CN102443638B - Internal reference for detecting miRNA (micro Ribonucleic Acid) in serum/blood plasma and application of internal reference - Google Patents
Internal reference for detecting miRNA (micro Ribonucleic Acid) in serum/blood plasma and application of internal reference Download PDFInfo
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Abstract
The invention belongs to the fields of genetic engineering and oncology and discloses an internal reference for detecting miRNA (micro Ribonucleic Acid) in serum/blood plasma and application of the internal reference. The internal reference is separate miR-484 or miR-191 or a combination of the miR-191 and the miR-484. The internal reference and a primer of the internal reference can be used for preparing an internal reference detection kit which is used for detecting the internal reference of the miRNA in serum/blood plasma.
Description
Invention field
The invention belongs to genetically engineered and oncology, relate to a kind of internal reference and application thereof detecting for serum/plasma miRNA serum.
Background technology
MicroRNAs (being miRNAs) is a focus of oncomolecularbiology research field in recent years.Ripe miRNA is the small molecules non-coding RNA that a class is about 21-24 Nucleotide, in karyon, first miRNA is expressed as long original (the primary miRNA of hundreds of Nucleotide, pri-miRNA), then by Drosha nuclease, processed into about long precursor (the precursor miRNA of 100 Nucleotide, pre-miRNA), and the mechanism relying on by Exportin-5 be transported to endochylema; In endochylema, further by Dicer nuclease, be processed into ripe miRNA.The expression that ripe miRNA mainly matches regulatory gene by the base complementrity with said target mrna 3 '-non-translational region (UTR), can cause the degraded of said target mrna or the inhibition of translation.Information biology research shows, single miRNA molecule can combine and bring into play regulating effect with the said target mrna of hundreds of Various Functions, participation comprises the most pathophysiological processeses such as growth, metabolism, cell enlargement, apoptosis, differentiation of stem cells, exists closely and contacts with generation, the development of numerous disease.Research also shows that the express spectra of miRNA can distinguish tumor tissue and non-tumor tissue, and has tumour-specific; And the limited vicious transformation that can cause cell of the maturation of miRNA integral body.The number of current found mankind miRNA is still increasing gradually, and its vital role aspect post-transcriptional control is also just revealed, and miRNA has become the important candidate markers of the various diseases morbidity, treatment and the prognosis that comprise tumour.
Previously the research of miRNAs is mainly concentrated on to them in intracellular activity.2008, Sino-U.S. Science man synchronously found stable existence miRNA in serum/plasma.These miRNA are all insensitive to RNase, acid or alkali environment, long-time (> 24 hours) room temperature placement and multigelation, no significant difference in blood plasma and serum.About the source of serum/plasma miRNA serum, also do not come to a conclusion at present, most investigators think that the miRNA in Serum of Cancer Patients/blood plasma derives from target tissue, but its expression changes and the consistence of target tissue need to inquire into; Also have investigator to think that miRNA can be enriched to by selectivity in " microparticles (MPs) " or " exosomes ", the mode being come off with " microvesicles microvesicle " by cell is secreting outside initiatively.In any case, serum/plasma miRNA serum possesses the potential quality that becomes high-quality biomarker, be rich content and stable in properties, have and take the not available speciality of traditional biological mark that protein is representative, without antibody preparation and be easy to accurate quantification, and there is disease specific, for biomarker has been opened up frontier.
Existing serum/plasma miRNA serum detection technique is comparatively ripe, as biochip technology, high throughput sequencing technologies and the most frequently used and effective real-time fluorescence quantitative PCR method, makes the acquisition of serum/plasma miRNA serum express spectra become possibility.Yet, still lack and can be used for the stable internal reference that serum/plasma miRNA serum detects at present.MiRNA is different from tissue/cell, and U6 expresses very low in serum/plasma, the internal reference that cannot express as serum/plasma miRNA serum.The conventional two kinds of methods of investigator are controlled experimental differences at present: the one, in each step of experiment, all process isopyknic " in right amount " serum/plasma, the 2nd, when miRNA extracts, after adding Trizol to make protein denaturation, at once add the non-human miRNA of synthetic (as nematode-39, cel-miR-39).
