CN103740853A

CN103740853A - Hsa-miR-134 (Human Serum Albumin-Micro Ribonucleic Acid-134) detection kit and hsa-miR-134 detection method based on AllGlo probe fluorescent quantitative PCR (Polymerase Chain Reaction)

Info

Publication number: CN103740853A
Application number: CN201410041222.8A
Authority: CN
Inventors: 林凌青; 李娟�; 张忠英
Original assignee: Zhongshan Hospital Xiamen University
Current assignee: Zhongshan Hospital Xiamen University
Priority date: 2014-01-28
Filing date: 2014-01-28
Publication date: 2014-04-23
Anticipated expiration: 2034-01-28
Also published as: CN103740853B

Abstract

The invention discloses an hsa-miR-134 (Human Serum Albumin-Micro Ribonucleic Acid-134) detection kit and an hsa-miR-134 detection method based on AllGlo probe fluorescent quantitative PCR (Polymerase Chain Reaction) and relates to MicroRNA (Micro Ribonucleic Acid). The kit is provided with a kit body, a partition, an exogenous reference bottle, a stem-loop reverse transcription reagent bottle and a real-time fluorescent quantitative PCR reagent bottle. The detection method comprises the steps of extracting miRNA from a sample, adding 5 microliters of exogenous reference cel-miR-39 (Caenorhabditis Elegans-Micro Ribonucleic Acid-39) at a concentration of 5n mol provided by the kit after full lysis of the sample, and swirling and shaking in case of a sample of serum/plasma sample or other body fluids, adding no exogenous reference cel-miR-39 in case of a sample of a cell or a tissue, performing reverse transcription on miRNA to form cDNA (Complementary Deoxyribonucleic Acid) with a stem-loop reverse transcription reagent provided by the kit, performing real-time PCR amplification on cDNA with a real-time fluorescent quantitative PCR reagent provided by the kit, and setting a reasonable threshold and a baseline for result analysis by synthesizing various data given by an analytical instrument.

Description

Hsa-miR-134 detection kit and detection method thereof based on AllGlo fluorescence probe quantitative PCR

Technical field

The present invention relates to MicroRNA, especially relate to a kind of hsa-miR-134 detection kit and detection method thereof based on AllGlo fluorescence probe quantitative PCR.

Background technology

MicroRNA (miRNA) is that a class is extensively present in the non-coding RNA molecule that is about 22 Nucleotide in eukaryotic cell, many pathological processes in organism have been participated in, by regulatory gene, express, in cell proliferation, apoptosis, grow, cytodifferentiation, in the processes such as metabolism, play a significant role, be embodied in miRNA process in nucleus and form initial primary transcribe pri-miRNA to pre-miRNA, after transport out nucleus, by shearing, form ripe miRNA, by forming with Ago albumen etc. the silencing complex that RISC(RNA induces), part suppresses or degraded said target mrna sequence, participate in gene expression regulation (Bartel DP.MicroRNAs:genomics, biogenesis, mechanism, and function[J] .Cell, 2004, 116 (2): 281-297).

Because miRNA is playing the part of key player under generation, development, prognosis judgement and many other diseases states of tumour, accurately detect its expression and be significant for the effect of research miRNA.At present, the method for detection miRNA mainly contains northern blot technology, Real-Time Fluorescent Quantitative PCR Technique, hybridization in situ technique, micro-array chip technology etc.Wherein, northern blot technology is one of early stage means of RNA detection, but its specificity and susceptibility are low, if sample rna content is low or have degraded, may can't detect; Hybridization in situ technique, for detection of tissue and the distribution situation of cell levels miRNA, carries out half-quantitative detection to miRNA simultaneously, the miRNA of place is accurately to detect low expression that it is not enough; The problems such as micro-array chip technology is a kind of high-throughout detection technique, can detect a large amount of miRNA of one or more samples simultaneously, but exist chip manufacturing time-consuming, and effort and mark cost are high, and detection sensitivity and specificity are low.

