplification unit length that the part of two exons is constituted is suitble to efficient amplification.The base number of First Intron need to less than 300,
If its base number, which is greater than 300, may cause PCR amplification imbalance, if First Intron can be 75 bases.It is above-mentioned can specificity
The amplimer for being annealed to the First Exon and the nucleic acid sequence of Second Exon is located at the Bu Tong outer of a gene
On aobvious son, gDNA and cDNA template can be expanded, the efficient expansion that the clip size of the amplicon of generation is reacted in quantitative PCR
Increase in range.Above-mentioned first hybridization probe and the second hybridization probe are respectively positioned on the amplification subrange that positive and negative amplimer is defined
It is interior.During reverse transcription quantitative PCR, the first hybridization probe acts on the intron sequences of gene, only detectable gDNA signal,
Second hybridization probe acts on the exon sequence of gene, detectable gDNA and cDNA signal summation (describing is RT_VIC).
Pair for amplification primer in kit and two specific probes through the invention, can be used reverse transcription quantitative PCR
GDNA signal and gDNA and cDNA signal summation are measured simultaneously, it is quantitative while to realize to DNA and cDNA.
The preferred embodiment of one embodiment as kit of the present invention, kit of the present invention also include PCR reaction solution and
Inverse transcription reaction liquid.It is highly preferred that the PCR reaction solution includes archaeal dna polymerase, deoxynucleotide, inorganic salts and pH buffer；
The inverse transcription reaction liquid includes reverse transcriptase, deoxynucleotide, inorganic salts and pH buffer.Further, the kit
It further include random primer or the poly-dT polynucleotide primer complementary with mRNA polyA tail.
A kind of above-mentioned dependenc RNA of reverse transcriptase is template archaeal dna polymerase.In transcriptive process,reversed, the reaction reagent that uses for
Above-mentioned inverse transcription reaction liquid and Oligonucleolide primers.Oligonucleolide primers can be gene-specific primer, such as can be special
One in the amplimer for the nucleic acid sequence that property is annealed to the First Exon and Second Exon, it is also possible to or at random
Primer, or the poly-dT polynucleotide primer complementary with mRNA polyA tail.
PH buffer in above-mentioned PCR reaction solution is used to adjust the pH value of PCR reaction system, inverse transcription reaction liquid pH buffering
Liquid is used to adjust the pH value of reverse transcription system.Those skilled in the art can be selected according to Conventional wisdom suitable pH buffer and
Inorganic salts, such as pH buffer select the buffer solution including trishydroxymethylaminomethane (Tris), such as Tris-HCl；It is inorganic
Salt selects magnesium chloride etc..The all the components of PCR reaction system and reverse transcription system can be bought or close from biological reagent producer
At acquisition.
In kit of the invention, the marker on hybridization probe is the signaling molecule that possible be detected, and be can be
Fluorescent marker is also possible to other markers, such as radioactive isotope (usually using phosphorus -32) etc..It is fixed for the ease of fluorescence
Amount analysis, the marker that the present invention selects are luminophore, i.e. fluorescent marker.In order to enable two probes generate respectively it is different
Signal, so that the fluorescence quantitative PCR instrument that two signals can be detected multiple wavelength detects simultaneously, first hybridization probe and
With the luminophore that emission wavelength is different on second hybridization probe.Preferably, the luminophore be FAM, ROX, HEX,
CY5, TET or VIC.It is highly preferred that the marker having on the first hybridization probe is FAM, the mark having on the second hybridization probe
Note object is VIC.FAM probe and VIC probe, such as Thermo Fisher, can order synthesis from biological reagent producer.
Preferably, any one of following (a)~(e):
(a) nucleic acid includes genomic DNA or its segment；
(b) nucleic acid includes RNA；
(c) nucleic acid includes cDNA or its segment, it is preferable that the cDNA passes through reverse transcription total serum IgE, mRNA, miRNA
Or other non-coding RNAs obtain；
(d) nucleic acid includes genomic DNA and RNA；
(e) nucleic acid includes genomic DNA and cDNA.
Kit of the present invention can be used for the various nucleic acid in sample.In some embodiments, nucleic acid includes
RNA.In some embodiments, nucleic acid includes the mixture of genomic DNA and RNA.
In some embodiments, nucleic acid-templated source (such as, fragmentation) in length be more than NGS method or platform most
The nucleic acid of good read length, such as overall length chromosomal DNA or full length mRNA.
In some embodiments, nucleic acid includes cDNA or its segment.In some embodiments, total by reverse transcription
RNA or part thereof (such as, mRNA, miRNA or other non-coding RNAs) obtains cDNA.In some embodiments, cDNA is
Single-stranded, such as at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of cDNA
Any one or more is single-stranded.In some embodiments, nucleic acid includes double-strand cDNA.
