CN101538615B - Detecting method of nucleotide sequence for detecting mircoRNA content in samples - Google Patents
Detecting method of nucleotide sequence for detecting mircoRNA content in samples Download PDFInfo
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- CN101538615B CN101538615B CN2009101357566A CN200910135756A CN101538615B CN 101538615 B CN101538615 B CN 101538615B CN 2009101357566 A CN2009101357566 A CN 2009101357566A CN 200910135756 A CN200910135756 A CN 200910135756A CN 101538615 B CN101538615 B CN 101538615B
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Abstract
The invention provides a nucleotide sequence and a kit for detecting mircoRNA content in samples as well as a mircoRNA detecting method which is based on the complementary combination of nucleic acid base pairing identification and utilizes silver-staining enhanced nano gold to mark a probe, the method is simple and practicable and can obtain detection signals with high sensitivity and high specificity without expensive equipment of radioactive labels, fluorescence and the like.
Description
Technical field
The present invention relates to be used for detecting the nucleotide sequence and the detection method of sample microRNAs content, relate in particular to a kind of method that adopts microRNAs in the gold and silver Nano microsphere technology for detection sample.
Background technology
Cardiovascular disorder has become the No.1 killer of human health and life.Acute myocardial infarction is the common anxious critical illness of emergency department; But it is not ideal enough for this sick result of treatment; One of them major reason is in time to make right judgement to the prognosis of myocardial infarction and the generation of complication, to such an extent as to can not take suitably regimen and measure timely.Malignant arrhythmia promptly is one of acute myocardial infarction severe complication, is the difficult point of clinical treatment, also is the focus of this research field; Consequence is serious, and case fatality rate is high, if can early discovery; Early stage treatment can be saved patient's life effectively, and is significant to reducing the myocardial infarction case fatality rate.The diagnosing acute myocardial infarction is mainly leaned on the detection of Electrocardioscopy and myocardial enzymes, but the accuracy rate of Electrocardioscopy has only 50%, and there have 30% cardiac to show as electrocardiogram(ECG to be normal, diagnoses thereby can not make accurately; And the detection of myocardial enzymes is also not ideal enough; Creatine kinase MB (CK-MB) and troponin lack susceptibility; The 6-8 hour concentration just rises gradually after the pectoralgia, and the rising lack of specific of myohaemoglobin, so present detection means still can not be made early diagnosis to acute myocardial infarction; Be difficult for to find the progress of the state of an illness, delay treatment opportunity.Therefore the biochemical indicator that presses for a kind of new high specific and susceptibility comes the generation of auxiliary diagnosis myocardial infarction; The more important thing is the severity of judging acute myocardial infarction as a kind of warning index, the generation of its prognosis and complication is in time made effectively judged and prevent and treat.