Real time fluorescent quantitative technology (RT-qPCR) is one of technology the most generally detecting for genetic expression at present, have easy fast, the feature such as the high and specificity of susceptibility is good.At present, comprise the quantitative fluorescent PCR two portions in miRNA reverse transcription and downstream for detection of the real time fluorescence quantifying PCR method of miRNA, wherein reverse transcription is divided into two kinds according to the difference of reverse transcription primer: homopolymeric tailing method and stem ring reverse transcription method; The detection of quantitative fluorescent PCR is divided into dye method and probe method, the probe that wherein detects at present miRNA mostly is Taqman-MGB probe (a kind of fluorescent probe of the short-movie section that is suitable for increasing), because ripe miRNA has the short and small feature of fragment, want special detection, probe method more has superiority, and in susceptibility and specificity, is better than dye method.Reverse transcriptase primer energy specific recognition based on loop-stem structure, the ripe miRNA sequence that increases, and the precursor of miRNA is not amplified, Taqman-MGB probe in detecting miRNA shortcoming is synthetic price, is unfavorable for promoting.

At present, AllGlo probe has been successfully applied to and has detected simplexvirus, hepatitis B virogene sudden change (Feng, Z.L.Yu, X.Y.Lu, Z.M.Geng, D.Y.Zhang, L.Chen, S.J.Rapid detection of the hepatitis B virus YMDD mutant using AllGlo ^tMprobes[J] .Clin Chim Acta, 2011,412 (11-12): 1018-1021), invasive aspergillosis (Wu, D.S.Shen, J.Z.Zhou, X.Q.Shen, S.F.Wu, X.M.The establishment and evaluation of diagnostic accuracy of AllGlo (TM) probe-based techniques for invasive aspergillosis[J] .Zhong hua Nei Ke Za Zhi, 2010,49 (2): 142-145), the detection such as K-ras transgenation, ankylosing spondylitis.

Studies have reported that miR-134 up-regulated specifically in brain relative disease.It can affect the form of dendritic spine, thereby causes encephalopathy, for example, and the generation of epilepsy.Disturb the expression of miR-134, can improve the outbreak situation of epilepsy.(Jimenez-Mateos, E.M., T.Engel, et al. (2012) .Silencing microRNA-134produces neuroprotective and prolonged seizure-suppressive effects.Nat Med18 (7): 1087-1094) in addition, miR-134 can be by acting on wwox gene, produce tumorigenesis and promote the effect of breeding, promote the generation of tumor of head and neck.（Liu,C.J.,W.G.Shen,et?al.(2013).miR-134induces?oncogenicity?and?metastasis?in?head?and?neck?carcinoma?through?targeting?WWOX?gene.Int?J?Cancer3(10):28358）。Can also affect by affecting the expression of an embryonic stem cell key protein propagation and the invasive ability of the pernicious glioma of brain.（Niu,C.S.,Y.Yang,et?al.(2013).MiR-134regulates?the?proliferation?and?invasion?of?glioblastoma?cells?by?reducing?Nanog?expression.Int?J?Oncol42(5):1533-1540）

Summary of the invention

One of object of the present invention is, for the test kit using in existing detection miRNA method and the above-mentioned defect of detection method existence, to provide the detection kit of the hsa-miR-134 based on AllGlo fluorescence probe quantitative PCR.

Two of object of the present invention is to provide the detection method of the hsa-miR-134 based on AllGlo fluorescence probe quantitative PCR.

Described hsa-miR-134 is a kind of humanized miRNA, and its nucleotides sequence is classified as: SEQ ID NO.15 ＇-UGUGACUGGUUGACCAGAGGGG-3 ＇.