Although by above-mentioned two kinds of methods, can effectively control experimental differences, but the biology difference of uncontrollable individuality itself, the shortage of special and stable serum/plasma miRNA serum internal reference is still restricting the comparison of achievement in research between the clinical conversion of serum/plasma miRNA serum achievement in research and different experiments chamber.Therefore, if can filter out, can be used for the internal reference that serum/plasma miRNA serum s detects, and develop corresponding detection kit, to the clinic diagnosis level of the diseases such as the clinical conversion of serum/plasma miRNA serum correlative study achievement and raising tumour, will be once strong promotion.
Summary of the invention
Primary and foremost purpose of the present invention is for above-mentioned technical problem, proposes a kind of internal reference detecting for serum/plasma miRNA serum.
Second object of the present invention is to provide the primer that above-mentioned serum/plasma miRNA serum detects internal reference.
The 3rd object of the present invention is to provide above-mentioned serum/plasma miRNA serum and detects internal reference and the application of primer in preparation serum/plasma miRNA serum internal reference detection kit thereof.
The 4th object of the present invention is to provide the test kit that serum/plasma miRNA serum internal reference detects.
Contriver is by miRNAs separated and in the different tumour patients of research and normal healthy controls serum/plasma, find the internal reference of one group of stably express between various disease proterties and healthy person, and develop the internal reference detection kit that can be convenient to clinical application, for achievement in research between the clinical conversion of serum/plasma miRNA serum achievement in research and different experiments chamber, relatively provide Data support.
The object of the invention is to realize by following technical proposal:
The internal reference detecting for serum/plasma miRNA serum, this internal reference is independent miR-484 or the combination of miR-191 and miR-484 formation.
The primer of described serum/plasma miRNA serum internal reference, these primers are:
The primer of miR-191 is SEQ ID No.1 and SEQ ID No.2; The primer of miR-484 is SEQ ID No.5 and SEQ ID No.6.
The application of described serum/plasma miRNA serum internal reference in preparation serum/plasma miRNA serum internal reference detection kit.
The application of the primer of described serum/plasma miRNA serum internal reference in preparation serum/plasma miRNA serum internal reference detection kit.
A serum/plasma miRNA serum internal reference detection kit, this test kit is for detection of the combination of miR-484 in serum/plasma or miR-191 and miR-484 formation.
Described detection kit, the primer that this test kit contains miR-191 and miR-484 in serum/plasma miRNA serum or contain separately the primer of miR-484;
Wherein:
The primer of miR-191 is SEQ ID No.1 and SEQ ID No.2; The primer of miR-484 is SEQ ID No.5 and SEQ ID No.6.
Described detection kit can also comprise that PCR reacts conventional enzyme and reagent, as reversed transcriptive enzyme, and damping fluid, dNTPs, MgCl2, DEPC water and Taq enzyme etc.; Can also contain standard substance and/or reference substance.
Specifically, the technical scheme that the present invention deals with problems comprises: (1) gathers standard compliant blood sample with Standard operation procedure SOP (SOP).(2) serum/plasma miRNA serum expression pattern analysis: select lung cancer, mammary cancer, cancer of the stomach, liver cancer, Patients with Cervical Cancer, men's health contrast and women's health contrast, detect its serum/plasma miRNA serum express spectra and content, analyze the stability that between different tumor cases and contrast, serum/plasma miRNA serum is expressed, screening stably express miRNAs, be candidate's internal reference miRNA, further verify.(3) serum/plasma candidate's internal reference miRNAs of the stably express filtering out is carried out to quantitative analysis in the esophageal carcinoma, colon and rectum carcinoma, carcinoma of the pancreas, oral carcinoma, cancer of the stomach, lung cancer, mammary cancer, liver cancer and normal healthy controls, determine and can be used for the internal reference that serum/plasma miRNA serum s detects.(4) development of serum/plasma miRNA serum internal reference detection kit: according to the serum/plasma miRNA serum of the stably express of different tumor cases and normal healthy controls, exploitation serum/plasma miRNA serum s internal reference detection kit, for comparing the correlative study result between different experiments chamber and realizing its clinical conversion.
The inventor gathers standard compliant blood sample with Standard operation procedure SOP (SOP), and has adopted RT-PCR, Real-timePCR method, Solexa sequencing technologies, TaqMan Low Density Array (TLDA) chip detection etc.