In some embodiments, nucleic acid includes gDNA or its segment.In some embodiments, gDNA is single-stranded,
Such as gDNA any one of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or
It is more single-stranded.In some embodiments, nucleic acid includes double-strand gDNA.
In some embodiments, nucleic acid includes the mixture of cDNA and gDNA.In some embodiments, cDNA with
Weight ratio between gDNA is more than about any one of 1:10,1:5,1:3,1:2,1:1,2:1,3:1,5:1,10:1 or more
Greatly.
In some embodiments, nucleic acid include no more than about 1000ng, 500ng, 200ng, 100ng, 50ng, 40ng,
Any one of 30ng, 25ng, 20ng, 15ng, 10ng, 5ng, 4ng, 3ng, 2ng, 1ng or less are nucleic acid-templated (such as
CDNA, gDNA, RNA, their combination or total nucleic acid).
Preferably, the samples sources are in blood sample, cell sample, tissue samples, food sample, environmental samples or life
Object sample.
In some embodiments, sample of the present invention derives from cell or tissue sample.In some embodiments, this hair
Bright samples sources are in cell line sample or from culture cell.In some embodiments, sample of the present invention derives from gene
Engineering cell system.In some embodiments, sample of the present invention derives from tumour cell.
In some embodiments, sample of the present invention is obtained from food sample, environmental samples or biological sample.Some
In embodiment, sample of the present invention derives from the biological sample from individual.In some embodiments, sample of the present invention source
In the biological sample for needing disease (such as cancer) to treat.In some embodiments, sample of the present invention is obtained from individual
Diagnostic sample.In some embodiments, sample of the present invention derives from the biological sample from healthy individuals.In some implementations
In scheme, sample of the present invention derives from genetic engineering animal (such as mouse, rat or non-human primates).
In some embodiments, biological sample also include protein, cell, fluid, biofluid, preservative and/or
Other substances.As non-limiting example, sample can be cheek swab, blood, serum, blood plasma, phlegm, cerebrospinal fluid, urine, tear
Liquid, alveolar isolate, liquor pleurae, pericardial fluid, cystic fluid, tumor tissues, tissue, living tissue, saliva, aspirate or they
Combination.In some embodiments, biological sample is obtained by excision or biopsy.
In some embodiments, blood sample of the sample of the present invention from individual.In some embodiments, this hair
Bright samples sources are in peripheral blood mononuclear cells (PMBC) sample of individual.In some embodiments, sample of the present invention derives from
A part of immunocyte (such as T cell, NK cell or B cell) in individual blood sample.In some embodiments, core
Acid template is Cell-free DNA.In some embodiments, the nucleic acid-templated Cell-free DNA for being derived from individual blood sample.?
In some embodiments, nucleic acid-templated is Circulating tumor DNA (i.e. ctDNA).In some embodiments, nucleic acid-templated to derive from
The circulating tumor cell of individual blood sample.
In some embodiments, sample of the present invention derives from individual biopsy sample.In some embodiments, of the invention
Biopsy of the samples sources after tumor biopsy, such as untreated biopsy or treatment.In some embodiments, originally
Invention samples sources are fixed and/or the biopsy of paraffin embedding in the formalin of individual.
In some embodiments, biological sample from need to treat associated with genetic change disease (such as, cancer or
Genetic disease) individual in obtain.In some embodiments, it is known that target sequence is present in the relevant gene of disease.?
In some embodiments, biological sample is obtained from the individual for need treating cancer.In some embodiments, biological sample packet
Tumour cell containing tumor locus one or more in individual.
In some embodiments, biological sample fresh collection from individual.In some embodiments, biological sample exists
For a period of time, such as at least about 1 day, 1 week, 1 month, 3 months, 6 months, 1 to be stored in method described herein before
Any one of year is more long.In some embodiments, biological sample is fixed paraffin embedding (FFPE) sample of formalin.
In some embodiments, biological sample is directly used as the sample of nucleic acid in methods described herein.In some embodiments, raw
Object sample is pre-processed by diluting and/or suspending in the solution.In some embodiments, biological sample is from subject
It is middle to obtain and be saved or process before in method described herein.For example, biological sample can be embedded in paraffin
In, refrigerated or freezed.The biological sample of freezing can use preceding defrosting.Other exemplary process or processing packet of biological sample
Include but be not limited to centrifugation, filtering, ultrasonic treatment, homogenizing, heating, freeze thawing, with preservative (for example, anti-coagulants or nuclease inhibit
Agent) contact and any combination of them.In some embodiments, biological sample chemistry and/or biological reagent processing.