The described hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR, is provided with box body, dividing plate, exogenous with reference to bottle, stem ring reverse transcription reagent bottle, real-time fluorescence quantitative PCR reagent bottle; Dividing plate is located in box body, exogenous reference bottle, stem ring reverse transcription reagent bottle, real-time fluorescence quantitative PCR reagent bottle are inserted on dividing plate, exogenous with reference in bottle, exogenous reference being housed, stem ring reverse transcription reagent is housed in stem ring reverse transcription reagent bottle, real-time fluorescence quantitative PCR reagent is housed in real-time fluorescence quantitative PCR reagent bottle.

Described exogenous reference can adopt cel-miR-39 etc., and described cel-miR-39 is a kind of threadworms miRNA, and its nucleotides sequence is classified as: SEQ ID NO.25 ＇-UCACCGGGUGUAAAUCAGCUUG-3 ＇; Its working concentration can be 5nmol.

Described stem ring reverse transcription reagent comprises following component: (described 5 × RT damping fluid is by 250m mol Tris-HCl, 375m mol KCl, 15m mol MgCl for the MMLV enzyme of 200 units/μ L, 10m mol dNTP mixed solution, 5 × RT damping fluid ₂, 50mmol DTT composition), the RNA enzyme inhibitors of 40 units/μ L, the MgCl of 10m mol ₂, 1 μ mol reverse transcriptase primer, the miRNA standard substance (the miRNA standard substance here refer to the miRNA of synthetic, and concentration is 100n mol) of special miRNA.

The special reverse transcriptase primer of described miRNA is comprised of with reference to the special reverse transcriptase primer of U6 the special reverse transcriptase primer of hsa-miR-134, the exogenous special reverse transcriptase primer with reference to cel-miR-39 and endogenous:

The nucleotides sequence of the special reverse transcriptase primer of described hsa-miR-134 is classified as: SEQ ID NO.35 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCCCCTCTG-3 ＇;

The nucleotides sequence of the described exogenous special reverse transcriptase primer with reference to cel-miR-39 is classified as: SEQ ID NO.45 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCTGTTCCT-3 ＇;

Described endogenous is classified as with reference to the nucleotides sequence of the special reverse transcriptase primer of U6: SEQ ID NO.55 ＇-AACGCTTCACGAATTTGCGT-3 ＇.

Described real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid (10 × Taq damping fluid comprises 100m mol Tris-HCl, 500m mol KCl), 10m mol ₂, 5 units/μ L Taq polysaccharase, 10m moldNTP mixed solution, 100mL nuclease free water, the special forward primer of 10 μ mol, the general reverse primer of 10 μ mol and AllGlo probe.

Described special forward primer is comprised of with reference to the special primer of U6 the special primer of hsa-miR-134, the exogenous special primer with reference to cel-miR-39 and endogenous:

The nucleotides sequence of the special primer of described hsa-miR-134 is classified as:

SEQ?ID?NO.65＇-TCCTTCTGTGACTGGTTGAC-3＇；

The nucleotides sequence of the described exogenous special primer with reference to cel-miR-39 is classified as:

SEQ?ID?NO.75＇-CAGAGTCACCGGGTGTAAAT-3＇；

Described endogenous is classified as with reference to the nucleotides sequence of the special primer of U6:

SEQ?ID?NO.85＇-CTCGCTTCGGCAGCACA-3＇。

The nucleotides sequence of described general reverse primer is classified as:

SEQ?ID?NO.95＇-CAGTGCGTGTCGTGGAGT-3＇。

Described AllGlo probe is comprised of with reference to the specific probe of U6 and exogenous specific probe with reference to cel-miR-39 the specific probe of hsa-miR-134, endogenous;

The nucleotides sequence of the specific probe of described hsa-miR-134 is classified as:

SEQ?ID?NO.10MAR-TTGCACTGGATACGACCCCCTCTG-MAR；

Described endogenous is classified as with reference to the nucleotides sequence of the specific probe of U6:

SEQ?ID?NO.11JUP-CGATACAGAGAAGATTAGCATGGCCCC-JUP；

The nucleotides sequence of the described exogenous specific probe with reference to cel-miR-39 is classified as:

