CN105039544A - Internal reference for real-time quantitative PCR detection of serum and plasma miRNA - Google Patents
Internal reference for real-time quantitative PCR detection of serum and plasma miRNA Download PDFInfo
- Publication number
- CN105039544A CN105039544A CN201510437551.9A CN201510437551A CN105039544A CN 105039544 A CN105039544 A CN 105039544A CN 201510437551 A CN201510437551 A CN 201510437551A CN 105039544 A CN105039544 A CN 105039544A
- Authority
- CN
- China
- Prior art keywords
- mir
- mirna
- serum
- internal reference
- real
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to an internal reference for real-time quantitative PCR detection of serum and plasma miRNA, and belongs to the technical field of biology. The internal reference for the real-time quantitative PCR detection of the serum and plasma miRNA is miR-30e or miR-30a. The invention discloses primers for detecting the internal reference. The sequences of a primer pair for amplifying miR-30e are shown in SEO ID NO:1 and SEQ ID NO:2, and the sequences of a primer pair for amplifying miR-30a are shown in SEO ID NO:3 and SEQ ID NO:2. The internal reference and the primers can be used for preparing a detection kit for the internal reference of serum and plasma miRNA and a labelled molecule kit for identifying prostate cancer prognosis miRNA. Expressions of the internal reference miR-30a and miR-30e are most stable among different individuals, the expressive abundance is high, and the internal reference is suitable for miRNA quantitative reference standard applied in serologic detection.
Description
Technical field
The present invention relates to a kind of internal reference detected for serum/plasma miRNA serum real-time quantitative PCR, be specifically related to a kind of internal reference miR-30e/a detected for serum/plasma miRNA serum real-time quantitative PCR, belong to biological technical field.
Background technology
Length, in the strand miRNA molecule of 21-23bp, can be incorporated into mRNA non-translational region with the mode target of partial complementarity, guides the translation of its degraded or suppression mRNA, makes target gene generation post-transcriptional silencing.MiRNA all plays the part of important regulating and controlling effect in numerous physiology and pathogenesis, almost participates in the pathologic process of all malignant tumours of the mankind.Confirm at present, the miRNA in blood has potential using value in the assessment of the diagnosis of Several Kinds of Malignancy, Treatment and Prognosis, has expedited the emergence of the new paragon based on detecting miRNA contamination and diagnosing tumour and assess.Once the specific miRNAs expression characteristic in body fluid is widely accepted, the method for so this detection miRNA is just the clinical detection mode providing the disease-related biomarker of a kind of non-intruding or minimum intrusive mood.And SD standard, once set up, namely possesses the feature of ubiquity, mandatory and objectivity as biochemical detection, can get rid of the interference of subjective factor on to greatest extent, and then improves diagnosis efficiency.But same miRNA expression characteristic is widely different in different research, this not only shows in the body fluid sample of blood plasma and the such different sources of urine, and shows to be study in the difference research of sample equally with blood.Such as, the expression level all showing miR-141 in serum of patients with prostate cancer and blood plasma in a lot of report increases.But also there is the expression of the same miRNA of a small amount of report display in patients serum to reduce, even cannot detect.Report is also had to point out that the content of this miRNA in prostate cancer, bladder cancer and patients with renal cell carcinoma serum does not have difference.For another example miR-16 presents low expression status in prostate cancer tissue, but but cannot differentiate patient and normal control population in serum.There is the reason of above conflicting result, no doubt have the factor of samples sources, sample process etc. aspect, but the more important thing is that different research and commercial test kit are applied caused by different qualitative reference systems.
When identifying and verify the miRNA of disease-related; usually two kinds of methods can be used; be the biomarker first being determined candidate by microarray or DNA sequencing technology of future generation, then use the method for real-time quantitative PCR to continue qualification with the reliability determining this biomarker.In the process, stdn occupies very consequence, because one, correct stdn can removal system bias and experimental variation, its two, stdn accurately can guarantee the detection to difference biological between different sample.For evaluating the therapeutic effect of miRNA in vitro, in body or even in clinical trial process, stdn is even more important.There is the stdn of bias that original significant result can be made to be misread, and then occur the conclusion of mistake.At present, diversified miRNA quantitative criterion seriously constrains the horizontal comparability of each result of study.
In the process of microarray, stdn can adopt loess method usually, miRNA kind and the absolute magnitude of this method hypothesis process LAN and the identical of low expression.And in DNA sequencing process of future generation, standardized method to suppose in different sample that the content of specific miRNA in total miRNA is consistent, thus specific miRNA relative expression quantity generally adopts the total copy number of the copy value of this miRNA and library compare and draw.The method of this population mean and other many standards merge the pattern of application and are not suitable for the research of small-scale, especially be not suitable for clinical applicability to detect, because in the miRNA dosing process being object with single sample or a small amount of gene, iff selection microarray helpless in order to stdn or DNA sequencing of future generation, this can cause the serious wasting of resources.So in scientific research and clinical practice, under real-time quantitative PCR detects and remains the background of the main flow means of current miRNA quantitative examination and application, a suitable qualitative reference greatly can promote the suitability of miRNA content detection from scientific research to clinical application.At present, the quantitative criterion that miRNA be widely accepted is special is still at issue.Bone of contention concentrates on the reasonableness of existing reference standard and the stability aspect of expression.
With tissue samples be at present miRNA quantitative examination many employings ribosome-RNA(rRNA) (5SrRNA) of object and small nucleolar RNA (smallnucleolarRNA) as RNU6, RNU43, RNU44, and RNU48.In addition, RNU43 may be used solely to, as with reference to standard, also to merge application as qualitative reference with RNU1-4, can increase its stability during merging.But there is obvious reasonableness problem in these quantitative criterions above.Function and the miRNA of ribosome-RNA(rRNA) and little nucleolar RNA exist obviously different.Three does not belong to the RNA of same type.Moreover, the length of small nucleolar RNA is between 60-300nt, obviously different from the 19-23nt of miRNA, and the difference on this molecular weight may cause the reactivity of small nucleolar RNA to leaching process and enzymatic chemical reaction to be different from miRNA.The more important thing is in addition, core is little to be mainly distributed in nucleus with small nucleolar RNA, and the miRNA of maturation is mainly distributed in the lipid bilayer corpusculum secreted by tenuigenin and cell, and there is remarkable difference the distributed areas of the two.In view of this, the little and small nucleolar RNA of core is directly brought as miRNA qualitative reference and improper.