The experimental technique of research mainly comprises following components specifically:
1. the selection of research sample
(1) tumour patient of clarifying a diagnosis through pathology, the lung cancer, mammary cancer, cancer of the stomach, liver cancer and the cases of cervical cancer that comprise internal reference screening stage, and the esophageal carcinoma of internal reference Qualify Phase, colon and rectum carcinoma, carcinoma of the pancreas, oral carcinoma, cancer of the stomach, lung cancer, mammary cancer and liver cancer case
(2) before blood sampling, without crossing, perform the operation and chemicotherapy, without chemicotherapy before operation
(3) healthy male, women's contrast
This research adopts 354 routine standard compliant samples to study altogether.
2.Trizol reagent (Invitrogen, Carlsbad, CA) and miRNeasy Mini Kit (QIAGEN company) extract the total RNA of serum/plasma, according to a conventional method operation.Conventionally can obtain～5 μ g RNA/50ml serum or blood plasma.
3.Solexa order-checking
(1) total RNA carries out PAGE electrophoresis recovery 17-27nt RNA molecule
(2) will link primer (adaptor prime, Solexa check order universal primer) enzyme be associated in 3 of small RNA molecular ' with 5 ' end
(3) carry out checking order after RT-PCR reaction
(4) data analysis and processing
4.TLDA (Applied Biosystems company) chip detection
(1) total RNA obtains cDNA sample by reverse transcription reaction
(2) cDNA sample carries out pre-amplified reaction
(3) pre-amplified production carries out TLDA chip detection, obtains the express spectra of miRNA
(4) data analysis and processing
5.Real-time RT-PCR (qRT-PCR) method
(1) get experimenter's the total RNA of serum/plasma, by RNA reverse transcription reaction, obtain cDNA sample;
(2) design primer;
(3) add fluorescent probe or dyestuff to carry out PCR reaction;
(4) stability of the amount of miRNA in detection more different tumour patient and normal healthy controls serum/plasma sample.
6. internal reference detection reagent box preparation method
In different tumour patients and normal healthy controls, the equal stable miRNA of copy number and expression level expresses one group of stable serum/plasma miRNA serum, the internal reference detecting as serum/plasma miRNA serum in different tumour patients and normal healthy controls by qRT-PCR technology screening.The serum/plasma miRNA serum internal reference finally filtering out forms detection kit (combination that independent miR-484 or miR-191 and miR-484 form).Detection kit can comprise the primer of these serum/plasma micro RNA combinations, probe, and the reagent such as Taq enzyme, dNTP.
7. statistical analysis technique
Use variation coefficient CV to analyze the degree of variation of each miRNA between different samples.Use Normfinder and quantitative expression data reference gene on-line analysis instrument (http://www.1eonxie.com/referencegene.php) to analyze the stability of each miRNA in different samples.MiRNA stationary value is lower, and its stability is higher.
We are in screening stage sample population, comprise 60 routine lung cancer (30 long lifetime of example and 30 routine short lifetimes), 48 routine mammary cancer (24 one group of example), 40 routine cancer of the stomach (20 one group of example), 30 routine liver cancer, 30 routine cases of cervical cancer and 48 routine healthy male contrasts, 48 routine healthy women contrasts, the result of study of integrated use Solexa order-checking and TLDA chip finds that there is 3 kinds of miRNAs can stably express between each sample.The miRNAs of stably express is further verified in the other 5 routine esophageal carcinoma, 5 routine colorectal carcinomas, the 5 routine rectum cancer, 5 routine carcinoma of the pancreas, 5 routine oral carcinoma, 5 routine cancer of the stomach, 5 routine lung cancer, 5 routine mammary cancer, 5 routine liver cancer cases and 5 routine normal healthy controls, and then observe the degree of stability of this result of study.
Below further instruction of the present invention:
In above-mentioned screening stage sample, we obtain correlated results by this 10 combination sample through Solexa order-checking test and TLDA chip detection.