SEQ?ID?NO.12JUP-TGCACTGGATACGACCAAGCT-JUP。

The detection method of the described hsa-miR-134 based on AllGlo fluorescence probe quantitative PCR, its step is as follows:

1) adopt ordinary method to extract miRNA in sample, if blood serum/blood plasma or other body fluid samples, the concentration that adds the described hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR to provide after the abundant cracking of sample is the exogenous with reference to cel-miR-395 μ L of 5n mol, carries out immediately whirlpool concussion 15s; If cell or tissue sample is exogenous with reference to cel-miR-39 without adding;

2) the stem ring reverse transcription reagent reverse transcription miRNA that adopts the described hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR to provide is cDNA;

3) the real-time fluorescence quantitative PCR reagent that adopts the described hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR to provide carries out PCR in real time amplification by cDNA;

4) every data that composite analyser provides, set rational threshold value (Threshold) and baseline (Baseline), carry out interpretation of result.

Described AllGlo probe can adopt the fluorescent quantitation probe of U.S. A1leLogic Biosciences Corporation company, it has general T aqman, the all advantages of Taqman-MGB and molecular beacon (molecular beacon) probe, overcome the maximum drawback of current these several probes, the restriction that it has broken reporter group one end, traditional Taqman one end quenching group, utilized the special fluorescence dye of the several frequently seen wavelength of U.S. A1leLogic Biosciences Corporation company development, be marked at above oligonucleotide each other reporter group and the essence group that goes out, and contain the special chemical group that can improve Tm value (annealing temperature) above, improve probe specificity, hybridization specificity improves greatly, after hybridization hydrolysis, the dyestuff of two ends mark all becomes again reporter group, improve fluorescence increment.

Described AllGlo probe has following advantage: 1. improve Tm value (can reach more than 10 ℃), probe is shorter can reach 15～16 bases, can be suitable for A, the sequences Design probe that T content is higher; 2. strengthened the selection of multiple fluorescence quantitative, because every kind of dyestuff is that reporter group is again quenching group, the Taqman probe that breaks traditions, because of wavelength reason Marker selection difficulty, is not limited by quenching group wavelength; 3. greatly improve signal to noise ratio, without background signal, space length is near, better cancellation effect; 4. cost is lower, and price is only equivalent to the half of Taqman-MGB probe, has MGB probe and has superiority.

The present invention has adopted AllGlo probe technique, has set up the method that can detect the real-time fluorescence quantitative PCR of miRNA, and compared with taqman-MGB probe method, linearity range all can reach 7 orders of magnitude.The minimum 10 copies/μ L that reaches of real-time fluorescence quantitative PCR sensitivity that the present invention sets up, reaches as high as 10 ⁷copy/μ L.

The present invention has adopted AllGlo probe technique, Auele Specific Primer and corresponding AllGlo probe have been designed, probe is 2 kinds of special fluorophors of mark (MAR, JUP) respectively, and this technology reaction conditions is optimized, a kind of set up AllGlo fluorescence probe quantitative PCR technology for detection hsa-miR-134 method, has a extensive future.

Beneficial effect of the present invention is as follows:

The invention provides a kind of hsa-miR-134 detection kit and detection method based on AllGlo probe RT-qPCR, its specificity and susceptibility are high, can quick and precisely detect the content of miRNA in sample, compared with prior art have following advantage:

1. with northern blot, miRNA chip is compared, and it is high that the specificity of AllGlo probe in detecting and susceptibility are all wanted, and it is cheap that cost ratio miRNA chip is wanted;

2. compared with taqman-MGB probe, AllGlo probe adopts identical fluorophor, reporter group and quenching group each other, and the fluorescent signal discharging is on the one hand stronger, and background signal is lower on the other hand; And synthesis program is simple, price is only equivalent to the half of Taqman-MGB.