Except endogenic RNA qualitative reference, the miRNA in the inhuman source of some synthetic is also used as insert qualitative reference.The method be by the non-human miRNA of the synthetic of equivalent RNA extract in add in different sample, when carrying out real-time quantitative PCR by detect this outer miRNAs content and by miRNA sample standardization.Conventional insert qualitative reference has the cel-miR-39/43/54/238 coming from C. Elegans Automatic Screening.The method lack in the sample conventional use quantitative time, especially often use in blood testing.But the method Problems existing is also apparent, first, the meaning that it exists only is technical difference criteriaization and the systematic error reducing experiment in RNA extraction and following detection step.System before any biological difference and RNA extract and human error all cannot accomplish effective stdn.Secondly, the possible human error that this insert qualitative reference newly adds again in operation, the most direct like this effect causes misreading of result.In addition, this qualitative reference very easily causes the outer miRNAs being difficult to eliminate to pollute in operation.
Lack the application that effective endogenous qualitative reference constrains miRNA to a certain extent.The different standards that each institute adopts illustrates at present up to the further precise verification of candidate criteria these with the quantitative potential of miRNA on the one hand, point out the qualitative reference of a certain type only can play useful effect to a certain particular system and cannot be transplanted in other system on the other hand, this situation can make the blanket quantitative criterion of searching more difficult.
At present, there is research display miR-191 and miR-103 all to present in numerous normal and tumor tissues and be obviously better than the expression of 5SrRNA and RNU6B high level of homogeneity, this result have also been obtained further confirmation, and this research finds that miR-103 is the most stable at the cells of all human adipose and different background.Other miRNA internal reference standard has miR-130b, miR-16 and miR-345.Wherein miR-16 is frequently used to as endogenous qualitative reference.But these endogenous are with reference to also having problems.Such as, evidence suggests miR-191 down-regulated expression inside prostate cancer tissue.MiR-103 is down-regulated expression in the gastric adenocarcinoma cells of multidrug resistance.Separately have research display, the content of miR-16 in red corpuscle is very high, once blood sampling is that haemolysis occurs, a large amount of miR-16 will be released, and like this in follow-up Serologic detection, miR-16 is just not suitable as endogenous reference and uses.
Based on above background, 192 the individual serum miRNA that come from that inventor gets equivalent are respectively prepared into library, utilize the DNA sequencing detection of platform of future generation expression level of each known miRNA, find that wherein the expression of miR-30a and miR-30e between Different Individual is the most stable.Following inventor applies the method for real-time quantitative PCR, determine miR-30a and miR-30e optimal stability in several candidate's internal reference, and gene expression abundance is high, thus has identified the miRNA qualitative reference standard of applicable Serologic detection application.
Summary of the invention
For there is at present referential deficiency, heterogeneousization in miRNA relative quantification process, stability is not enough and the problems such as pollution between planting, the invention provides a kind of internal reference miR-30e or miR-30a detected for serum/plasma miRNA serum real-time quantitative PCR, overcome shortcoming and the mistaken ideas of existing endogenous and exogenous internal reference standard, provide the internal reference of the better serum miRNA content standard of a kind of relative ease, effect.
The present invention is achieved by the following technical solutions:
For the internal reference that serum/plasma miRNA serum real-time quantitative PCR detects, described internal reference is miR-30e or miR-30a.
The primer of the internal reference for the detection of serum/plasma miRNA serum real-time quantitative PCR described in detection, the primer pair sequence of amplification miR-30e is for shown in SEOIDNO:1 and SEQIDNO:2; The primer pair sequence of amplification miR-30a is for shown in SEOIDNO:3 and SEQIDNO:2.
The described application of internal reference in preparation serum/plasma miRNA serum internal reference detection kit detected for serum/plasma miRNA serum real-time quantitative PCR.
A kind of serum/plasma miRNA serum internal reference detection kit, this test kit contains the primer of described miR-30e or the primer containing described miR-30a.
The described application of internal reference in characterization prostate cancer prognosis miRNA tagged molecule test kit detected for serum/plasma miRNA serum real-time quantitative PCR.And
In the present invention, preferably, described prostate cancer prognosis miRNA tagged molecule is miR-375 or miR-1290.
The application of primer in characterization prostate cancer prognosis miRNA tagged molecule test kit of the described internal reference for the detection of serum/plasma miRNA serum real-time quantitative PCR.
In the present invention, preferably, described prostate cancer prognosis miRNA tagged molecule is miR-375 or miR-1290.
Technical scheme of the present invention is made up of following 4 steps, prepared by (1) serum/plasma; (2) miRNA extracts; (3) miRNA is quantitative; (4) miRNA reverse transcription; (5) real-time quantitative PCR; (6) miR-30a/miR-30e expression stability analyzes (Fig. 1).Free and technical continuous relationship between each step.This technical sequential can not be changed.Concrete steps are:
(1) serum/plasma preparation: that gets equivalent comes from 192 individual serum/plasma; (2) miRNA extracts: the miRNeasySerummiRNA adopting QIAGEN company to produce extracts the miRNA in test kit extraction serum/plasma sample; (3) miRNA is quantitative: detect the smallRNA of extracting from serum with AgilentSmallRNAChip; (4) miRNA reverse transcription: use LifeTechnologies's respectively
the MiScriptIIRT test kit that MicroRNAReverseTranscriptionKit test kit and QIAGEN produce is to miRNA reverse transcription; (5) real-time quantitative PCR: application LifeTechnologies company produces
the miScriptSYBRGreenPCR test kit that UniversalPCRMasterMixII, NoUNG and QIAGEN company produces carries out real-time quantitative PCR; (6) miR-30a/miR-30e expression stability is analyzed: 1) by sequence expressing information input stability evaluating software Normfinder and BestKeeper that obtain for research material with equivalent miRNA, and software provides stability numerical value by for the copy number of each miRNA molecule in different sample; 2) be that the miRNA of 2ng is for after transcription templates carries out reverse transcription with total amount, to increase interested miRNA molecule for each sample again, by in the Ct value of Real-timePCR input stability evaluating software Normfinder and BestKeeper, software provides stability numerical value by for the Ct value of each miRNA molecule in different sample; 3) after output stability reference value, for each miRNA molecule, stability degree of variation in different sample can show with the form of column diagram, it should be noted that: the stability reference value that Normfinder exports is less, show that the stability of this molecule in different sample is higher.