Chemistry and/or biological reagent can be used for that the biological sample being included in is protected and/or maintained during processing and/or storage
Or nucleic acid-templated stability.In some embodiments, chemistry and/or biological reagent can be used for from biological sample other
It is discharged in component nucleic acid-templated.As non-limiting example, blood sample can be for obtaining in method described herein
Sample of nucleic acid before handled with anti-coagulants.Technical staff is perfectly clear the method for processing, saving or handle biological sample
And process, and method of the nucleic acid to carry out foranalysis of nucleic acids is separated from biological sample or cell sample.
In some embodiments, nucleic acid-templated in biological sample or sample of nucleic acid can be used for method described herein
In before by separation, enrichment or purifying.The appropriate method of separation, enrichment or purification of nucleic acid from sample can be used.
In order to facilitate kit of the invention is used, the kit further includes the operation instructions of kit.
Kit of the invention also may include one or more additional components such as container, co-factor, or additional
Reagent such as denaturant.Reagent constituents can be packaged together.
In one embodiment of the invention, the present invention provides a kind of using mentioned reagent box to the sample containing nucleic acid
The method of middle DNA and RNA simultaneous quantitative comprising following steps:
(1) house-keeping gene is chosen, reverse transcription is carried out by template of the nucleic acid, is converted into the RNA in nucleic acid
cDNA；
(2) using the product after step (1) reverse transcription as template, using the amplimer as primer, while described is added
One hybridization probe and the second hybridization probe carry out PCR amplification, obtain amplicon；
(3) signal caused by marker on the first hybridization probe and the second hybridization probe is detected, to the sample containing nucleic acid
DNA and RNA are quantified in this, quantitative method are as follows: record marker on the first hybridization probe and the second hybridization probe respectively
Corresponding Ct value passes through the quality of DNA and RNA in the absolute value and differential analysis sample of Ct value；Wherein, the Ct value is mark
Signal produced by note object reaches corresponding amplification cycles number when setting detection threshold value.
In the step of above method of the present invention (1), the mixing nucleic acid containing genomic DNA (gDNA) and RNA by reverse transcription,
Reverse transcription reaction is caused by combining with the oligonucleotides of RNA template, is catalyzed by reverse transcriptase, is reaction with deoxynucleotide
Substrate copies the DNA of complementary series, i.e., RNA therein is converted into complementary DNA (cDNA), the product after obtained reverse transcription
The as mixture of genomic DNA and cDNA.The difference of gDNA and cDNA is that gDNA contains exon and intron sequences, and
CDNA only contains exon sequence.Wherein, following condition can be selected in reverse transcription: by the component of reverse transcription system and to quantitative nucleic acid
Mixing, be placed in it is certain at a temperature of (such as 42 to 55 degrees Celsius), incubate certain time (such as 5 minutes to two hours), can be obtained
The complementary DNA (cDNA) of RNA template.Then, using pair for amplification primer and two not isolabeling probe (such as FAM probe and
VIC probe) carry out quantitative amplification reaction (such as qPCR).In quantitative amplification reaction process, a pair of positive and negative amplimer is located at
On the different exons of one gene, reaction is drawn by the amplimer for being incorporated into DNA template (including gDNA and cDNA template)
Hair, by archaeal dna polymerase enzymatic, using deoxynucleotide as reaction substrate, copies new DNA chain.New DNA chain becomes again
The template of next round reaction, so that reaction product expands the specific fragment of target double-stranded DNA, the expansion of generation in a manner of geometric progression
Increase the clip size of son within the scope of the efficient amplification that quantitative PCR reacts.Wherein, the first hybridization probe acts on introne, only
GDNA signal is detected, the second hybridization probe acts on exon, detection gDNA and cDNA signal summation (RT_VIC).
In some embodiments, can be after the completion of reverse transcription, then add reagent (the i.e. PCR reaction of quantitative PCR system
Liquid, amplimer, the first hybridization probe and the second hybridization probe).It in other embodiments, can also be in reverse transcription system
It is pre-mixed the component (i.e. PCR reaction solution, amplimer, the first hybridization probe and the second hybridization probe) of quantitative PCR system, is claimed
It is well-known to those skilled in the art for " one-step method reverse transcription PCR "." one-step method reverse transcription PCR " system is first placed in suitable
Under the conditions of the temperature and time for closing reverse transcription, such as 42 to 55 degrees Celsius, 5 minutes to two hours, the complementary DNA of RNA template is obtained
(cDNA).Under the conditions of this, the hot resistant DNA polymerase for quantitative PCR is not had an effect.System heating is then entered into PCR
It recycles, the thermal melting point (such as 80 to 100 degrees Celsius) under cycling condition can be such that reverse transcriptase inactivates, and terminate reverse transcription reaction.