3. that compares current generally employing detects miRNA method based on SYBGreen dyestuff, and the reaction times based on AllGlo probe in detecting miRNA shortens greatly, and sensitivity and specificity will be apparently higher than the method that detects miRNA based on SYBGreen;

4. the present invention has set up the method that detects hsa-miR-134 based on the RT-qPCR of AllGlo probe, is suitable for the clinical sample checking as screening miRNA, for the effect that further research miRNA plays the part of in relative disease plays a role in promoting.

Accompanying drawing explanation

Fig. 1 is the structure composition schematic diagram that the present invention is based on the hsa-miR-134 detection kit embodiment of AllGlo fluorescence probe quantitative PCR.

Embodiment

Embodiment 1

Referring to Fig. 1, the hsa-miR-134 detection kit embodiment based on AllGlo fluorescence probe quantitative PCR of the present invention, is provided with box body 1, dividing plate 2, exogenous with reference to bottle 3, stem ring reverse transcription reagent bottle 4, real-time fluorescence quantitative PCR reagent bottle 5; Dividing plate 2 is located in box body 1, exogenous reference bottle 3, stem ring reverse transcription reagent bottle 4, real-time fluorescence quantitative PCR reagent bottle 5 are inserted on dividing plate 2, exogenous with reference in bottle 3, exogenous reference being housed, stem ring reverse transcription reagent is housed in stem ring reverse transcription reagent bottle 4, real-time fluorescence quantitative PCR reagent is housed in real-time fluorescence quantitative PCR reagent bottle 5.

1. described exogenous reference can adopt cel-miR-39 etc., and described cel-miR-39 is a kind of threadworms miRNA, and its working concentration can be 5n mol, its role is to monitor the reaction efficiency that extracts follow-up real-time fluorescence quantitative PCR whole process from miRNA;

2. stem ring reverse transcription reagent comprises following component: (5 × RT damping fluid is by 250m mol Tris-HCl, 375m mol KCl, 15m mol MgCl for the MMLV enzyme of 200 units/μ L, 10m mol dNTP mixed solution, 5 × RT damping fluid ₂50m mol DTT composition), reverse transcriptase primer, the miRNA standard substance (the miRNA standard substance here refer to the miRNA of synthetic, and concentration is 100n mol) of the special miRNA of MgCl2, the 1 μ mol of the RNA enzyme inhibitors of 40 units/μ L, 10m mol;

3. real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid (10 × Taq damping fluid comprises 100m mol Tris-HCl, 500m mol KCl), 10m mol ₂, 5 units/μ L Taq polysaccharase, 10m moldNTP mixed solution, 100mL nuclease free water, the special forward primer of 10 μ mol, the general reverse primer of 10 μ mol and AllGlo probe.

Embodiment 2

The detection of serum or blood plasma hsa-miR-134, comprises the following steps:

1. collect blood plasma or serum:

Extract that fresh blood sample 2mL is loaded on EDTA anticoagulant tube or without in antithrombotics pipe, immediately above-mentioned test tube is put upside down and mixed 6～8 times, then above-mentioned test tube is placed in to the centrifugal 10min of whizzer 3000r, after finishing, take out and be placed on test-tube stand, careful absorption supernatant is placed in the centrifuge tube without RNA enzyme of new 1.5mL, the centrifuge tube of the 1.5mL that supernatant is housed is placed in to the centrifugal 10min of whizzer 13000r, and after finishing, by upper plasma or serum transfers, to the centrifuge tube of new 1.5mL without RNA enzyme, (this step notes not being drawn to the cell precipitation of lower floor.), every pipe packing 400～500 μ L, get 200 μ L and extract for next step, and all the other blood plasma or serum are frozen in-80 ℃.