The representative collection of illustrative plates when detecting with AgilentSmallRNAChip of the smallRNA of extracting from serum can draw, miRNA is in the interval range of 19-25nt, records concentration between 3 ~ 6ng/uL.Detect collection of illustrative plates based on this can determine: method of the present invention can be stablized and in serum, extract miRNA efficiently.
By the copy number input Normfinder of each candidate molecules of order-checking gained, after calculating, draw the stability that each candidate molecules is expressed in 192 samples and degree of variation.6 selected candidate molecules are respectively miR-30e, miR-30a, miR-99a, miR-16, miR-125 and let-7c.Result shows, when miRNA original bulk is homogeneous, miR-30e, miR-30a and miR-99a show the most stable candidate molecules.Real-timePCR shows the qualification of internal reference candidate molecules and the result, and the stability of the arithmetical av (GM30a/e) of miR-30e and miR-30a is best, and the distribution inside colony is the most homogeneous.When applying QIAGENReal-timeqPCR test kit, the degree of variation of miRNA candidate internal reference in 100 samples and stability and TaqManReal-timeqPCR method acquired results similar, the stability of the arithmetical av (GM30a/e) of miR-30e and miR-30a will be much better than other internal reference candidate molecules.
Based on above-mentioned experimental result, the invention provides a kind of serum/plasma miRNA serum internal reference detection kit, this test kit contains the primer of described miR-30e or the primer containing described miR-30a, and this test kit can also contain the conventional reagent of round pcr simultaneously.
MiR-30e and miR-30a is as internal reference for the present invention's application, and successful identification miR-375 and miR-1290 two miRNA molecule are to the diagnostic significance of prostate cancer prognosis.In 100 selected hormone-sensitives and insensitive patient, the method for application real-timePCR, using the Ct value of miR-30e and miR-30a as internal reference, the Δ Ct that the Ct value of target molecule obtains after deducting with it is that this molecule is at this intraindividual relative expression quantity.The height of 100 patients' foundation relative expression quantities is divided into two groups, and after Kaplan-Meier analyzes, we find, only have miR-375 and 1,290 two molecule relevant with overall patient's survival rate.The miR-375 of serum high expression level is relevant with short survival with 1290 (p=0.00020.0001).Another one does not then find associating between overall survival by inspection molecule miR-1246.By miR-375 compared with combining both 1290 and predicting with unit molecule, its diagnostic strength stronger (p=4.9 × 10-5).
Based on above-mentioned experimental result, the invention provides the described application of internal reference in characterization prostate cancer prognosis miRNA tagged molecule test kit detected for serum/plasma miRNA serum real-time quantitative PCR.And
The application of primer in characterization prostate cancer prognosis miRNA tagged molecule test kit of the described internal reference for the detection of serum/plasma miRNA serum real-time quantitative PCR.
"/" in the present invention in " serum/plasma " represents " with " and the relation of "or".
Compared with prior art, beneficial effect of the present invention is embodied in:
So far, we apply the serum miRNA qualitative reference experiment that quantitative criterion miR-30e/a has completed nearly 300 clinical various disease type patients of example, and result shows this standard can play effective standardized effect really.In addition, because this standard belongs to endogenous qualitative reference, it is much better than that its reasonableness compares small nuclear RNA and nucleolar RNA and insert qualitative reference.Moreover this standard expression amount is high, show special, stable in the detection means of real-time quantitative PCR.Finally, this quantitative criterion seems to migrate to other system and even applies in species, is not restricted to the quality standard of serum human miRNA.
The endogenous quantitative criterion that the present invention determines at least possesses the following feature of a qualified internal reference: same type; High conservative between different plant species; High abundance; Height comparability in histological types, most important, not because of species diversity, morbid state and process difference and stablize and express widely.
Current microRNA transforms to clinical application gradually as the diagnostic flag of disease, and its detection sensitivity and specific degree have obtained the confirmation of many research institutions.Therefore it is expected to this diagnostic flag can be widely used clinical in the short period of time.There is suitable endogenous qualitative reference, testing cost can have been saved to the full extent, relieve patient ' s burden.Simultaneously because early diagnosis kit can be prepared easily, disease can be found that this morning carries out clinical intervention in bud after being applied, like this can the health level of significant increase compatriots, alleviate the psychological pressure of patient to the full extent, strengthen its general well-being, therefore there is obvious social benefit.