According to Solexa sequence measurement, the inventor detects (the average copy number > 50 of stably express in the serum/plasma in different tumours and normal healthy controls group, variation coefficient < 100%) miRNA comprise hsa-miR-191, hsa-miR-320, hsa-miR-484, hsa-miR-222, hsa-let-7a, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f, hsa-miR-103, hsa-miR-107, hsa-miR-122, hsa-miR-125a-5p, hsa-miR-139-3p, hsa-miR-140-3p, hsa-miR-192, hsa-miR-197, hsa-miR-221, hsa-miR-23a, hsa-miR-25, hsa-miR-30d, hsa-miR-339-5p, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-451, hsa-miR-486-3p, hsa-miR-486-5p, hsa-miR-532-3p, hsa-miR-584, hsa-miR-744, hsa-miR-99a.
According to TLDA chip detection, the miRNA that the inventor detects stably express in the serum/plasma in different tumours and normal healthy controls group (average Ct value < 25, variation coefficient < 5%) comprises: hsa-miR-191, hsa-miR-320, hsa-miR-484, hsa-miR-126, hsa-miR-150, hsa-miR-24, hsa-miR-20a, hsa-miR-17, hsa-miR-106a, hsa-miR-92a, hsa-miR-19b, hsa-miR-146a.
The result detecting according to above-mentioned two kinds of methods, the miRNAs that is chosen in equal stably express in Solexa order-checking and TLDA chip further verifies by qRT-PCR method:
The miRNAs that meets above-mentioned condition comprises: miR-191, miR-320 and miR-484.
According to the above results, these 3 candidate's internal reference miRNAs are further verified with probe method and dye method qRT-PCR respectively in the other 5 routine esophageal carcinoma, 5 routine colorectal carcinomas, the 5 routine rectum cancer, 5 routine carcinoma of the pancreas, 5 routine oral carcinoma, 5 routine cancer of the stomach, 5 routine lung cancer, 5 routine mammary cancer, 5 routine liver cancer cases and 5 routine normal healthy controls.Normfinder and internal reference on-line analysis software all shows, with regard to single miRNA in serum/plasma, the expression of miR-484 is the most stable, and its stability of the combination of miR-191 and miR-484 has surpassed single miR-484.Probe method is consistent with dye method result.
According to above-mentioned experimental result, the inventor has prepared a kind of test kit that can detect for serum/plasma miRNA serum internal reference, comprises primer and other detection reagent of measuring stable existence in experimenter's serum/plasma and detectable ripe miR-191 and miR-484.
Particularly, miR-484 separately and the combination that forms of miR-191 and miR-484, or the internal reference detection kit that the primer of the primer that miR-484 is independent and miR-191 and miR-484 combination forms, the clinical conversion of serum/plasma miRNA serum correlative study result and the comparison of different experiments chamber result of study have been promoted, for clinician quick and precisely grasps patient's morbid state and coincident with severity degree of condition, take in time the scheme of preventing and treating of more personalized to provide support.
"/" in the application in " serum/plasma " represent " with " and the relation of "or".
Beneficial effect of the present invention:
The advantage that serum/plasma miRNA provided by the invention (microRNAs/miRNAs) detects internal reference is:
(1) serum/plasma miRNA serum s is a kind of new bio mark, be different from traditional biological mark, not only stable, Wicresoft, be easy to detect, and quantitatively accurate, susceptibility and the specificity of medical diagnosis on disease will be improved greatly, the clinical conversion that the successful research of such microRNA internal reference contributes to serum/plasma miRNA serum s to be worth at the aspect such as disease early diagnosis and prognosis judgement, and the comparison of result of study between different experiments chamber.
(2) serum/plasma miRNA serum s internal reference detection kit can be used for detecting the internal reference miRNAs in different experimenter's serum/plasma, contribute to reflect the baseline expression level of different experimenter's internal reference miRNA, for controlling the miRNA expression level difference that idiobiology difference causes, to highlight the differential expression miRNA of real and disease-related.
(3) adopt tight design and appraisement system, inventor's initial stage adopts Solexa genome sequencing technology to carry out direct Sequencing to serum/plasma miRNA serum s, adopt TLDA chip detection to obtain the serum/plasma miRNA serum s of stably express, and the method for applying qRT-PCR has been carried out single sample checking simultaneously; Above method and tactful application acceleration and the application that has guaranteed serum/plasma miRNA serum s internal reference detection kit.