MicroRNA promptly came to light in 1993, was Nucleotide (RNA) molecule that is about 19-25, was the little RNA of non-coding.It can be through combining with specific mRNA, and the translation process that suppresses the mRNA coded protein comes regulate gene expression.Confirmed microRNA has nearly 800 in the human genome at present, possibly participate in 30% human protein's expression regulation.Function assessment research shows that microRNA participates in regulating many pathophysiological processes such as the increment that comprises cell, differentiation, apoptosis, tumour generation.In recent years, the effect of microRNA in irregular pulse obtained the achievement in research that attracts people's attention, and a series of research confirms that microRNA participates in the expression of the multiple cardiac electrical activity GAP-associated protein GAP of regulation and control, is potential irregular pulse action target spot.Wherein studying more is miR-1 and the miR-133 that muscle specific is expressed.The contriver finds that miR-1 is the key factor that causes ischemic arrhythmia to take place, and is in close relations with various irregular pulse.The expression level of experiment proof miR-1 in patients with coronary heart disease cardiac muscular tissue is 2.8 times of normal people cardiac muscular tissue; In rat experiment property myocardial infarction and ischemia model, observe same result: ischemic region cardiac muscle miR-1 expression amount is higher 2.6 times than normal control group behind the ischemic 12h.When being applied in the body rotaring dyeing technology with miR-1 specific antisense oligomerization (deoxidation) Nucleotide (AMO-1) importing ischemic myocardium, incidence of arrhythmia obviously reduces.In addition, we find that microRNA328 can cause the generation of atrial fibrillation, and miR-124-1 and heart failure and myocardial fibrosis are relevant, and microRNA21 can cause myocardial hypertrophy.Therefore, we imagine the change that myocardial ischemia causes that some miRNAs expresses, like microRNA1; MicroRNA133, microRNA328, miR-124-1; Whether microRNA21 can also be detected in blood, and has dependency with the progress of some blood circulation diseases.If this supposes to set up; These detections to these miRNA promptly can be used as early warning signal, and through the detection to the circulation peripheral blood, just can judge the prognosis of disease provides foundation; For the early discovery of complication such as myocardial infarction patient generation malignant arrhythmia provides foundation; So that irregular pulse is made early diagnosis and processing, save patient's life, have clinical meaning and the good prospects for application and the economical, societal benefits of reality.
Measuring Northern blot at present is " gold standard " method that generally acknowledged in the world miRNAs detects; A lot of deriving methods have also been developed; But detect a large amount of total RNA sample of preparation in advance with these class methods, and process is tediously long, technological relative complex; Higher relatively to the requirement of testing staff's specialty degree, so possibly limit its application.People such as He Wenlei (biological chemistry and biophysics progress; 2008; 35 (11): 1332～1338) utilize the nanometer gold silver dying enhancement techniques to set up a kind of quantivative approach of microRNA simply fast;, as capture probe, target sequence is captured on the solid phase carrier enzyme mark hole as signal probe with the oligonucleotide molecules of nano gold mark with the biotin labeling oligonucleotide molecules through Streptavidin-vitamin H effect; Then the silver through nano gold catalysis dyes enhancing scale effect generation sensitive recognition signal, realizes the quantitative of microRNA molecule thereby write down its absorbance.It is used for detecting mouse liver, cerebral tissue miR-122a and miR-128 content separately and synthetic miR-122a, and (lowest detection is limited to 10fmol/L in the 10pmol/L～10fmol/L) in good linear range.This method is simple, need not expensive device such as radio-labeling, fluorescence.Yet its sensitivity and specific degree still remain to be improved.
Summary of the invention
In order to address the above problem, the object of the present invention is to provide the method for microRNA in the nucleotide sequence that is used for measuring sample microRNA and a kind of novel easy, quick, the quantitative measurment sample.
In order to achieve the above object, the present invention provides three kinds of nucleotide sequences, can check the content of microRNA in the sample (comprising serum, whole blood, lysis, tissue etc.) quickly and accurately through these three kinds of nucleotide sequence combining nano technology for gold, and it specifically comprises:
The present invention provide a kind of be used for detecting sample mircoRNA content by biotin labeled nucleotide sequence I, it is characterized in that said sequence does
5’-(y)
n-(CH
2)
a-3’，
Wherein a is 3-20; (y) n representes a kind of and holds 6-16 base length complementary nucleotide sequence from miRNAs5 ', and said biotin labeling is at 3 ' end; The sequence of said miRNAs is selected from the arbitrary sequence among the SEQ ID NO:1-3.
Preferably, said is 5 '-tttacattcca-(CH by biotin labeled nucleotide sequence I
2)
6-3 ' or 5 '-gagagggcctg-(CH
2)
6-3 ' or 5 '-gcgtgcctta-(CH
2)
8Arbitrary sequence in-3 '.