Accompanying drawing explanation
Fig. 1 is the technology of the present invention operational flowchart;
Fig. 2 is the representative collection of illustrative plates when detecting with AgilentSmallRNAChip of the smallRNA of extracting from serum; Wherein, figure A is the detection collection of illustrative plates of RNA sample, and figure B is the peak figure corresponding with each track;
Fig. 3 is the estimation of stability result figures of 6 candidate miRNA reference molecules (miR-30e, miR-30a, miR-99a, miR-16, miR-125 and let-7c) after order-checking qualification; Wherein, A figure is that Normfinder analyzes 6 the stability result figures of candidate molecules in colony, figure B is 6 its general stabilities of candidate miRNA reference molecules, organizes stability and degree of variation between interior and group, and figure C is the distribution plans of 6 candidate molecules copy numbers that system records when sequencing inside colony;
Fig. 4 is that Real-timePCR is to the qualification of internal reference candidate molecules and the result figure; Wherein, A figure and B figure is respectively in 10 combination of primers, degree of variation figure in other 100 samples of 5 miRNA candidate internal references and stability diagram, C figure and D figure be respectively when primer form be reduced to 4 by 10 time, the degree of variation figure of each miRNA candidate internal reference in same 100 samples and stability diagram;
Fig. 5 is when applying QIAGENReal-timeqPCR test kit, degree of variation figure in 100 samples of 4 miRNA candidate internal references and stability diagram; Wherein, A figure and B figure is respectively the degree of variation figure of 4 miRNA candidate internal references in 100 samples and stability diagram;
Fig. 6 is for application miR-30e and miR-30a is as internal reference, and qualification miR-375 and miR-1290 two miRNA molecule are on the impact of prostate cancer diagnosis; Wherein, A figure and B figure is respectively the miR-375 of serum high expression level and the correlation diagram between 1290 and overall survival, C figure is the correlation diagram that another one is subject between inspection molecule miR-1246 and overall survival, and D figure is the correlation diagram after both miR-375 and 1290 combination and between overall survival.
Embodiment
Below by experiment, also the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiments only for the object of illustration, never limit the scope of the invention.
Technical scheme of the present invention is made up of following 4 steps, prepared by (1) serum/plasma; (2) miRNA extracts; (3) miRNA is quantitative; (4) miRNA reverse transcription; (5) real-time quantitative PCR; (6) miR-30a/miR-30e expression stability analyzes (Fig. 1).Free and technical continuous relationship between each step.This technical sequential can not be changed.Be specifically described as follows:
Prepared by embodiment 1 serum/plasma
1) spare unit: vacuum Sodium Citrate or EDTA anti-freezing collection tube or vacuum are without antithrombotics blood collection tube 1, and blood taking needle 1 overlaps, tourniquet, the tincture of iodine, 75% ethanol.1, whizzer, pipettor 1 overlaps, aseptic liquid transfer gun head and tissue freezing pipe some.
2) check before intravenous blood collection that blood sampling spare unit have N/D, guarantee that collection tube is without breakage, anti-freezing liquid does not go bad, without muddy and foreign matter, and within the quality guaranteed period.Need before blood sampling to shake blood collection tube, make antithrombotics fully coat tube wall.
3) venous blood is gathered: selection is thick and straight, the vein of good springiness, first iodine disinfection, alcohol disinfecting after 1 minute, then prick tourniquet, with blood taking needle inclined-plane upwards, miter angle inserting needle, needle point intravasation is parallel inserting needle just, treats pin intravasation, fix blood collection needles, the other end is thrust the rubber cap of vacuum collection pipe, when blood flow volume reaches about 5 milliliters, separated and collected pipe and blood taking needle, put upside down collection tube gently several times, blood and antithrombotics are fully mixed.As serum need be extracted, then antithrombotics can not be had in heparin tube.
4) preparation of serum: the blood of acquisition can not anti-freezing, in collection tube, leave standstill 1 hour or put short its in 37 DEG C of environment and solidify, after blood coagulation, the centrifugal 10min of 3000g, be transferred in new tissue freezing pipe with the careful Aspirate supernatant of pipettor (i.e. serum composition), note will drawing out cellular constituent scarcely, for avoiding cellular component to pollute, the serum that can remain about 0.5cm height in collection tube does not shift.By serum component according to the packing of 0.25mL/ pipe ,-80 DEG C for subsequent use.
5) blood plasma preparation: by the anticoagulant tube with blood after room temperature leaves standstill 30min, trim, then in 3000g, 4 DEG C of centrifugal 30min, the supernatant liquor of gained is blood plasma.Draw blood plasma, be dispensed in clean 1.5mL plastic centrifuge tube with 0.25mL/ pipe ,-80 DEG C save backup.For guaranteeing not contain cellular constituent in serum, in centrifuge tube, at least retain the high supernatant of 0.5cm.
Embodiment 2 serum/plasma RNA extracts
1) spare unit: without enzyme 15mL and 1.5mL centrifuge tube, dehydrated alcohol, chloroform, without each 1 box of enzyme 1mL, 200uL, 10uL liquid transfer gun head, centrifuge tube shelf 1, pipettor 1 overlaps, desktop type refrigerated centrifuge 1.The miRNeasySerummiRNA that QIAGEN company produces extracts test kit 1 and overlaps (article No.: 217184).RNaseZap1 bottle, aseptic mouth mask and each 1 pair of gloves.
2) taken out from refrigerator by serum/plasma sample, thaw on ice, period wears masks and gloves, with RNaseZap spraying operation table top, pipettor and relevant extraction consumptive material, and deactivation RNA enzyme.
3) draw 200uL sample with pipettor and be transferred to a new 1.5mL without in enzyme centrifuge tube.Add 1mLQIAzolLysisReagent.Vortex or piping and druming mixing.Room temperature (15 – 25 DEG C) hatches 5min.
4) in mixture, add 200uL chloroform, acutely rock 15s, fully incubated at room 2-3min after mixing.
5) 4 DEG C, the centrifugal 10min of 12,000g.
6) by upper water phase transition to new collection tube, the dehydrated alcohol of 1.5 times of volumes is then added, piping and druming mixing.
7) draw the mixture that 700uL comprises any precipitation, proceed in a RNeasyMinElute centrifugal column be pre-installed on collection tube, cover lid, with the centrifugal 15s of the rotating speed of >8000g, discards effluent liquid.
8) repeat previous step, remaining sample is crossed post and discards effluent liquid.
9) drawing 700uLBufferRWT is added in centrifugal column, and cover lid, with the centrifugal 15s of the rotating speed of >8000g, discards effluent liquid.
10) drawing 500uLBufferRPE is added in centrifugal column, and cover lid, with the centrifugal 15s of the rotating speed of >8000g, discards effluent liquid.
11) drawing 500uL80% ethanol is added in centrifugal column, and cover lid, with the centrifugal 2min of the rotating speed of >8000g, discards effluent liquid and collection tube.