In ideal quantitative PCR, Ct value and DNA content are inversely proportional with 2 for the logarithm at bottom, i.e., amount of DNA often turns over
Then Ct value reduces by 1 again.In practical applications, probe is to the joint efficiency of template, luminous intensity, the effect of amplification of different fluorophors
Rate, accidental factor etc. can all impact Ct value.Generally, it still can determine whether that the opposite of two nucleic acid contains by comparing Ct value
Amount." Ct value is low " of term is equal to " signal is strong " herein, and vice versa.
In method of the present invention to DNA in the sample containing nucleic acid and RNA simultaneous quantitative, the signal of the first hybridization probe
GDN in sample can be assessed, the difference of the signal of the signal of the second hybridization probe and the first hybridization probe can assess RNA in sample.Example
Such as, if using FAM group the first hybridization probe of label, VIC group the second hybridization probe of label then passes through reverse transcription quantitative PCR
Two Ct values are obtained, are described as RT_FAM and RT_VIC.RT_FAM reflects the signal that DNA template generates, RT_VIC and RT_FAM
Difference reflection RNA generate signal.The absolute value of signal and the threshold decision of difference, can be set by data accumulation and experience
It is fixed:
In some samples, RT_FAM signal is strong, and RT_VIC value is significantly lower than RT_FAM, illustrates DNA and RNA quality all
It is good；
In some samples, RT_FAM signal is strong, and the difference of RT_VIC and RT_FAM is unobvious, illustrates that DNA quality is good,
And RNA poor quality；
In some samples, RT_FAM signal is weak, and the difference of RT_VIC and RT_FAM is unobvious, illustrates DNA and RNA product
Matter is all poor；
In some samples, RT_FAM signal is weak, and RT_VIC value is significantly lower than RT_FAM, illustrates that RNA is high-quality；For
It is not affected by the sample of nucleic acid of pro-active intervention (be such as subjected to the degradation treatment of DNA nuclease, or only extract RNA), because of the stability of RNA
Lower than DNA, if RNA well can extrapolated sample DNA it is good, and it is due to competitive by strong RNA that RT_FAM signal is weak
Caused by inhibiting.
In one embodiment of the invention, the present invention provides another kinds to use mentioned reagent box to the sample containing nucleic acid
The method of DNA and RNA simultaneous quantitative in this comprising following steps:
(1) house-keeping gene is chosen, reverse transcription is carried out by template of the nucleic acid, is converted into the RNA in nucleic acid
cDNA；
(2) using the product after step (1) reverse transcription as template, using the amplimer as primer, while described is added
One hybridization probe and the second hybridization probe carry out PCR amplification, obtain amplicon；
(3) using the nucleic acid as template, using the amplimer as primer, while first hybridization probe and is added
Two hybridization probes carry out PCR amplification；
(4) respectively in detecting step (2) and step (3) on the first hybridization probe and the second hybridization probe produced by marker
Signal, DNA in the sample containing nucleic acid and RNA quantify, quantitative method are as follows: distinguish recording step (2) and step
(3) the corresponding Ct value of marker on the first hybridization probe and the second hybridization probe, passes through the first hybridization probe in step (3) in
And/or second marker on hybridization probe Ct value analysis sample in DNA quality, pass through the first hybridization probe in step (2)
The difference of the Ct value of marker analyses the quality of RNA in sample on the first hybridization probe in the Ct value of upper marker and step (3)；Its
In, the Ct value is that signal produced by marker reaches corresponding amplification cycles number when setting detection threshold value.
Above-mentioned steps (3) are the quantitative PCR reaction systems without reverse transcription.Quantitative PCR system can be only added in this step
Reagent (i.e. PCR reaction solution, amplimer, the first hybridization probe and the second hybridization probe), PCR system can also be added simultaneously
Reagent and part or all of reverse transcription needed for reagent.It, can be anti-by omitting when the reagent needed for reverse transcription is added simultaneously
Transcriptase or the other necessary components of change or omission reverse transcription incubation step directly heat into PCR cycle and reach, so that RNA
Not by reverse transcription.Quantitative PCR obtained signal value of the step (3) without reverse transcription is all from gDNA, can be used for assessing in sample
gDNA；Step (1) and gDNA the and cDNA signal summation of (2) reverse transcription quantitative PCR are identical without reverse transcription reaction pipe as step (3)
The difference of the gDNA value of probe can assess RNA in sample.
In another kind provided by the invention using mentioned reagent box to DNA in the sample containing nucleic acid and RNA simultaneous quantitative
Preferred method in, if using FAM group the first hybridization probe of label, VIC group the second hybridization probe of label passes through step
(1) and step (2) reverse transcription quantitative PCR obtains two Ct values, describes as RT_FAM and RT_VIC, by step (3) without reversion
The quantitative PCR of record can get two additional Ct values, describe as NR_FAM and NR_VIC, then:
It can illustrate that DNA quality is good with the DNA in NR_FAM and/or NR_VIC measurement sample, lower Ct value.This signal is not
Inhibited interference by RNA.