The present invention also provides a kind of nucleotide sequence II that is used for detecting sample mircoRNA content, it is characterized in that said sequence does
5’-(A)
b-(Y)
m-3’，
Wherein b is 8-50; (Y)
mRepresent that arbitrary site a kind of and from the 7th to 17 base of miRNAs5 ' end extends to the base length complementary nucleotide sequence of 3 ' end, wherein said nucleotide sequence can be combined with the full length sequence of said miRNAs is complementary by biotin labeled nucleotide sequence I with aforementioned simultaneously; A is a VITAMIN B4.
Preferably, said nucleotide sequence II is 5 '-(A)
30-ATACACACTTC-3 ' or 5 '-(A)
25-ACGGAAGGGCA-3 ' or 5 '-(A)
15Arbitrary sequence among the-GGCATTCACC-3 '.
The present invention further provides a kind of nucleotide sequence III that is used for detecting sample mircoRNA content, it is characterized in that said sequence does
[0016]?5’-(T)
d-(CH
2)
e-SH-3’，
[0017]Wherein e is 3-20; D equates with b among the said nucleotide sequence II, and said sequence can combine with said nucleotide sequence II is complementary; T is a thymus pyrimidine; SH is a sulfydryl.
Preferably, said nucleotide sequence III is 5 '-(T)
30-(CH
2)
6-SH-3 ' or 5 '-(T)
25-(CH
2)
6-SH-3 ' or 5 '-(T)
15-(CH
2)
6Arbitrary sequence among the-SH-3 '.
The present invention also provides a kind of test kit that detects microRNA content in the sample; It is said by biotin labeled nucleotide sequence I to it is characterized in that it comprises; Said nucleotide sequence II and said nucleotide sequence III, gene extracts material, and nano-Au solution and silver strengthen liquid.
The present invention also provides a kind of and is combined into the basis with nucleic acid base pairing identification complementation, and the microRNAs detection method through silver dyes enhanced nano gold mark probe is characterized in that its key step comprises:
(1) preparation of sample and the extraction of RNA always;
(2) hybridization: sample, capture probe, signal probe are mixed with the nano gold mark probe, this mixture system is transferred in the enzyme plate hole that each Streptavidin encapsulates hatched; Wherein, Said capture probe is described by biotin labeled nucleotide sequence I; Said signal probe is described nucleotide sequence II, and said nano gold mark probe is that general T probe mixes with nano-Au solution and obtains, and said general T probe is described nucleotide sequence III;
(3) the nano gold mark probe quantitative detects microRNA: the every hole of aforementioned enzyme plate adds silver and strengthens liquid; Reaction is to occurring washing termination reaction with ultrapure water behind the grey scale signal under the room temperature lucifuge condition; Enzyme plate is inserted in the ELIASA, and single length scanning detects the A value of solution in real time through ELIASA.
Preferably, said is 5 '-tttacattcca-(CH by biotin labeled nucleotide sequence I
2)
6-3 ' or 5 '-gagagggcctg-(CH
2)
6-3 ' or 5 '-gcgtgcctta-(CH
2)
8Arbitrary sequence in-3 '; Said nucleotide sequence II is 5 '-(A)
30-ATACACACTTC-3 ' or 5 '-(A)
25-ACGGAAGGGCA-3 or 5 '-(A)
15Arbitrary sequence among the-GGCATTCACC-3 ';
Said nucleotide sequence III is 5 '-(T)
30-(CH
2)
6-SH-3 ' or 5 '-(T)
25-(CH
2)
6-SH-3 ' or 5 '-(T)
15-(CH
2)
6Arbitrary sequence among the-SH-3 '.
Said nanometer gold can be bought from commercial sources and (for example available from Sino-American Biotechnology Co., SABC), also can adopt following method preparation: with the 1.0mM HAuCl of 20ml
4Place vessel in heating to boiling, add two water citric acid trisodium (Na of 2ml 1%
3C
6H
5O
72H
2O), under refluxad vigorous stirring 15min forms gold sol until the further change that does not observe color then, filters and promptly gets nano-Au solution of the present invention.Nanometer gold with this method preparation has maximum absorption band at the 518nm place, mean diameter 12-13nm.