12) centrifugal column is placed on a new 2mL collection tube, uncap, at full speed (>20,000g) centrifugal 5min.Discard collection tube and effluent liquid.
13) centrifugal column is placed on a new 1.5mL collection tube, draws 14uL and be directly added on the film of post central position without enzyme deionized water.Cover lid, (>20,000g) centrifugal 1min is to gather in the crops RNA elute soln at full speed.-80 DEG C save backup.
Embodiment 3RNA detection by quantitative
1) spare unit: AgilentSmallRNAChip, without enzyme 15mL and 1.5mL centrifuge tube, without enzyme deionized water, RNaseZap1 bottle, without each 1 box of enzyme 1mL, 200uL, 10uL liquid transfer gun head, centrifuge tube shelf 1, pipettor 1 overlaps, desktop type refrigerated centrifuge 1, AgilentBioanalyzer2100 detector 1, the chip prep stand (Agilentprimingstation) with 1mL syringe is a set of.
2) instrument prepares: first draw 500uLRNaseZap and join in the cleaning chip provided in one piece of AgilentSmallRNAChip test kit, avoid occurring bubble when noting liquid feeding.Be placed on the chip erecting stage of detector by cleaning chip, close the cover, soak probe after 5 minutes, change other one piece and be added with the chip of 500uL without enzyme deionized water in advance, soaking and washing probe is to detecting beginning.
3) test kit before using needs the liquid glue of about 650uL to be transferred to (test kit provides) in centrifugal chimney filter, with the centrifugal 15min of centrifugal force room temperature of 10000g ± 20%, and impurity screening.The liquid glue filtered can preserve 4 weeks under refrigerated conditions.
4) from refrigeration, AgilentSmallRNAChip is taken out before detecting, balance to room temperature.By be stamped blue cap concentrated dyestuff vortex oscillation 10sec after simply centrifugal, then draw 2uL and join a 0.5mL without in enzyme centrifuge tube (test kit provides).
5) add in centrifuge tube 40uL filter after liquid glue, repeatedly inhale beat or attack until estimate fluorescence dye be uniformly distributed completely.13,000g, after the centrifugal 10min of room temperature, lucifuge is for subsequent use.
6) be placed on operator's console by chip prep stand, guarantee: draw-in groove is positioned at C position, the syringe piston of connection is in 1mL position, and fixing handle card is on the injector in lowest order.
7) chip is placed in the draw-in groove of prep stand, in " G " hole indicating dark-background, adds the glue-dye mixture of 9uL.Close the upper cover of prep stand, at the uniform velocity presses down piston until handle card is by stuck piston.Timing 1min, release handle card, when waiting for piston clear-cutting forestland to more than 0.7mL, manually recovers piston to 1mL position, opens upper cover.
8) in two " G " holes indicating light background, add the glue-dye mixture of 9uL respectively.Next in the hole indicating " C ", 9uL condition damping fluid (conditioningsolution) is added.
9) in all the other 12 holes, add 5uL label solution (MarkerSolution) respectively, absorption 1uLLadder joins the contiguous lower right corner and indicates in the hole of ladder-shaped.
10) draw 1uLRNA sample, join all the other 11 respectively and be added with in the hole of label solution.If sample number is less than 11, then add as an alternative with 1uL label solution.
11) take out chip from prep stand, be placed on horizontal vortex vortex mixer and shake after 1 minute, upper machine testing.
The representative collection of illustrative plates when detecting with AgilentSmallRNAChip of the smallRNA of extracting from serum as shown in Figure 2, Fig. 2 A is the detection collection of illustrative plates of RNA sample, the RNA sample extracted from 4 different serum samples respectively gets 1uL, detects the gel electrophoresis spectrum formed through Bioanalyzer.Fig. 2 B is the peak figure corresponding with each track.MiRNA is in the interval range of 19-25nt, records concentration between 3 ~ 6ng/uL.Detect collection of illustrative plates based on this can determine: method of the present invention can be stablized and in serum, extract miRNA efficiently.
Embodiment 4miRNA reverse transcription
Reverse transcription adopts the test kit from Liang Ge company respectively, and one is LifeTechnologies
microRNAReverseTranscriptionKit test kit, one is the MiScriptIIRT test kit that QIAGEN produces, and is described below:
1) apply
microRNAReverseTranscriptionKit (article No.: 4366596)
1. spare unit:
microRNAReverseTranscriptionKit,
microRNAAssaycontainsone5 × RTprimer, without enzyme deionized water, TE damping fluid, without enzyme 1.5mL centrifuge tube and 200uLPCR thin-walled reaction tubes and lid, without each 1 box of enzyme liquid transfer gun head.
2. from refrigerator, take out RNA sample, Reverse Transcriptase kit and miR-30a, miR-30e reverse transcriptase primer (RTprimer).After thawed on ice, by stand-by for reverse transcriptase primer vortex concussion mixing.Period RNaseZap spray treatment operating table surface and pipettor.
3. draw 10uL5 × miRNA reverse transcriptase primer to join in clean 1.5mL centrifuge tube, then add 980uL1 × TE, concentrated RT primer is diluted 100 times.
4. get one without the 200uLPCR reaction tubes of enzyme, add 2ngmiRNA as original template (volume is no more than 4.01uL, as being less than 4.01uL, not enough volume H
2o polishing), according to the Establishing reverse transcription reaction of table 1, for 15uL reaction system:
Table 1
| Component | Application of sample amount (uL) |
| 100mM dNTPs(with dTTP) | 0.30 |
| MultiScribe TM Reverse Transcriptase,50U/μL | 3.00 |
| 10×Reverse Transcription Buffer | 1.50 |
| RNase Inhibitor,20U/μL | 0.19 |
| RT primer mix | 6.00 |
| RNA sample+Nuclease-free water | 4.01 |
| Cumulative volume | 15.00 |
5. cover lid, repeatedly puts upside down gently and mixes each component 6 times, and the of short duration centrifugal reaction mixture that makes flows to bottom PCR reaction tubes.Centrifuge tube is placed in and hatches 5min on ice, then go up machine reverse transcription.