The RNA in the difference judgement sample of RT_VIC and NR_VIC can be used, if RT_VIC value significantly lower than RNA if NR_VIC
Quality is good.RNA quality can carry out auxiliary judgment with the difference of RT_FAM and NR_FAM.Its RT_VIC of preferable RNA can be to RT_
FAM causes to inhibit, its reading is caused to be higher than NR_FAM.
In the amplification reaction, the RNA of reverse transcription forms competitive type to DNA signal and inhibits, therefore the enhancing meeting of RT_VIC signal
With the decrease of RT_FAM, the opposite RNA mass weakened in aided assessment sample of RT_FAM and NR_FAM can be passed through.Synchronizing makes
Can get more data points with reverse transcription quantitative PCR and without reverse transcription quantitative PCR, but need to pay more reagent costs and
Operating cost may be unnecessary in some cases.
Fourth aspect, the present invention provides a kind of method for carrying out Quality Control to DNA in the sample containing nucleic acid and RNA,
The following steps are included:
A reference gene is chosen from nucleic acid, the reference gene includes First Intron of the base number less than 300, alkali
Radix greater than 300 intron 2, two exons adjacent with the First Intron and with the intron 2 phase
Two adjacent exons；Or two reference genes are chosen from nucleic acid, one of reference gene includes base number less than 300
First Intron and two exons adjacent with the First Intron, another reference gene include base number be greater than
300 intron 2 and two exons adjacent with the intron 2；Wherein, two adjacent with First Intron
Exon is respectively First Exon and Second Exon, and two exons adjacent with intron 2 are respectively aobvious outside third
Son and the 4th exon；
To contain the reference gene of First Intron as house-keeping gene, using any one of claim 10~13 side
Method is to DNA in the sample containing nucleic acid and RNA simultaneous quantitative；
One of third exon edge builds library primer for building library by the single-ended anchoring of the RNA of template of the nucleic acid；With
The DNA cloning product of the base of intron 2 and third exon junctional area sequencing reading metering reference gene, outside the 4th
The RNA amplification product of the sequencing reading metering reference gene of aobvious son, the RNA in the library NGS is judged with the ratio of two sequencing readings
Build library quality；
According to the result of DNA and RNA simultaneous quantitative and judge the library NGS RNA build library quality as a result, comprehensive analysis
The quality of DNA and RNA in sample.
In the above-mentioned method for carrying out Quality Control to DNA in the sample containing nucleic acid and RNA, First Intron is shorter includes
Son, intron 2 are longer introne.First Intron is the portion that shorter introne refers to itself and two adjacent exons
Constituted amplification unit length is divided to be suitble to efficient amplification, the base number of First Intron need to be less than 300, if its base number is greater than
300 may cause PCR amplification imbalance, if First Intron can be 75 bases.Intron 2 is that longer introne refers to it
Length is more than the nucleic acid average fragment length of two generations sequencing, and such as more than 300 bases, concretely 1608 base, second is included
If large fragment DNA reading and RNA reading may cannot be distinguished in sub shorter than 300 bases.If First Intron and intron 2 position
In on the same gene, they can be adjacent or non-conterminous.
The present invention carries out DNA in the sample containing nucleic acid and RNA in the method for Quality Control, and quantitative PCR and NGS sequencing are read
Associated guide's future position and subsequent analysis point are respectively become, the Quality Control mode of RNA library construction is collectively formed.The present invention into
The method of row Quality Control can be used for carrying out sample genetic test (such as gene sequencing or all kinds of PCR) preceding Quality Control for carrying out nucleic acid and comment
Estimate.Such genetic test relates generally to the duplication of nucleic acid template, requires to nucleic acid content and quality, and uses spectrometer
Nucleic acid quantification method only can determine nucleic acid content but cannot assess Nucleic acid quality.The method that the present invention carries out Quality Control is then needed to nucleic acid
Duplication amplification is carried out, if nucleic acid content is high and integrality is good, expanding effect is good.Such as built in library in two generations sequencing (NGS)
Using are as follows:
A) the clip size range of amplicon is suitable for high-flux sequence, thus can be used for NGS library construction
Initial nucleic acid does the Quality Control of DNA and/or RNA.
If b) the DNA signal of sample is good, illustrate that nucleic acid is complete, is then suitble to construct the library NGS by template of DNA.
If c) the extracted rna content of sample comparatively fresh is high and more complete, it is suitble to build library by the NGS of template of RNA；
Otherwise rna content is low or has degraded, and discomfort builds library jointly.