The preparation method of said nano gold mark probe is following: with the centrifugal supernatant of abandoning of above-mentioned nano-Au solution, add general T probe (MST-probe) redispersion nanometer gold, it is centrifugal to get supernatant after the adding damping fluid is hatched; Abandon supernatant,, abandon supernatant once more with centrifugal behind the damping fluid washing precipitate; Twice of washing precipitate is to remove unnecessary strand oligonucleoside on unmarked; At last, adding the resuspended throw out of damping fluid promptly gets.
The preparation that said silver strengthens liquid (SES) is following: (0.085g is dissolved in 1.5ml H with quinhydrones
2Among the O), (0.255g Hydrocerol A and 0.235g trisodium citrate are dissolved in 1ml H to the Hydrocerol A saline solution
2Among the O, pH3.8), Silver Nitrate (AgNO
3, 0.25g is dissolved in 1ml H
2Among the O) transparent with 75: 25: 3 ratio mixing until solution becomes.
The principle of the inventive method is following: the dna probe of alkane sulfydryl modification can adsorb the surface that combines to be fixed on nm gold particles under certain conditions; Form Nano-Au probe; After hatching certain hour under certain salt ionic concentration and the temperature condition; Hybridization forms one one end marking nano gold grain between nano gold mark probe, signal probe, biotin labeled capture probe and the complementary target miRNAs, and an end is marked with the heteroduplex of vitamin H.Shift this hybridization product subsequently in the enzyme plate that Streptavidin encapsulates; 37 ℃ hatch 30min after; The hybridization product is fixed on the enzyme plate through Streptavidin-vitamin H specific recognition system; Through PBS, PBN solution washing, remove free Nano-Au probe in the enzyme plate, with strengthening coupling reaction amplification nano-gold signal after silver dyes.ELIASA dyes photoabsorption A value or the optical density(OD) A value that strengthens liquid at 518nm wavelength record silver, and through the analysis to target miRNAs concentration and corresponding A value, concentration and the silver of drawing out miRNAs dye the dose-effect relationship curve (Fig. 1) that strengthens between the liquid A value.
The inventive method is compared with existing document, has accuracy, highly sensitive characteristics.The nucleotide sequence that gained is satisfied in the design of n and m in the probe of the present invention can combine with the miRNA that is detected is specific, has improved sensitivity; One group of A and one group of T are not easy to form mispairing from the pair relationhip in addition, and nanometer gold directly is connected on the general T probe, and this has just increased the accuracy of inspection signal; And detection method of the present invention is simple, need not the detection signal that expensive device such as radio-labeling, fluorescence just can obtain highly sensitive, high specific degree, for new way has been opened up in the research of miRNAs, can play conveniently effect.
Description of drawings
Fig. 1 is the schematic flow sheet of preferred embodiment of the present invention;
The content graphic representation of miRNA-1 among total RNA that the peripheral blood that Fig. 2 records for employing the inventive method extracts;
Fig. 3 is concentration logarithmic value and the A value graph of a relation of the miRNA-1 that adopts the inventive method and record.
The invention provides three kinds of nucleotide sequences, classify the content that probe and combining nano technology for gold can be checked microRNA in the sample (comprising serum, whole blood, lysis, tissue etc.) quickly and accurately as with these three kinds of nucleotides sequences, it specifically comprises:
Capture probe: promptly be by biotin labeled nucleotide sequence I, its sequence is 5 '-(y)
n-(CH
2)
a-3 ', wherein a is 3-20; (y)
nRepresent a kind of with from 6-16 base length complementary nucleotide sequence of miRNAs5 ' end, said biotin labeling is held 3 '; The sequence of said miRNAs is selected from the arbitrary sequence among the SEQ ID NO:1-3.
