CN101538615A - Nucleotide sequence for detecting mircoRNA content in samples and detecting method - Google Patents
Nucleotide sequence for detecting mircoRNA content in samples and detecting method Download PDFInfo
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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 treatment plan 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 the case fatality rate height 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 by 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, thereby can not make diagnosis 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 of myohaemoglobin lacks specificity, 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 was found in 1993, was Nucleotide (RNA) molecule that is about 19-25, was the little RNA of non-coding.It can be by 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, may participate in 30% human protein's expression regulation.Function assessment studies show that microRNA participate in to regulate 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 microRNA that studies confirm that participates in the expression of the multiple cardiac electrical activity associated protein 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.Experimental results show that the expression level of miR-1 in patients with coronary heart disease cardiac muscular tissue is 2.8 times of normal people cardiac muscular tissue; Observe same result in the rat experiment myocardial infarction and ischemia model: 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, as microRNA1, microRNA133, microRNA328, miR-124-1, whether microRNA21 can also be detected in blood, and has dependency with the progress of some circulation system disease.If this supposes to set up, these detections at these miRNA promptly can be used as early warning signal, by detection to the circulation peripheral blood, just can judge foundation is provided the prognosis of disease, 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, the technology relative complex, higher relatively to the requirement of testing staff's specialty degree, so may 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, with the oligonucleotide molecules of nano gold mark as signal probe, with the biotin labeling oligonucleotide molecules as capture probe, target sequence is captured on the solid phase carrier enzyme mark hole through Streptavidin-vitamin H effect, then the silver by 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 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 by these three kinds of nucleotide sequence combining nano technology for gold, it specifically comprises:
The invention provides a kind of be used for detecting sample mircoRNA content by biotin labeled nucleotide sequence I, it is characterized in that described 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 miRNAs 5 ' end, described biotin labeling is held 3 '; The sequence of described miRNAs is selected from the arbitrary sequence among the SEQ ID NO:1-3.
Preferably, described 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 described 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 miRNAs 5 ' end extends to 3 ' terminal base length complementary nucleotide sequence, wherein said nucleotide sequence can be combined with the full length sequence of described miRNAs is complementary by biotin labeled nucleotide sequence I simultaneously with aforementioned; A is a VITAMIN B4.
Preferably, described 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 described sequence is
5’-(T)
d-(CH
2)
e-SH-3’，
Wherein e is 3-20; D equates with b among the described nucleotide sequence II, and described sequence can combine with described nucleotide sequence II is complementary; T is a thymus pyrimidine; SH is a sulfydryl.
Preferably, described 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 described by biotin labeled nucleotide sequence I to it is characterized in that it comprises, described nucleotide sequence II and described 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, through the microRNAs detection method that silver dyes enhanced nano gold mark probe, it 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 of each Streptavidin bag quilt and hatched; Wherein, described capture probe is described by biotin labeled nucleotide sequence I, described signal probe is described nucleotide sequence II, and described nano gold mark probe is that general T probe mixes with nano-Au solution and obtains, and described 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 microplate reader, and single length scanning detects the A value of solution in real time by microplate reader.
Preferably, described 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 '; Described nucleotide sequence II is 5 '-(A)
30-ATACACACTTC-3 ' or 5 '-(A)
25-ACGGAAGGGCA-3 or 5 '-(A)
15Arbitrary sequence among the-GGCATTCACC-3 ';
Described 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 '.
Described nanometer gold can be bought from commercial channels and (for example available from Sino-American BiotechnologyCo., 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 described nano gold mark probe is as follows: 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, add the resuspended throw out of damping fluid promptly.
Described silver strengthens being prepared as follows of liquid (SES): (0.085g is dissolved in 1.5ml H with quinhydrones
2Among the O), (0.255g citric acid and 0.235g trisodium citrate are dissolved in 1ml H to the citric acid 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 as follows: the dna probe of alkane sulfydryl modification can adsorb under certain conditions in conjunction with the surface that is fixed on nm gold particles, 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 of Streptavidin bag quilt, 37 ℃ hatch 30min after, the hybridization product is fixed on the enzyme plate by Streptavidin-vitamin H specific recognition system, through PBS, PBN solution washing, remove free Nano-Au probe in the enzyme plate, with strengthening color reaction amplification nano-gold signal after silver dyes.Microplate reader is dyed photoabsorption A value or the optical density(OD) A value that strengthens liquid at 518nm wavelength place record silver, and by 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 checking 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.
Embodiment
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 miRNAs 5 ' end, described biotin labeling is held 3 '; The sequence of described miRNAs is selected from the arbitrary sequence among the SEQ ID NO:1-3.
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 miRNAs 5 ' end extends to 3 ' terminal base length complementary nucleotide sequence, wherein said nucleotide sequence can be combined with the full length sequence of described miRNAs is complementary by biotin labeled nucleotide sequence 1 simultaneously with aforementioned; 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 described nucleotide sequence II, and described sequence can combine with described 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 ℃ standby.
A kind of test kit of measuring microRNA content in the sample of the present invention, it comprises that described capture probe, described 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 described 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 of each Streptavidin bag quilt and hatched, described 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 microplate reader, and single length scanning detects the A value of solution in real time by microplate reader.