It should be noted that the present invention is to bases such as the result of DNA in the sample containing nucleic acid and RNA simultaneous quantitative and NGS
Because the quality of testing result has strong correlation, it is not intended that the two fits like a glove.The present invention carries out the method judgement of Quality Control
For low-quality nucleic acid it is possible to obtaining good testing result, vice versa, and therefore, the method that the present invention carries out Quality Control is suitable
For quickly and easily assessing sample qualities if appropriate for carrying out operation and cost requires that higher high-flux sequence gene is examined
It surveys.
The method that the present invention carries out Quality Control to DNA in the sample containing nucleic acid and RNA is as shown in Figure 1.
Embodiment 1
Present embodiment describes the present invention to the reagent of DNA in the sample containing nucleic acid and the kit of RNA simultaneous quantitative
Box.Wherein, the house-keeping gene of selection be mankind CHMP2A gene, gene number be NM_014453, choose CHMP2A gene in compared with
Short introne is No. 3 intrones (base number is 75), and longer introne is No. 2 intrones (base number is 1608).Of the invention
The composition of kit is as shown in table 1, and the amplimer in kit of the invention and probe sequence are as shown in table 2.Wherein, it expands
Primer 1 and amplimer 2 can be annealed to the nucleic acid sequence of two exon adjacent with No. 3 intrones in CHMP2A gene.
The composition of 1 kit of table.
The sequence of table 2 amplimer and probe
Embodiment 2
Present embodiment describes kits described in use embodiment 1 to DNA in the sample containing nucleic acid and RNA simultaneous quantitative
Method, i.e. the application method of kit described in embodiment 1.The equipment that the present embodiment the method uses is quantitative fluorescent PCR
Instrument, the specific steps are as follows:
Reagent pretreatment before use for the first time: 2 50 × Primer&Probe of pipe Mix are separately added into 2 × PreQC DNA
It is mixed with 2 × PreQC RNA, is labeled as 2 × PreQC DNA Mix and 2 × PreQC RNA Mix.
1) it according to the following table 3 and 4, prepares and mixes reagent:
Quantitative PCR system of the table 3 without reverse transcription
| Component | Volume |
| 2×PreQC DNA Mix | 5.0μL |
| Nuclease free deionized water | 4μL |
| Sample of nucleic acid to be checked | 1μL |
| Total volume | 10.0μL |
4 reverse transcription quantitative PCR system of table
| Component | Volume |
| 2×PreQC RNA Mix | 5.0μL |
| Nuclease free deionized water | 4μL |
| Sample of nucleic acid to be checked | 1μL |
| Total volume | 10.0μL |
2) brief centrifugation, coded program such as the following table 5
Table 5
3) parameter setting: by taking 3 instrument of QuantStudio as an example, the threshold value of FAM is set as the threshold value setting of 20000, VIC
It is 10000.
Embodiment 3
Present embodiment describes the nucleic acid to a collection of clinical tumor tissue samples to carry out quality evaluation process: sample 244
The total nucleic acid that a tumor tissues extract uses kits quantitative PCR value described in embodiment 1 respectively, and uses Thermo
The Qubit of Fisher company production quantifies DNA and RNA.Nucleic acid be used to target by the single-ended anchor titration of template of RNA and DNA
NGS builds library, and detection range includes a rich content and stable gene of expression is as internal reference in all types of tissues.The base
The amplicon of cause is since the marginal zone of an exon, with letter of the sequencing reading measurement from DNA template for adjoining introne
Number, the signal from RNA template is measured with the sequencing reading for the exon for being located at the introne other end, with the ratio of two signals
Measure the RNA detection quality that NGS builds library.
By the Ct difference (x-axis, RT_VIC-RT_FAM) of the exon probe of reverse transcription quantitative PCR and introne probe with
Reference gene RNA is sequenced to DNA reading ratio (y-axis, NGS:RNA2DNA) mapping, as a result as shown in Figure 2 in NGS.Data acquisition is certainly
The uneven sample of data is removed side by side in the library NGS of 244 clinical samples.In Fig. 2, NGS reference gene RNA reads ratio to DNA
In 0.3 or more (on the right side of vertical solid line) it is believed that the detection quality of RNA is preferable.From Figure 2 it can be seen that when RT_VIC is low with RT_FAM difference
Most of sample is fallen on the right side of vertical solid line (below horizontal dotted line) when -0.5, and the sample that NGS reference gene is read entirely without RNA
Point (dotted-line ellipse is irised out in figure) be entirely located in horizontal dotted line top or boundary and to be judged as nucleic acid unqualified, illustrate that it can do
Guide's quality control index of quality is detected for NGS.