Embodiment
Signal probe: be nucleotide sequence II, its sequence is 5 '-(A)
b-(Y)
m-3 ', wherein b is 8-50; (Y)
mRepresent that arbitrary site a kind of and from the 7th to 17 base of miRNAs5 ' end extends to the base length complementary nucleotide sequence of 3 ' end, wherein said nucleotide sequence can be combined with the full length sequence of said miRNAs is complementary by biotin labeled nucleotide sequence 1 with aforementioned simultaneously; A is a VITAMIN B4.
General T probe: be nucleotide sequence III, its sequence is 5 '-(T)
d-(CH
2)
e-SH-3 ', wherein e is 3-20; D equates with b among the said nucleotide sequence II, and said sequence can combine with said nucleotide sequence II is complementary; T is a thymus pyrimidine; SH is a sulfydryl.
For example, for miR-1 (SEQ ID NO:1): 5 '-uggaauguaaaGAAGUGUGUAU-3 ',
Capture probe is: 5 '-tttacattcca-(CH
2)
6-Bio-3 ',
Signal probe is: 5 '-(A)
30-ATACACACTTC-3 ',
General T probe is: 5 '-(T)
30-(CH
2)
6-SH-3 '.
Again for example, for miR-328 (SEQ ID NO:2): 5 '-cuggcccucucUGCCCUUCCGU-3 ',
Capture probe is: 5 '-gagagggcctg-(CH
2)
6-Bio-3 ',
Signal probe is: 5 '-(A)
25-ACGGAAGGGCA-3 ',
General T probe is: 5 '-(T)
25-(CH
2)
6-SH-3 '.
Again for example, for miR-124-1 (SEQ ID NO:3): 5 '-uaaggcacgcGGUGAAUGCC-3 ',
Capture probe is: 5 '-gcgtgcctta-(CH
2)
8-Bio-3 ',
Signal probe is: 5 '-(A)
15-GGCATTCACC-3 ',
General T probe is: 5 '-(T)
15-(CH
2)
6-SH-3 '.
Three kinds of nucleotide sequences of the present invention are the deoxy-oligonucleotide probes that adopt the synthetic HPLC level of ordinary method, and all oligonucleotide are dissolved in the TE damping fluid, packing, be stored in-80 ℃ subsequent use.
A kind of test kit of measuring microRNA content in the sample of the present invention, it comprises that said capture probe, said signal probe, nano gold mark probe and silver strengthen liquid, wherein the nano gold mark probe is to be mixed with nano-Au solution by said general T probe to obtain.
Of the present inventionly a kind ofly be combined into the basis with nucleic acid base pairing identification complementation, through the miRNAs detection method that silver dyes enhanced nano gold mark probe, its key step comprises:
1. the preparation of sample and the extraction of RNA always is to adopt ordinary method and reagent to extract;
2. hybridization: sample, capture probe, signal probe are mixed with the nano gold mark probe, this mixture system is transferred in the enzyme plate hole that each Streptavidin encapsulates hatched, said nano gold mark probe is that general T probe mixes with nano-Au solution and obtains;
3. the nano gold mark probe quantitative detects microRNA: the every hole of aforementioned enzyme plate adds silver and strengthens liquid; Reaction is to occurring washing termination reaction with ultrapure water behind the grey scale signal under the room temperature lucifuge condition; Enzyme plate is inserted in the ELIASA, and single length scanning detects the A value of solution in real time through ELIASA.
Wherein, said nanometer gold can be bought from commercial sources and (for example available from Sino-American Biotechnology Co., SABC), also can adopt following method preparation: with the 1.0mM HAuCl of 20ml
4Place vessel in heating to boiling, add two water citric acid trisodium (Na of 2ml 1%
3C
6H
5O
72H
2O), under refluxad vigorous stirring 15min forms gold sol until the further change that does not observe color then, filters and promptly gets nano-Au solution of the present invention.Nanometer gold with this method preparation has maximum absorption band at the 518nm place, mean diameter 12-13nm.