Wherein, described nanometer gold can be bought from commercial channels (for example available from Sino-AmericanBiotechnology Co., SABC), also can adopt following method preparation: place vessel in heating to boiling the 1.0mM HAuCl4 of 20ml, 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.
Described 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, add the resuspended throw out of damping fluid promptly.
Described silver strengthens being prepared as follows of liquid (SES): (0.085g is dissolved in 1.5ml H with quinhydrones
2Among the O), (0.255g citric acid and 0.235g trisodium citrate are dissolved in 1ml H to the citric acid 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.).
The invention will be further described below in conjunction with embodiment, 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. microplate reader
2. spectrophotometer
Three, reagent
1. Streptavidin
2. Silver Nitrate (AgNO
3, 0.25g is dissolved in 1ml H
2Among the O)
3. citric acid
4. trisodium citrate (Na
3C
6H
5O
72H
2O)
5. citric acid saline solution: 0.255g citric acid 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 in advance as being stored in the brown bottle.
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 ℃ standby.
For miR-1:5 '-uggaauguaaaGAAGUGUGUAU-3 '
MSB-probe: 5 '-(CH
2)
6-tttacattcca-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 citric acid 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 solution is to boiling;
(2) two water citric acid trisodium (Na of adding 2ml1% 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 citric acid and 0.235g trisodium citrate are dissolved in 1ml H to the citric acid 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 1000xg;
(3) with 1ml PBS (no MgCl
2And CaCl
2, available from Invitrogen) and washing is once;
(4) the centrifugal 3min sedimentation cell of 1000xg;
(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 1000xg 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 instrument to measure the RNA concentration of extraction (supposition 1A260=40 μ g/mL);
(3)-80 ℃ stored samples is standby;
(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 l100nM MSB-probe;
1 μ l100nM MPA-probe;
20 μ lTE 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 of each Streptavidin bag quilt, hatched 30min for 25 ℃;
(5) 1xPBS damping fluid washing 3min;
(6) 2xPBN (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 microplate reader, and single length scanning detects the A value of solution in real time by microplate reader.
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 be seen from Figure 3, the lowest detectable limit 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 only is 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.
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 (10)
- One kind be used for detecting sample mircoRNA content by biotin labeled nucleotide sequence, it is characterized in that described sequence is5’-(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 miRNAs 5 ' end, described biotin labeling is held 3 '; The sequence of described miRNAs is selected from the arbitrary sequence among the SEQ ID NO:1-3.
- 2. nucleotide sequence according to claim 1 is characterized in that, described sequence is 5 '-tttacattcca-(CH 2) 6-3 ' or 5 '-gagagggcctg-(CH 2) 6-3 ' or 5 '-gcgtgcctta-(CH 2) 8Arbitrary sequence in-3 '.
- 3. a nucleotide sequence that is used for detecting sample mircoRNA content is characterized in that, described sequence is5’-(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 miRNAs 5 ' end extends to 3 ' terminal base length complementary nucleotide sequence, wherein said nucleotide sequence can combine with the full length sequence of described miRNAs is complementary simultaneously with the described nucleotide sequence of claim 1; A is a VITAMIN B4.
- 4. nucleotide sequence according to claim 3 is characterized in that, described sequence is 5 '-(A) 30-ATACACACTTC-3 ' or 5 '-(A) 25-ACGGAAGGGCA-3 ' or 5 '-(A) 15Arbitrary sequence among the-GGCATTCACC-3 '.
- 5. a nucleotide sequence that is used for detecting sample mircoRNA content is characterized in that, described sequence is5’-(T) d-(CH 2) e-SH-3’，Wherein e is 3-20; D equates with the described b of claim 3, and described sequence can combine with the described nucleotide sequence of claim 3 is complementary; T is a thymus pyrimidine; SH is a sulfydryl.
- 6. nucleotide sequence according to claim 5 is characterized in that, described sequence 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 '.
- 7. a test kit that detects microRNA content in the sample is characterized in that it comprises claim 1,3 and 5 described nucleotide sequences, and gene extracts material, and nano-Au solution and silver strengthen liquid.
- 8. a test kit that detects microRNA content in the sample is characterized in that it comprises claim 2,4 and 6 described nucleotide sequences, and gene extracts material, and nano-Au solution and silver strengthen liquid.
- 9. one kind is combined into the basis with nucleic acid base pairing identification complementation, through the microRNAs detection method that silver dyes enhanced nano gold mark probe, it 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 of each Streptavidin bag quilt and hatched; Wherein, described capture probe is the described nucleotide sequence of claim 1, described signal probe is the described nucleotide sequence of claim 3, and described nano gold mark probe is that general T probe mixes with nano-Au solution and obtains, and described general T probe is the described nucleotide sequence of claim 5;(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 microplate reader, and single length scanning detects the A value of solution in real time by microplate reader.
- 10. detection method according to claim 9 is characterized in that described capture probe is the described nucleotide sequence of claim 2, and described signal probe is the described nucleotide sequence of claim 4; Described nano gold mark probe is that general T probe mixes with nano-Au solution and obtains, and described general T probe is the described nucleotide sequence of claim 6, and described nano-Au solution adopts following method to prepare: with the 1.0mMHAuCl 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 promptly, and the nanometer gold for preparing with this method has maximum absorption band at the 518nm place, mean diameter 12-13nm.
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