By Ct difference (x-axis, the RT_VIC-NR_ of reverse transcription quantitative PCR and the exon probe of no reverse transcription quantitative PCR
VIC) with NGS sequencing reference gene RNA to DNA reading ratio (y-axis, NGS:RNA2DNA) mapping, as a result as shown in Figure 3.Data
Acquisition removes the uneven sample of data from the library NGS of 244 clinical samples side by side.NGS reference gene RNA reads ratio to DNA
In 0.3 or more (on the right side of vertical solid line) it is believed that the detection quality of RNA is preferable.As seen from Figure 3, when RT_VIC is low with NR_VIC difference
Most of sample is fallen on the right side of vertical solid line (below horizontal dotted line) when -0.5, and the sample that NGS reference gene is read entirely without RNA
Point (dotted-line ellipse is irised out in figure) be entirely located in horizontal dotted line top or boundary and to be judged as nucleic acid unqualified, illustrate that it can do
Guide's quality control index of quality is detected for NGS.
Internal reference base is sequenced in the ratio (x-axis, Qubit:RNA/DNA) of the RNA and DNA concentration that use Qubit quantitative and NGS
Because RNA is to DNA reading ratio (y-axis, NGS:RNA2DNA) mapping, as a result as shown in Figure 4.Data are acquired from 244 clinical samples
The library NGS remove the uneven sample of data side by side.0.3 or more, (vertical solid line is right to DNA reading ratio by NGS reference gene RNA
Side) it is believed that the detection quality of RNA is preferable.By Fig. 4 figure as it can be seen that can not make a horizontal line in figure makes major part above it
Sample is fallen on the right side of vertical solid line with respect to most of sample of lower section, and the sample point (figure that NGS reference gene is read entirely without RNA
Middle dotted-line ellipse is irised out) intersperse among y-axis to and cannot judge whether nucleic acid qualified, it is only fixed by the ratio of RNA and DNA concentration to illustrate
It measures without considering that Nucleic acid quality can not detect guide's quality control index of quality as NGS.
The RNA concentration for using Qubit quantitative (x-axis, Qubit:RNA (ng/ul)) and NGS are sequenced RNA pairs of reference gene
DNA reads ratio (y-axis, NGS:RNA2DNA) mapping, as a result as shown in Figure 5.NGS text of the data acquisition from 244 clinical samples
The uneven sample of data is removed side by side in library.NGS reference gene RNA is believed that DNA reading ratio 0.3 or more (on the right side of vertical solid line)
The detection quality of RNA is preferable.As seen from Figure 5, visible in figure to make a horizontal line most of sample below is fallen
On the right side of vertical solid line, and the sample point (dotted-line ellipse is irised out in figure) that NGS reference gene is read entirely without RNA intersperse among y-axis to
And cannot judge whether nucleic acid is qualified, illustrate only by RNA concentration quantitative without considering that Nucleic acid quality can not be detected as NGS RNA
Guide's quality control index of quality.
Three samples of selection are analyzed, the results are shown in Table 6.By table 6 as it can be seen that the Qubit of three samples is quantified
It is normal: Qubit:RNA/DNA > 1, Qubit:DNA > 10ng/ul, Qubit:RNA > 10ng/ul.However the first two library L18-
The NGS RNA detection effect of 00149T and L18-00152T is good (NGS:RNA/DNA > 1), third library L18-00194T's
NGS RNA detection effect is bad (NGS:RNA/DNA=0.14), illustrates that NGS and Qubit is quantitatively misfitted, and surveys with this method
Measure result to coincide: RT_VIC-RT_FAM and the RT_VIC-NR_VIC value in the first two library are below the threshold value (- 0.5) of setting,
RT_VIC-RT_FAM and the RT_VIC-NR_VIC value in third library are above -0.5.
Table 6
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
SEQUENCE LISTING
<110>Shenzhen perseverance Te Jiyin Co., Ltd
<120>kit, method and the quality control method of a kind of DNA and RNA simultaneous quantitative
<160> 4
<170> PatentIn version 3.3
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<211> 21
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<213>artificial synthesized
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tcccccttcc cactcccacg a 21
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Claims (10)
1. a kind of kit to DNA in the sample containing nucleic acid and RNA simultaneous quantitative, which is characterized in that the nucleic acid includes
House-keeping gene, the house-keeping gene include base number less than 300 First Intron and adjacent with the First Intron two
A exon, two exons are respectively First Exon and Second Exon；The kit includes being capable of specificity
First that is annealed to the amplimer of the nucleic acid sequence of the First Exon and Second Exon, can hybridize with First Intron
Hybridization probe and the second hybridization probe that can hybridize with First Exon or Second Exon, first hybridization probe and
Different markers is had on two hybridization probes.
2. kit as described in claim 1, which is characterized in that described also comprising PCR reaction solution and inverse transcription reaction liquid
PCR reaction solution includes archaeal dna polymerase, deoxynucleotide, inorganic salts and pH buffer；The inverse transcription reaction liquid includes reverse transcription
Enzyme, deoxynucleotide, inorganic salts and pH buffer.