Said nano gold mark probe can adopt following method to prepare: with the centrifugal supernatant of abandoning of above-mentioned nano-Au solution, add general T probe (MST-probe) redispersion nanometer gold, it is centrifugal to get supernatant after the adding damping fluid is hatched; Abandon supernatant,, abandon supernatant once more with centrifugal behind the damping fluid washing precipitate; Twice of washing precipitate is to remove unnecessary strand oligonucleoside on unmarked; At last, adding the resuspended throw out of damping fluid promptly gets.
The preparation that said silver strengthens liquid (SES) is following: (0.085g is dissolved in 1.5ml H with quinhydrones
2Among the O), (0.255g Hydrocerol A and 0.235g trisodium citrate are dissolved in 1ml H to the Hydrocerol A saline solution
2Among the O, pH3.8), Silver Nitrate (AgNO
3, 0.25g is dissolved in 1ml H
2Among the O) transparent with 75: 25: 3 ratio mixing until solution becomes.
Three kinds of nucleotide sequences, test kit and detection methods provided by the invention can be at the middle microRNA content of cardiovascular field test sample (comprising serum, whole blood, lysis, tissue etc.).
Below in conjunction with embodiment the present invention is described further, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.The protection domain of this patent is as the criterion with claim.
One, material
1. enzyme-linked immunoassay plate (enzyme plate; Available from Genetimes Technology, Inc)
Two, instrument
1. ELIASA
2. spectrophotometer
Three, reagent
1. Streptavidin
2. Silver Nitrate (AgNO
3, 0.25g is dissolved in 1ml H
2Among the O)
3. Hydrocerol A
4. trisodium citrate (Na
3C
6H
5O
72H
2O)
5. Hydrocerol A saline solution: 0.255g Hydrocerol A and 0.235g trisodium citrate are dissolved in 1ml H
2Among the O, pH3.8
6. (0.085g is dissolved in 1.5ml H to quinhydrones
2Among the O)
7.Trizol lysate
8.TE damping fluid: 10mM Tris-HCl, 1mM EDTA, pH7.4
9.PBN damping fluid: 0.3M NaNO
3With 10mM Na
2HPO
4/ NaH
2PO
4Damping fluid, pH7.0
10.1mM HAuCl
4(tetra chlorauric acid): 0.1g HAuCl
4(U.S. Aldrich company, article No. G4022) is dissolved in the 500ml deionized water.Trivalent gold ion storage liquid can be prepared as being stored in the brown bottle in advance.
11.NaCl solution
12. lysate: 20mM Tris-HCl (pH 8.5), 0.5%NP-40,20 μ g/mL tRNA (not adding divalent cation)
13.PBS (no MgCl
2And CaCl
2, available from Invitrogen)
(pH7.5) 14.MOPS (available from U.S. Sigma company)
Four, preparation method
1. the preparation of capture probe (MSB-probe), signal probe (MPA-probe) and general T probe is the deoxy-oligonucleotide probe that adopts the synthetic HPLC level of ordinary method, and all oligonucleotide are dissolved in the TE damping fluid, packing, be stored in-80 ℃ subsequent use.
For miR-1:5 '-uggaauguaaaGAAGUGUGUAU-3 '
MSB-probe: 5 '-tttacattcca-(CH
2)
6-Bio-3 '
MPA-probe: 5 '-(A)
30-ATACACACTTC-3 '
General T probe: 5 '-(T)
30-(CH
2)
6-SH-3 '.
2. the preparation of nanometer gold (GNP) solution
The formation of nanometer gold can be observed by change in color, and little nm gold particles is red.Surface adsorption one deck Hydrocerol A negatively charged ion of nano particle can make particle separation.The existence of suspension colloid can be detected by the reflection of particle to laser beam.Become littler negatively charged ion and can make particle more approaching, and observe the change of another color.