The present invention, by the miRNA of stably express in research serum/plasma, seeks can be used for the internal reference that serum/plasma miRNA serum detects.Therefore, the present invention has obtained the internal reference miRNA of serum/plasma miRNA serum s expression database and stably express; By the development and application of serum/plasma miRNA serum s internal reference test kit, the comparison of result of study between the clinical conversion of promotion serum/plasma miRNA serum s correlative study result and different experiments chamber, for clinician quick and precisely grasps conditions of patients, for clinical therapeutic efficacy evaluation lays the foundation, and for finding that the new small molecule drug targets with potential therapeutic value offers help.
Accompanying drawing explanation
Fig. 1. show 3 kinds of candidate's internal reference miRNA of different tumour patients and contrast and cel-miR-39 expression broken line graph.
Embodiment
The invention will be further elaborated by the following examples.
The collection of embodiment 1 sample and the arrangement of sample data
Contriver from July, 2003 from Jiangsu inside the province a plurality of Grade III Class A hospitals collected each a large amount of tumour patients and normal healthy controls serum/plasma sample, for the case studied and check sample, be and collect the same period, sampling, packing, preservation condition homogeneous, by the arrangement to sample data, the laboratory sample that the sample that contriver has therefrom selected 354 examples to meet following standard is verified as Solexa order-checking, TLDA chip detection and follow-up a series of qRT-PCR:
The tumour patient of 1, clarifying a diagnosis through pathology, the lung cancer, mammary cancer, cancer of the stomach, liver cancer and the cases of cervical cancer that comprise internal reference screening stage, and the esophageal carcinoma of internal reference Qualify Phase, colon and rectum carcinoma, carcinoma of the pancreas, oral carcinoma, cancer of the stomach, lung cancer, mammary cancer and liver cancer case.
2, before blood sampling, without crossing, perform the operation and chemicotherapy, without chemicotherapy before operation.
3, healthy male, women's contrast.
And system acquisition the situations such as the demography data of these samples, clinical data.
The Solexa of miRNA order-checking experiment in embodiment 2 serum/plasma
Above-mentioned qualified sample comprises 60 routine lung cancer (30 example long lifetimes of groups and 30 routine short lifetime group), 48 routine mammary cancer (24 examples/group), 40 routine cancer of the stomach (20 examples/group), 30 routine liver cancer, 30 routine cases of cervical cancer and 48 routine healthy males contrasts, and 48 routine healthy womens contrast.These 10 groups of crowds are obtained to correlated results through Solexa order-checking test (test kit is purchased from ABI company).Concrete steps are:
1, every group of sample got serum/plasma 50ml, adds isopyknic Trizol reagent;
2, be separated: room temperature is placed 15min, then by the volume ratio of 0.2ml chloroform/1ml Trizol reagent, adds chloroform, concussion 15s, room temperature 15min, 12,000g, 4 ℃, centrifugal 15min;
3, water is transferred to the centrifuge tube of new 50ml, 3 step phenol/chloroform are except Deproteinization phase;
4, RNA precipitation: water is transferred in new centrifuge tube, added Virahol by 0.5ml Virahol/1ml Trizol reagent volume, preserve 60min, 12,000g, 4 ℃, centrifugal 60min for-20 ℃;
5, by the resuspended precipitation of 1ml Trizol reagent, suspension is transferred in the centrifuge tube of new 1.5ml;
6, repeat 2,4 steps (the centrifugal 15min that changes into of the 4th step);
7, RNA washing: remove supernatant, add 75% ethanol, 12,000g, 4 ℃ of centrifugal 5min;
8, measure concentration: conventionally can obtain～5 μ g RNA/50ml serum/plasma;
9, total RNA carries out PAGE electrophoresis recovery 17-27nt RNA molecule;
10, will link primer (adaptor prime, Solexa check order universal primer) enzyme be associated in 3 of small RNA molecular ' with 5 ' end;
11, carrying out RT-PCR reacts rear and checks order;
12, data analysis and processing: in every group, use the miRNA of the serum/plasma stably express of Solexa sequence measurement discovery to enumerate out hereinbefore.