2. extract microRNA

Adopt the miRNA of the production of Tian Gen bio tech ltd to extract test kit (DP501), according to process specifications, carrying out sample (blood plasma or serum) miRNA extracts, wherein 200 μ L blood plasma or serum are adding standing 5min after the lysate concuss 30s of equal volume, then add exogenous with reference to cel-mir-39 (its working concentration is 5n mol) 5 μ L, after to specifications operation carry out, finally use the stoning enzyme water dissolution miRNA of 30 μ L, on nucleic acid spectrophotometric instrument Nano Drop2000, measure respectively concentration and purity, get purity A260/A280 ratio between 1.6～2.2, get the miRNA having extracted of concentration 2～20ng/ μ L for the reverse transcription in downstream, remaining miRNA is all frozen in-80 ℃.

The reverse transcription of 3.miRNA:

3.1 dissolve required reverse transcription composition under room temperature, and concussion mixes and is placed on ice; Please according to table 1, prepare reverse transcription reaction liquid.

Table 1

Component	Add volume (μ L)
		MMLV reversed transcriptive enzyme (200 units/μ L) (promega company)	0.4
MMLV5 times of reversed transcriptive enzyme damping fluid (promega company)	8
		Deoxynucleoside mixed solution (10mL)	1.6
RNA enzyme inhibitors (40 units/μ L)	0.4
		Specificity stem ring reverse transcriptase primer (1 μ L)	Each 1.2, totally 2.4
Nuclease free water	25.2
		Cumulative volume	38

Note: in table 1, the each 1.2 μ L of specificity reverse transcriptase primer refer to that respectively object hsa-miR-134's is special contrary

Transcribe primer and the exogenous special primer with reference to cel-miR-39.

3.2 are sub-packed in the 38 μ L mixed solutions that prepare in the PCR pipe without RNA enzyme, after add the 2 μ L miRNA solution of Extraction and enrichment, with fully mix (generation that bubble is avoided in this operation) without RNA enzyme rifle head as far as possible, to be placed on regular-PCR instrument (production of Biorad company) upper to managing the end for of short duration centrifugal drying, and reverse transcription reaction condition is as table 2.

Table 2

Step	Condition
			1	25℃5min
2	42℃60min
		3	70℃15min

Reaction finishes to be placed on 4 ℃ or on ice.

4. real-time fluorescence quantitative PCR detects hsa-miR-134

4.1 are placed in room temperature by each component of quantitative fluorescent PCR dissolves, and mixes and is placed on ice.

4.2 preparation PCR reaction mixtures are as table 3.

Table 3

Component	Every pipe volume (μ L)
		DNTP mixed solution (10m mol)	2
MgCl2(25m?mol)	2
		10 × Taq damping fluid is (without Mg ²⁺)	2
Taq (5 units/μ L) (takara company)	0.2
		Forward primer (10 μ mol)	0.4
Reverse primer (10 μ mol)	0.4
		Probe (10 μ mol)	0.2
Stoning enzyme water	10.8
		Cumulative volume	18

4.3 in 8 unions packing prepare PCR reaction mixture, every pipe adds the cDNA of 2 μ L, fully mixes, whole process is carried out on ice, avoids the generation of bubble.

4.4 quantitative fluorescent PCR reaction response conditions are as table 4.

Table 4

Detection is carried out on real-time fluorescence quantitative PCR instrument, can use ABI7300,7500(U.S. Applied Biosystems company) etc. multiple instrument.

5. data analysis and standardization: application ABI instrument carries software and Excel carries out data analysis, the CT value of hsa-miR-134 and exogenous contrast cel-mir-39 in the sample (serum or blood plasma) that can survey with aforesaid method, according to the CT value level of reference, try to achieve the relative content of hsa-miR-134 in serum or blood plasma, with 2 of relative quantification in qPCR ^{-Δ Ct}mode represents the level (Δ Ct is Ct value poor of hsa-miR-134 and outer source reference) of object hsa-miR-134 in serum or blood plasma.

Embodiment 3. organizes or cell miRNA detects

1. sample preparation:

A. tissue: will be organized in liquid nitrogen and grind.Every 30～50mg animal tissues or 100mg plant tissue add 1mL lysate MZ(Tian Gen bio tech ltd and produce), carry out homogenized with Syrup-homogenizing instrument.Sample volume should not exceed 10% of lysate MZ volume.