(1) adds the 1.0mM HAuCl of 20ml
4Go in the 50ml Erlenmeyer flask, Erlenmeyer flask is placed on the magnetic agitation hot plate, put into magnetic stirring bar, and heated soln is to boiling;
(2) two water citric acid trisodium (Na of adding 2ml 1% in the solution that boils
3C
6H
5O
72H
2O);
(3) under refluxad vigorous stirring 15min until the further change that does not observe color.Citrate trianion reduction trivalent gold forms gold sol gradually;
(4) filter colloidal solution with 0.2 μ m pore membrane, 4 ℃ of storages, autoclaving before using.
Nanometer gold with this method preparation has maximum absorption band at the 518nm place, mean diameter 12-13nm.
The existence of suspension colloid can be detected by the reflection of particle to laser beam.Because laser designator emission polarized light, it is invisible that light beam is shown.When laser beam was visible in a visual field, the vertical visual field at it was sightless so.Add 5-10 and drip 1M NaCl solution, note the variation of solution colour, show nano particle bonded tightness.
3. the preparation of nano gold mark probe.
(1) nano-Au solution 500 μ l, 4 ℃ of centrifugal 10min of 14000rpm abandon supernatant;
(2) add the general T probe 50 μ l redispersion nanometer gold of 300pM, hatch 12h for 4 ℃;
(3) add 50 μ l TE damping fluids again, hatch 24h for 4 ℃;
(4) carefully get supernatant, 4 ℃, the centrifugal 10min of 14000rpm;
(5) abandon supernatant, add 0.15M NaCl, 10mM phosphate (pH7.4) damping fluid washing precipitate;
(6) 4 ℃, the centrifugal 10min of 14000rpm abandons supernatant once more;
(7) twice of washing precipitate is to remove unnecessary strand oligonucleoside on unmarked;
(8) last, add 0.15M NaCl, the resuspended throw out of 10mM phosphate (pH8.5) damping fluid 50 μ l is put 4 ℃ and is stored for future use.
4. silver strengthens the preparation of liquid (SES)
(1) with the following composition of 75: 25: 3 mixed
(0.085g is dissolved in 1.5ml H to quinhydrones
2Among the O);
(0.255g Hydrocerol A and 0.235g trisodium citrate are dissolved in 1ml H to the Hydrocerol A saline solution
2Among the O, pH3.8);
Silver Nitrate (AgNO
3, 0.25g is dissolved in 1ml H
2Among the O);
(2) the vortex mixing is transparent until solution becomes.
Five, detection method
1. the preparation of cell pyrolysis liquid
(1) from cultivate or blood obtain cell suspension, density about 1 * 10
6
(2) the centrifugal 3min sedimentation cell of 1000x g;
(3) with 1ml PBS (no MgCl
2And CaCl
2, available from Invitrogen) and washing is once;
(4) the centrifugal 3min sedimentation cell of 1000x g;
(5) 10mM MOPS (pH7.5), 100mM KCl solution 100 μ l re-suspended cells;
(6) get 2 μ l cell suspensions, add 98 μ l lysates (20mM Tris-HCl (pH 8.5), 0.5%NP-40,20 μ g/mL tRNA (not adding divalent cation));
(7) sample is heated 15min at 80 ℃;
(8) the centrifugal 3min of 1000x g removes impurity such as cell debris.
2. the extraction of total RNA
(1) total RNA can be from culturing cell, extracts in hemocyte or the tissue, uses Trizol reagent, presses the shop instruction operation and gets final product;
(2) use the nucleic acid quantification appearance to measure the RNA concentration of extraction (supposition 1A260=40 μ g/mL);
(3)-80 ℃ stored samples is subsequent use;
(4) use before dilution RNA to final concentration be 20 μ g/ml.