6. reverse transcription reaction, according to table 2 condition setting PCR instrument.After setting reaction conditions, be placed on the heating module of PCR instrument by the PCR pipe of reverse transcription reaction mixed solution, cover the heat lid of PCR instrument, input reverse transcription system is 15uL, starts reverse transcription reaction.
Table 2
| Step | Temperature (DEG C) | Time (min) |
| Hold | 16 | 30 |
| Hold | 42 | 30 |
| Hold | 85 | 5 |
| Hold | 4 | ∞ |
7. react complete, take out PCR reaction tubes ,-20 DEG C save backup, and reaction mixture can stablize week age at-15 ~-20 DEG C the longest.
2) QIAGENMiScriptIIRT Reverse Transcriptase kit (article No.: 218160) is applied
1. spare unit: QIAGENMiScriptIIRTKit, without enzyme deionized water, 200uLPCR thin-walled reaction tubes and lid, without each 1 box of enzyme liquid transfer gun head.
2. before application of sample preparation work as aforementioned.From refrigerator, take out the components such as 10 × NucleicsMix in RNA template and test kit and 5 × miScriptHiSpecBuffer, be placed in thawed on ice, dissolve the mixing of rear vortex.The apparatuses such as the operating table surface used in process of reverse-transcription and pipettor carry out without the process of RNA enzyme by period.
3. set up reverse transcription reaction for 20uL reaction, in order each component is joined in 200uLPCR pipe.Concrete application of sample amount reference table 3:
Table 3
| Component | Application of sample amount (uL) |
| 5×miScript HiSpec Buffer or 5×miScript HiFlex Buffer | 4.00 |
| 10×Nucleics Mix | 2.00 |
| miScript Reverse Transcriptase Mix | 2.00 |
| RNase-free Water | Variable |
| RNA sample | Variable |
| Cumulative volume | 20.00 |
In upper table, the usage quantity of RNA template is 2ng.Add the deionized water polishing of volume not enough after template without enzyme to 20uL.Application of sample is complete, puts upside down mixing gently, to be of short durationly centrifugally placed on ice until upper machine.
4. according to the condition setting PCR instrument of table 4:
Table 4
| Step | Temperature (DEG C) | Time (min) |
| Hold | 37 | 60 |
| Hold | 95 | 5 |
| Hold | 4 | ∞ |
After setting reaction conditions, be placed on the heating module of PCR instrument by the PCR pipe of reverse transcription reaction mixed solution, cover the heat lid of PCR instrument, input reverse transcription system is 20uL, starts reverse transcription reaction.
5. react complete, take out PCR reaction tubes ,-20 DEG C save backup.
Embodiment 5 real-time quantitative PCR (RealtimeqPCR)
Real-time quantitative PCR still duration employing sets up reaction from the test kit of above Liang Ge company.
1) application LifeTechnologies company produces
universalPCRMasterMixII, NoUNG (article No.: 44400043)
1. spare unit:
universalPCRMasterMixII, NoUNG, without enzyme deionized water, TE damping fluid, without enzyme 200uLPCR thin-walled eight connecting leg and lid, or 96/384 hole PCR plate (operating for eight connecting legs below), without each 1 box of enzyme liquid transfer gun head.
2. 0.1 × TE is used by reverse transcription product with the dilution proportion of 1:20, after repeatedly putting upside down mixing, of short duration centrifugal for subsequent use.
3. in PCR eight connecting leg, set up real-time quantitative PCR reaction according to the design of table 5, the multiple hole of each reaction setting two is to reduce system and personal errors:
Table 5
| Component | Application of sample amount (uL) |
| 20×TaqMan13 MicroRNA Assays primer | 0.50 |
| TaqMan13 Universal Master Mix II,No UNG(2×) | 5.00 |
| Diluted RT Product | 2.00 |
| RNase-free Water | 2.50 |
| Cumulative volume | 10.00 |
Above each component is added good after, repeatedly put upside down pipe mixing, to be of short durationly centrifugally placed on ice.
4. start Real-timePCR instrument, open heat lid, reaction mixture is placed on the heating module of Real-timePCR instrument, covers tightly lid.Reaction conditions is set according to table 6:
Table 6
2) the miScriptSYBRGreenPCR test kit that QIAGEN company produces is applied
1. spare unit: miScriptSYBRGreenPCR test kit, without enzyme deionized water, without enzyme 200uLPCR thin-walled eight connecting leg and lid, or 96/384 hole PCR plate (operating for eight connecting legs below), without each 1 box of enzyme liquid transfer gun head.
2. 0.1 × TE is used by reverse transcription product with the dilution proportion of 1:20, after repeatedly putting upside down mixing, of short duration centrifugal for subsequent use.
3. in PCR eight connecting leg, set up real-time quantitative PCR reaction according to the design of table 7, the multiple hole of each reaction setting two is to reduce system and personal errors:
Table 7
| Component | Application of sample amount (uL) |
| 2x QuantiTect SYBR Green PCR Master Mix | 10.00 |
| miRNA primer | 2.00 |
| 10×miScript Universal Primer | 2.00 |
| Diluted RT Product | 2.00 |
| RNase-free Water | 4.00 |
| Cumulative volume | 20.00 |
Above each component is added good after, repeatedly put upside down pipe mixing, to be of short durationly centrifugally placed on ice.
4. start Real-timePCR instrument, open heat lid, reaction mixture is placed on the heating module of Real-timePCR instrument, covers tightly lid.Reaction conditions is set according to table 8:
Table 8
Embodiment 6 data analysis
1) by sequence expressing information input stability evaluating software Normfinder and BestKeeper that obtain for research material with equivalent miRNA, software provides stability numerical value by for the copy number of each miRNA molecule in different sample.