The TLDA chip detection of miRNA in embodiment 3 serum/plasma
Above-mentioned 10 groups of samples that carry out Solexa order-checking are obtained to correlated results through TLDA chip detection (test kit is purchased from ABI company).Concrete steps are:
1, get serum/plasma 600 μ l for every group, add the Trizol reagent of 3 times of volumes;
2, be separated: room temperature is placed 15min, add final concentration be the cel-miR-39 (TAKARA) of 10-4pmol/ μ l as internal reference, then add and the isopyknic chloroform of serum/plasma concussion 50s, room temperature 15min, 14,000rpm, 4 ℃, centrifugal 15min;
3, RNA precipitation: water is transferred to the centrifuge tube of new 15ml, added the dehydrated alcohol of 1.5 times of water volumes, fully mix;
4, with QIAGEN miRNeasy kit enrichment RNA: draw 700 μ l samples to centrifugal column, the centrifugal 15s of 14,000rpm, discards filtrate in collection tube, is repeated to sample standard deviation and crosses post at every turn; Add 700 μ l washing lotions 1,14, the centrifugal 15s of 000rpm, abandons filtrate in collection tube; Add 500 μ l washing lotions 2,14, the centrifugal 15s of 000rpm, abandons filtrate in collection tube; Add 500 μ l washing lotions 2,14, the centrifugal 2min of 000rpm, abandons filtrate in collection tube again; Centrifugal column is put back in empty collection tube, and the centrifugal 2min of 14,000rpm is to be dried centrifuge tube; Centrifuge tube is put into new 1.5ml pipe, add 50 μ l nuclease free water, the centrifugal 1min of 10,000rpm; Liquid in pipe is refunded in centrifugal column, and the centrifugal 1min of 14,000rpm, abandons centrifugal column;
5, measure concentration: conventionally can obtain～1250ng RNA/600 μ l serum/plasma;
6, with the supporting reverse transcription test kit of TLDA chip, by RNA reverse transcription reaction, obtain cDNA.The reaction system of reverse transcription comprises 0.8 μ l reverse transcriptase primer (10 *), 0.2 μ l 100mM dNTPs mixture, 1.5 μ l reversed transcriptive enzymes (50U/ μ L), 0.8 μ l 10 * reverse transcription damping fluid, 0.9 μ l 25mM magnesium chloride, 0.1 μ l RNA inhibitor and 0.2 μ l nuclease free water.The total RNA that adds 3 μ l (1-350ng).Reactions steps is 16 ℃ hatches 2 minutes, 42 ℃ of reactions 1 minute, and 50 ℃ of reactions 1 second, above-mentioned 3 steps are through 40 circulating reactions, then 85 ℃ hatch 5 minutes;
10, the specific miRNA of chip is carried out to pre-amplification to increase the amount of expressing required CNDA.The reaction system of pre-amplification comprises: 12.5 μ l increase in advance Master Mix (2 *), the pre-amplimer of 2.5 μ l (10 *), 7.5 μ l nuclease free water, 2.5 μ l CDNA.Reactions steps is: 95 ℃ 10 minutes → 55 ℃ 2 minutes → 72 ℃ 2 minutes → 95 ℃ 15 seconds, 60 ℃ 4 minutes, 12 circulation → 99.9 ℃ 10 minutes; After finishing, reaction adds 75 μ l 0.1X TE dilutions;
11, get the pre-amplified production after 9 μ l dilutions, add 450 μ l genetic expression Master Mix, 441 μ l nuclease free water after fully mixing, add 100 μ l/ holes on TLDA chip; The centrifugal 1min of 1000rpm, centrifugal 2 times.What the experiment of TLDA chip was used is ABI Prism 7900 quantitative real time PCR Instruments.Select the specific program of 384-well TaqMan Low DensityArray to react;
12, data analysis and processing: with RQ-Manger software, carry out data processing, C
tthreshold value is made as 0.2.In each group that utilization TLDA chip detection is found, the miRNA of serum/plasma stably express is enumerated out hereinbefore.