3. kit as claimed in claim 2, which is characterized in that further include random primer or complementary with mRNA polyA tail
Poly-dT polynucleotide primer.
4. kit as described in claim 1, which is characterized in that had on first hybridization probe and the second hybridization probe
The different luminophore of emission wavelength.
5. kit as claimed in claim 4, which is characterized in that the luminophore be FAM, ROX, HEX, CY5, TET or
VIC。
6. kit as described in claim 1, which is characterized in that any one of following (a)~(e):
(a) nucleic acid includes genomic DNA or its segment；
(b) nucleic acid includes RNA；
(c) nucleic acid include cDNA or its segment, it is preferable that the cDNA by reverse transcription total serum IgE, mRNA, miRNA or its
He obtains non-coding RNA；
(d) nucleic acid includes genomic DNA and RNA；
(e) nucleic acid includes genomic DNA and cDNA.
7. kit as described in claim 1, which is characterized in that the samples sources are in blood sample, cell sample, tissue
Sample, food sample, environmental samples or biological sample.
8. it is a kind of using any one of claim 1~7 kit to DNA in the sample containing nucleic acid and RNA simultaneous quantitative
Method, which comprises the following steps:
(1) house-keeping gene is chosen, reverse transcription is carried out by template of the nucleic acid, the RNA in nucleic acid is made to be converted into cDNA；
(2) using the product after step (1) reverse transcription as template, using the amplimer as primer, while it is miscellaneous to be added described first
Probe and the second hybridization probe are handed over, PCR amplification is carried out, obtains amplicon；
(3) signal caused by marker on the first hybridization probe and the second hybridization probe is detected, in the sample containing nucleic acid
DNA and RNA are quantified, quantitative method are as follows: it is corresponding to record marker on the first hybridization probe and the second hybridization probe respectively
Ct value, pass through the quality of DNA and RNA in the absolute value and differential analysis sample of Ct value；Wherein, the Ct value is marker
Produced signal reaches corresponding amplification cycles number when setting detection threshold value.
9. it is a kind of using any one of claim 1~7 kit to DNA in the sample containing nucleic acid and RNA simultaneous quantitative
Method, which comprises the following steps:
(1) house-keeping gene is chosen, reverse transcription is carried out by template of the nucleic acid, the RNA in nucleic acid is made to be converted into cDNA；
(2) using the product after step (1) reverse transcription as template, using the amplimer as primer, while it is miscellaneous to be added described first
Probe and the second hybridization probe are handed over, PCR amplification is carried out, obtains amplicon；
(3) using the nucleic acid as template, using the amplimer as primer, while first hybridization probe and second miscellaneous is added
Probe is handed over, PCR amplification is carried out；
(4) detecting step (2) believe with caused by marker on the first hybridization probe in step (3) and the second hybridization probe respectively
Number, DNA in the sample containing nucleic acid and RNA are quantified, quantitative method are as follows: respectively in recording step (2) and step (3)
The corresponding Ct value of marker on first hybridization probe and the second hybridization probe passes through the first hybridization probe in step (3) and/or
The Ct value of marker analyzes the quality of DNA in sample on two hybridization probes, passes through marker on the first hybridization probe in step (2)
Ct value and step (3) on the first hybridization probe in the difference analysis sample of the Ct value of marker RNA quality；Wherein, described
Ct value is that signal produced by marker reaches corresponding amplification cycles number when setting detection threshold value.
10. a kind of method for carrying out Quality Control to DNA in the sample containing nucleic acid and RNA, which comprises the following steps:
A reference gene is chosen from nucleic acid, the reference gene includes First Intron of the base number less than 300, base number
Greater than 300 intron 2, two exons adjacent with the First Intron and adjacent with the intron 2
Two exons；Or two reference genes are chosen from nucleic acid, one of reference gene includes the of base number less than 300
One introne and two exons adjacent with the First Intron, another reference gene include base number greater than 300
Intron 2 and two exons adjacent with the intron 2；Wherein, two adjacent with First Intron show outside
Son be respectively First Exon and Second Exon, two exons adjacent with intron 2 be respectively third exon and
4th exon；
To contain the reference gene of First Intron as house-keeping gene, using any one of claim 10~13 the method pair
DNA and RNA simultaneous quantitative in sample containing nucleic acid；
One of third exon edge builds library primer for building library by the single-ended anchoring of the RNA of template of the nucleic acid；With second
The DNA cloning product of the base of introne and third exon junctional area sequencing reading metering reference gene, with the 4th exon
Sequencing reading metering reference gene RNA amplification product, with two sequencing reading ratio judge that the RNA in the library NGS builds library
Quality；
According to the result of DNA and RNA simultaneous quantitative and judge the library NGS RNA build library quality as a result, comprehensive analysis sample
The quality of middle DNA and RNA.
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