6 candidate miRNA reference molecules are through checking order the estimation of stability after identifying as shown in Figure 3.By the copy number input Normfinder of each candidate molecules of order-checking gained, after calculating, draw the stability that each candidate molecules is expressed in 192 samples and degree of variation.6 selected candidate molecules are respectively miR-30e, miR-30a, miR-99a, miR-16, miR-125 and let-7c.192 samples are not divided into groups or is divided into disease group and normal group, the stability of these 6 candidate molecules in colony via Normfinder analyze after as shown in Figure 3A, in its general stability, group and between group, stability and degree of variation are as shown in 3B, and visible miR-30e, miR-30a and miR-99a are 3 the most stable candidate molecules.What Fig. 3 C represented is the distribution of 6 candidate molecules copy numbers that system records when sequencing inside colony, and as seen when miRNA original bulk is homogeneous, miR-30e, miR-30a and miR-99a show the most stable candidate molecules.
2) be that the miRNA of 2ng is for after transcription templates carries out reverse transcription with total amount, to increase interested miRNA molecule for each sample again, by in the Ct value of Real-timePCR input stability evaluating software Normfinder and BestKeeper, software provides stability numerical value by for the Ct value of each miRNA molecule in different sample.
Real-timePCR is to the qualification of internal reference candidate molecules and verify as shown in Figure 4, application TaqManReal-timeqPCR method, in 10 combination of primers (see table 9), degree of variation (4A) in other 100 samples of 5 miRNA candidate internal references and stability (4B).When primer composition is reduced to 4 by 10, the degree of variation (4C) of each miRNA candidate internal reference in same 100 samples significantly reduces, and what correspond is that stability (4D) obviously strengthens.The all data shown in scatter diagram are all by the order sequence that miR-16-5pCt value is descending.This shows miR-30e and miR-30a, especially the stability of the arithmetical av (GM30a/e) of miR-30e and miR-30a is best, and the distribution inside colony is the most homogeneous.
Table 9 is by the primer sequence information of inspection miRNA molecule
When applying QIAGENReal-timeqPCR test kit, the degree of variation of 4 miRNA candidates internal reference (table 9) in 100 samples (5A) and stability (5B) and TaqManReal-timeqPCR method acquired results similar.The stability of the arithmetical av (GM30a/e) of miR-30e and miR-30a, especially miR-30e and miR-30a will be much better than other internal reference candidate molecules.The all data shown in scatter diagram are all by the order sequence that miR-16-5pCt value is descending.
3) after output stability reference value, for each miRNA molecule, stability degree of variation in different sample can show with the form of column diagram, it should be noted that: the stability reference value that Normfinder exports is less, show that the stability of this molecule in different sample is higher.
MiR-30e and miR-30a is as internal reference in application, and our successful identification miR-375 and miR-1290 two miRNA molecule are to the diagnostic significance of prostate cancer prognosis.In 100 selected hormone-sensitives and insensitive patient, the method for application real-timePCR, using the Ct value of miR-30e and miR-30a as internal reference, the Δ Ct that the Ct value of target molecule obtains after deducting with it is that this molecule is at this intraindividual relative expression quantity.The height of 100 patients' foundation relative expression quantities is divided into two groups, and after Kaplan-Meier analyzes, we find, only have miR-375 and miR-1290 two molecules relevant with overall patient's survival rate.MiR-375 with miR-1290 relevant with short survival (p=0.00020.0001) (Fig. 6 A, Fig. 6 C) of serum high expression level. another one does not then find associating (Fig. 6 C) between overall survival by inspection molecule miR-1246.Both miR-375 with miR-1290 are combined compared with predicting with unit molecule, stronger (p=4.9 × 10-5) (Fig. 6 D) of its diagnostic strength.
Embodiment 7 is for the preparation of serum/plasma miRNA serum internal reference detection kit
The preparation of miRNA test kit and operating process are based on RT-PCR and real-timePCR technology.This test kit comprises serum/plasma miRNA serum primer, and (comprise the primer of miR-30e or the primer of miR-30a, wherein, the primer pair sequence of miR-30e is for shown in SEOIDNO:1 and SEQIDNO:2; The primer pair sequence of miR-30a is shown in SEOIDNO:3 and SEQIDNO:2), the conventional enzyme needed for corresponding PCR reaction and/or reagent can also be had, as: reversed transcriptive enzyme, damping fluid, dNTPs, MgCl
2, remove nuclease water, fluorescence dye or probe, Taq enzyme etc., can select according to the concrete experimental technique adopted, these conventional enzymes and/or reagent are well known to those skilled in the art.This test kit can detect the internal reference of serum/plasma miRNA serum, apply this internal reference test kit and can promote the clinical conversion of serum/plasma miRNA serum correlative study achievement and the comparison of different experiments room result of study, for clinician grasps conditions of patients fast, for clinical therapeutic efficacy evaluation lays the foundation.
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skill in the art understand, and can carry out many changes in the spirit and scope that the claims in the present invention limit to it, amendment, and even equivalence is changed, but all will fall within the scope of protection of the present invention.
Claims (8)
1., for the internal reference that serum/plasma miRNA serum real-time quantitative PCR detects, it is characterized in that, described internal reference is miR-30e or miR-30a.
2. test right requires the primer of the internal reference for the detection of serum/plasma miRNA serum real-time quantitative PCR described in 1, it is characterized in that, the primer pair sequence of amplification miR-30e is for shown in SEOIDNO:1 and SEQIDNO:2; The primer pair sequence of amplification miR-30a is for shown in SEOIDNO:3 and SEQIDNO:2.
3. the application of internal reference in preparation serum/plasma miRNA serum internal reference detection kit detected for serum/plasma miRNA serum real-time quantitative PCR according to claim 1.
4. a serum/plasma miRNA serum internal reference detection kit, is characterized in that, this test kit contains the primer of miR-30e according to claim 2 or the primer containing miR-30a according to claim 2.
5. the application of internal reference in characterization prostate cancer prognosis miRNA tagged molecule test kit detected for serum/plasma miRNA serum real-time quantitative PCR according to claim 1.
6. application according to claim 5, is characterized in that, described prostate cancer prognosis miRNA tagged molecule is miR-375 or miR-1290.
7. the application of primer in characterization prostate cancer prognosis miRNA tagged molecule test kit of the internal reference for the detection of serum/plasma miRNA serum real-time quantitative PCR according to claim 2.