The qRT-PCR of miRNA checking in embodiment 4 serum/plasma
According to above-mentioned Solexa and TLDA result, the miRNAs that selection meets following condition further verifies by qRT-PCR method: 1) these miRNAs average copy number > 50 in different tumours and normal healthy controls group in Solexa order-checking, and variation coefficient < 100%; 2) these miRNA average Ct value < 25 in different tumours and normal healthy controls group in TLDA chip detection, variation coefficient < 5%; 3) in Solexa order-checking and TLDA chip detection, all demonstrate stably express.Primer (in Table 2) to 3 miRNAs design reverse transcriptions such as selected miR-191, miR-320 and miR-484 and qRT-PCR.The stability of candidate's internal reference miRNA in Table 1, Fig. 1.In whole research process, all implement strict Quality Control.Each sample continuous detecting three times.All detections all adopt blind method, in the situation that not knowing sample background, complete to avoid bias.
(1) prepare RNA sample: a) get 100 μ l serum/plasma; B) the Trizol room temperature that adds 3 times of volumes is placed 15min, and adding final concentration is 10
-4the cel-miR-39 (TAKARA) of pmol/ μ l, as internal reference, then adds and the isopyknic chloroform of serum/plasma, concussion 50s, room temperature 15min, 14,000rpm, 4 ℃, centrifugal 15min; C) water is transferred to the centrifuge tube of new 15ml, added the dehydrated alcohol of 1.5 times of water volumes, fully mix; D) with the miRNeasy kit enrichment RNA of QIAGEN company: draw 700 μ l samples to centrifugal column, the centrifugal 15s of 14,000rpm, discards filtrate in collection tube, is repeated to sample standard deviation and crosses post at every turn; Add 700 μ l washing lotions 1,14, the centrifugal 15s of 000rpm, abandons filtrate in collection tube; Add 500 μ l washing lotions 2,14, the centrifugal 15s of 000rpm, abandons filtrate in collection tube; Add 500 μ l washing lotions 2,14, the centrifugal 2min of 000rpm, abandons filtrate in collection tube again; Centrifugal column is put back in empty collection tube, and the centrifugal 2min of 14,000rpm is to be dried centrifuge tube; Centrifuge tube is put into new 1.5ml pipe, add 50 μ l nuclease free water, the centrifugal 1min of 10,000rpm; Liquid in pipe is refunded in centrifugal column, and the centrifugal 1min of 14,000rpm, abandons centrifugal column, using the liquid in pipe as RNA sample;
(2) probe method: use ABI test kit.
A) by RNA reverse transcription reaction, obtain cDNA.The reverse transcription reaction system of probe method comprises 0.15 μ l 100mMdNTPs mixture, 1 μ l reversed transcriptive enzyme (50U/ μ L), 1.5 μ l 10X reverse transcription damping fluids, 0.19 μ l RNA inhibitor and 3 μ l 5 * reverse transcriptase primers (the reverse transcriptase primer mixture of miR-191, miR-320, miR-484 and cel-miR-39, every kind of miRNA adds 3/4 μ l reverse transcriptase primer).The total RNA that adds 9.16 μ l.Reactions steps is 16 ℃ hatches 30 minutes, and 42 ℃ are reacted 30 minutes, hatch 5 minutes for 85 ℃;
B) q-PCR: cDNA is added to 5 μ l water dilutions, get the cDNA after 1 μ l dilution, add respectively 0.25 μ l20 * MicroRNA detection probes (probe of single miRNA in miR-191, miR-320, miR-484 and cel-miR-39), 2.5 μ l 2 * genetic expression Master Mix, 1.25 μ l distilled waters, 5 μ l systems are carried out q-PCR.What instrument used is ABI Prism 7900 quantitative real time PCR Instruments, and the reaction conditions of PCR is: within 95 ℃, 5 minutes, carry out 1 circulation → 95 ℃, 15 seconds, within 60 ℃, 1 minute, carry out 45 circulations.