3. hybridization
(1) mix following solution:
Total RNA sample of 20 μ g/ml or enchylema (5 μ l) or serum (50 μ l);
1 μ l 100nM MSB-probe;
1 μ l 100nM MPA-probe;
20 μ l TE damping fluids (pH 8.5);
(2) mixture places 25 ℃, 10min;
(3) add above-mentioned nano gold mark probe 3 μ l, mixing, 25 ℃, 10min;
(4) this mixture system is transferred in the enzyme plate hole that each Streptavidin encapsulates, hatched 30min for 25 ℃;
(5) 1x PBS damping fluid washing 3min;
(6) 2x PBN (0.3M NaNO
3With 10mM Na
2HPO
4/ NaH
2PO
4Damping fluid, pH 7.0) wash each 3min 2 times.
4. the nano gold mark probe quantitative detects miRNA
(1) the every hole of aforementioned enzyme plate adds 100 μ l silver enhancing liquid;
(2) make reaction continue 150s under the room temperature lucifuge condition;
(3) reaction occurs washing termination reaction with ultrapure water behind the grey scale signal;
(4) enzyme plate is inserted in the ELIASA, and single length scanning detects the A value of solution in real time through ELIASA.
Six, experimental result:
Adopt method of the present invention to detect the content of miRNA-1 among the total RNA that peripheral blood extracts; As shown in Figure 2; Each concentration of specimens detects 3 times, and with the negative contrast of TE damping fluid, the result shows that the total RNA of the peripheral blood that only needs 2ng just can detect (Fig. 2) to target miRNA-1.
In addition; Synthetic miRNA-1 serial dilution is become different concns (from 1nmol/L to 1fmol/L); Detect with aforesaid method, as can beappreciated from fig. 3, the LDL that detects miRNA-1 is 1fmol/L; Linearity range is that (in the 100pmol/L～1fmol/L), the concentration logarithmic value of miRNA-1 and A value have good linear relation (R=0.9923).
The above is merely preferred embodiment of the present invention, only is illustrative for the purpose of the present invention, and nonrestrictive.After having read foregoing of the present invention, those skilled in the art can make various changes or modification to the present invention, even equivalence, fall within the appended claims of the application institute restricted portion equally.
KLPI090326_2.txt
Sequence table
< 110>Harbin university of TCM
< 120>be used for detecting the nucleotide sequence and the detection method of sample mircoRNAs content
<130>KLPI090
<160>3
<170>PatentIn?version?3.5
<210>1
<211>22
<212>RNA
<213>Homo?sapiens
<400>1
uggaauguaa?agaagugugu?au 22
<210>2
<211>22
<212>RNA
<213>Homo?sapiens
<400>2
cuggcccucu?cugcccuucc?gu 22
<210>3
<211>20
<212>RNA
<213>Homo?sapiens
<400>3
uaaggcacgc?ggugaaugcc 20
Claims (2)
1. be used for detecting the nucleotide sequence of sample mircoRNA content, it is characterized in that, said sequence is 5 '-tttacattcca-(CH
2)
6-3 ',
Represent a kind of with from 11 base length complementary nucleotide sequences of miRNAs 5 ' end, biotin labeling is held 3 '; The sequence of said miRNAs is SEQ ID NO:1;
With 5 '-(A)
30-ATACACACTTC-3 ',
Represent a kind of and the base length complementary nucleotide sequence that extends to 3 ' end from the 12nd base of miRNAs 5 ' end, with said 5 '-tttacattcca-(CH
2)
6-3 ' can combine with the full length sequence of said miRNAs is complementary simultaneously; A is a VITAMIN B4;
And 5 '-(T)
30-(CH
2)
6-SH-3 ',
Said sequence and said 5 '-(A)
30-ATACACACTTC-3 ' is complementary to be combined; T is a thymus pyrimidine; SH is a sulfydryl.
2. a test kit that detects microRNA content in the sample is characterized in that the nucleotide sequence that it comprises the described three kinds of cooperations of claim 1, and gene extracts material, and nano-Au solution and silver strengthen liquid.
Priority Applications (1)
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