8. application according to claim 7, is characterized in that, described prostate cancer prognosis miRNA tagged molecule is miR-375 or miR-1290.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510437551.9A CN105039544A (en) | 2015-07-23 | 2015-07-23 | Internal reference for real-time quantitative PCR detection of serum and plasma miRNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510437551.9A CN105039544A (en) | 2015-07-23 | 2015-07-23 | Internal reference for real-time quantitative PCR detection of serum and plasma miRNA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105039544A true CN105039544A (en) | 2015-11-11 |
Family
ID=54446510
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510437551.9A Pending CN105039544A (en) | 2015-07-23 | 2015-07-23 | Internal reference for real-time quantitative PCR detection of serum and plasma miRNA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105039544A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536502A (en) * | 2018-12-19 | 2019-03-29 | 浙江大学医学院附属妇产科医院 | A kind of PCR internal reference suitable for Trophoblastic Tumor blood plasma excretion body miRNA |
| CN109913555A (en) * | 2019-04-23 | 2019-06-21 | 武汉科技大学 | A kind of prostate cancer diagnosis marker, measuring method and its application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876676A (en) * | 2012-09-24 | 2013-01-16 | 南京医科大学 | Blood serum/blood plasma micro ribonucleic acid (miRNA) marker relevant with pancreatic cancer and application thereof |
| CN104531703A (en) * | 2014-12-01 | 2015-04-22 | 山西医科大学第一医院 | Application of miR-30 family molecule to schizophrenia diagnosis and treatment |
-
2015
- 2015-07-23 CN CN201510437551.9A patent/CN105039544A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102876676A (en) * | 2012-09-24 | 2013-01-16 | 南京医科大学 | Blood serum/blood plasma micro ribonucleic acid (miRNA) marker relevant with pancreatic cancer and application thereof |
| CN104531703A (en) * | 2014-12-01 | 2015-04-22 | 山西医科大学第一医院 | Application of miR-30 family molecule to schizophrenia diagnosis and treatment |
Non-Patent Citations (2)
| Title |
|---|
| XIAOYI HUANG等: "exosomal miR-1290 and miR-375 as prognostic markers in castration-resistant prostate cancer", 《EUROPEAN ASSOCIATION OF UROLOGY》 * |
| 凌晓辉: "miR-30c在前列腺癌中的临床意义及功能研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109536502A (en) * | 2018-12-19 | 2019-03-29 | 浙江大学医学院附属妇产科医院 | A kind of PCR internal reference suitable for Trophoblastic Tumor blood plasma excretion body miRNA |
| CN109536502B (en) * | 2018-12-19 | 2020-02-18 | 浙江大学医学院附属妇产科医院 | PCR (polymerase chain reaction) internal reference applicable to plasma exosome miRNA of patient with gestational trophoblastic tumor |
| CN109913555A (en) * | 2019-04-23 | 2019-06-21 | 武汉科技大学 | A kind of prostate cancer diagnosis marker, measuring method and its application |
| CN109913555B (en) * | 2019-04-23 | 2022-09-06 | 湖北民族大学 | Prostate cancer diagnosis marker, determination method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20160312310A1 (en) | Serum or plasma microrna as biomarkers for non-small cell lung cancer | |
| CN103003442B (en) | A method of passing through microrna expression proficiency assessment people's allograft situation | |
| US20150011414A1 (en) | Microrna for diagnosis of pancreatic cancer and/or prognosis of patients with pancreatic cancer by blood samples | |
| CN101988060A (en) | Marker for detecting colon and rectum cancer as well as detection method, kit and biological chip thereof | |
| CN110724741B (en) | Primer, probe and kit for detecting minimal residual leukemia related fusion gene | |
| CN101988061A (en) | Breast cancer detecting marker as well as detecting method, kit and biological chip thereof | |
| US20220127684A1 (en) | Serum miRNA Markers for Diagnosing Liver Cancer and Predicting Liver Cancer Metastasis and Detection Kit Thereof | |
| CN103173448A (en) | Early-stage lung adenocarcinoma miRNA (micro ribonucleic acid) specific expression profile and reverse transcription primer and application thereof | |
| CN108342464A (en) | One-step method detects the kit and its detection method of HER2/neu gene expression amounts | |
| CN101871004A (en) | Quantitative detection method of mature miRNA (micro Ribonucleic Acid) and reagent kit | |
| CN105039544A (en) | Internal reference for real-time quantitative PCR detection of serum and plasma miRNA | |
| CN101418343A (en) | Application of miRNA in predicting postoperative recurrence for early primary hepatocarcinoma patient and kit thereof | |
| CN101260428B (en) | Device of judging lymph node metastasis of stomach cancer | |
| CN105154541A (en) | Application of miRNA to diagnosis and treatment of acute myelogenous leukemia | |
| CN101988062A (en) | cervical cancer detection markers and detection method, kit and biochip thereof | |
| CN107312851A (en) | Myocardial infarction biomarker miR 1283 | |
| CN110396542A (en) | A kind of LncRNA marker and its application in diabetes | |
| CN102443638A (en) | Internal reference for detecting miRNA (micro Ribonucleic Acid) in serum/blood plasma and application of internal reference | |
| Bavykin | Circulating microRNAs in the identification of biological fluids: A new approach to standardization of expression-based diagnostics | |
| CN105349666A (en) | Cerebral-arterial-thrombosis miRNA marker | |
| CN102899390A (en) | Small cell lung cancer markers and their detection | |
| CN109266750B (en) | Biomarker for nasopharyngeal carcinoma diagnosis and application | |
| CN116287282A (en) | Exosome miRNA marker for early pregnancy diagnosis of sow and application thereof | |
| CN106676186A (en) | Reagent and kit for rapidly detecting free nucleic acid integrity of peripheral blood and application of reagent and kit | |
| CN103740852B (en) | Based on hsa-miR-137 detection kit and the detection method thereof of AllGlo fluorescence probe quantitative PCR |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151111 |