(3) dye method:
A) by RNA reverse transcription reaction, obtain cDNA.The reaction system of the reverse transcription of dye method comprises the mixture (the specific reverse primers mixture (in Table 2) of miR-191, miR-320, miR-484 and cel-miR-39, every kind adds 1.5/4 μ l) of 4 μ l 5 * AMV damping fluids, 2 μ l 10mM dNTP mixtures (Takara company), 0.5 μ l RNase inhibitor (Takara company), 2 μ lAMV (Takara company) and 1.5 μ l miRNA specific reverse primers.Reactions steps is 16 ℃ hatches 15 minutes, and 42 ℃ are reacted 1 hour, hatch 5 minutes for 85 ℃;
B) q-PCR: cDNA is pressed to 1/5 volume dilution, get the cDNA after 0.5 μ l dilution, add 0.15 μ l Taq enzyme (Takara company), 0.5 μ l 20 * EVAGREEN, 0.1 μ l 10 μ M forward primers a kind of (adopting respectively forward primer corresponding to above-mentioned single miRNA), the 0.1 μ l general reverse primer of 10 μ M (URP:TGGTGTCGTGGAGTCG), 0.6 μ l 25mM MgCl
2, 0.8 μ l 2.5mM dNTP mixture (Takara company), 1 μ l 10 * PCR damping fluid, 6.75 μ lH
2o, 10 μ l systems are carried out q-PCR.What instrument used is ABI Prism 7900 quantitative real time PCR Instruments, and the reaction conditions of PCR is: within 95 ℃, 5 minutes, carry out 1 circulation → 95 ℃, 15 seconds, within 60 ℃, 1 minute, carry out 45 circulations.
(4) data processing and analysis
The expression amount ratio of each sample blood plasma miRNA s can be used equation 2
-Δ Ctrepresent, wherein Δ Ct=C
t sample-C
t cel-miR-39, difference when we add the RNA of cel-miR-39 to control RNA extraction when each sample extraction, calculates relative expression quantity (primer sequence of cel-miR-39: SEQ ID No.7 and SEQ ID No.8).
Probe method qRT-PCR found that the stability of miR-484 is best in 50 routine samples, and its stability of the combination of miR-191 and miR-484 has surpassed single miR-484.Dye method result and probe method are similar unlisted.
Therefore the combination that, the inventor has proved miR-484 separately and miR-191 and miR-484 form can stably be expressed in each tumour patient and normal healthy controls.
The stability scoring of table 1. candidate internal reference
The table 2 miRNA primer information of being correlated with
Embodiment 5 is for the making of serum/plasma miRNA serum internal reference detection kit
The making of miRNA test kit and operating process are based on solexa order-checking, TLDA chip detection, the technology such as RT-PCR and real-time PCR.Test kit comprise serum/plasma miRNA serum primer (combination of primers that comprises the primer of following independent miR-484 or the primer of miR-484 and miR-191, wherein: the primer of miR-191 is SEQ ID No.1 and SEQ ID No.2; The primer of miR-484 is SEQ ID No.5 and SEQ ID No.6), can also there is corresponding PCR to react required conventional enzyme and/or reagent, as: reversed transcriptive enzyme, damping fluid, dNTPs, MgCl2, remove nuclease water, fluorescence dye or probe, Taq enzyme etc., can select according to the experimental technique of concrete employing, these conventional enzymes and/or reagent are well known to those skilled in the art, can also have in addition standard substance and contrast (as nematode mir-39 sample of quantitative mark etc.).The value of this test kit is to detect the internal reference of serum/plasma miRNA serum, for result of study between different experiments chamber, relatively provide foundation, to promote the clinical conversion of correlative study result, therefore this test kit is dropped into practice, can help to instruct clinical diagnosis and the more effective individualized treatment accurately made.
Claims (5)
1. independent miR-484 or miR-191 and miR-484 form be combined in serum/plasma miRNA serum detect in as the application of internal reference.
2. the application of the primer of miRNA in preparation serum/plasma miRNA serum internal reference detection kit described in claim 1;
Wherein, the primer of described miRNA is:
The primer of miR-191 is SEQ ID No.1 and SEQ ID No.2; The primer of miR-484 is SEQ ID No.5 and SEQ ID No.6.
3. a serum/plasma miRNA serum internal reference detection kit, is characterized in that the primer that this test kit contains miR-191 and miR-484 in serum/plasma miRNA serum or contains separately the primer of miR-484.
4. internal reference detection kit according to claim 3, is characterized in that described primer is:
The primer of miR-191 is SEQ ID No.1 and SEQ ID No.2; The primer of miR-484 is SEQ ID No.5 and SEQ ID No.6.
5. according to internal reference detection kit described in claim 3 or 4, it is characterized in that this test kit can also comprise the reagent that round pcr is conventional.
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