B. single-layer culturing cell: directly add lysate MZ lysing cell in culture plate, every 10cm ²area adds 1mLMZ.Lash several times with sampler.

C. cell suspension: centrifugal 800r5min gets cell, abandons supernatant.Add 1mL lysate MZ, vibrator vibration or pipettor are inhaled to beat and are mixed for several times.Before adding lysate MZ, do not want washed cell, in order to avoid degradation of rna.

2. tissue, cell miRNA extracts and enrichment

Adopt the miRNA of the production of Tian Gen bio tech ltd to extract test kit (DP501), according to process specifications, organize or cell miRNA extraction, finally use the stoning enzyme water dissolution RNA of 50 μ L, on nucleic acid spectrophotometric instrument Nano Drop2000, measure respectively concentration and purity

3. tissue, the reverse transcription of cell miRNA and enforcement fluorescent quantitation detect.

Subsequent step is with reference to step 2～4 of embodiment 2, but change below doing:

1. in embodiment 2 steps 2, in the mixed solution of reverse transcription reaction liquid, the exogenous special primer with reference to cel-miR-39 will change the special primer of U6, the concentration of primer and constancy of volume into;

2. in the PCR mixed solution of follow-up real time fluorescent quantitative, the exogenous special primer with reference to cel-miR-39 and probe will change special primer and the probe of U6 into.

Can range of application:

Hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR and detection method thereof can be applicable to the detection of cell hsa-miR-134 expression level in human tissue, hemocyte and body fluid, above demonstration and described ultimate principle of the present invention, principal character and advantage of the present invention.

Claims

1. the hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR, is characterized in that described hsa-miR-134 is a kind of humanized miRNA, and its nucleotides sequence is classified as: SEQ ID NO.15 ＇-UGUGACUGGUUGACCAGAGGGG-3 ＇;

2. the hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR as claimed in claim 1, it is characterized in that the described exogenous cel-miR-39 of adopting by reference, described cel-miR-39 is a kind of threadworms miRNA, and its nucleotides sequence is classified as: SEQ ID NO.25 ＇-UCACCGGGUGUAAAUCAGCUUG-3 ＇; Its working concentration is 5nmol.

3. the hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR as claimed in claim 1, is characterized in that described stem ring reverse transcription reagent comprises following component: the MMLV enzyme of 200 units/μ L, 10m mol dNTP mixed solution, 5 × RT damping fluid, the RNA enzyme inhibitors of 40 units/μ L, the MgCl of 10m mol ₂, 1 μ mol reverse transcriptase primer, the miRNA standard substance of special miRNA, concentration is 100n mol; Described 5 × RT damping fluid is by 250m mol Tris-HCl, 375mmol KCl, 15m mol MgCl ₂, 50m mol DTT composition.

4. the hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR as claimed in claim 3, is characterized in that the special reverse transcriptase primer of described miRNA is comprised of with reference to the special reverse transcriptase primer of U6 the special reverse transcriptase primer of hsa-miR-134, the exogenous special reverse transcriptase primer with reference to cel-miR-39 and endogenous;

5. the hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR as claimed in claim 1, is characterized in that described real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid, 10m mol ₂, 5 units/μ L Taq polysaccharase, 10m moldNTP mixed solution, 100mL nuclease free water, the special forward primer of 10 μ mol, the general reverse primer of 10 μ mol and AllGlo probe; Described 10 × Taq damping fluid comprises 100m mol Tris-HCl, 500m mol KCl.

6. the hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR as claimed in claim 5, is characterized in that described special forward primer is comprised of with reference to the special primer of U6 the special primer of hsa-miR-134, the exogenous special primer with reference to cel-miR-39 and endogenous;

SEQ?ID?NO.65＇-TCCTTCTGTGACTGGTTGAC-3＇；

SEQ?ID?NO.75＇-CAGAGTCACCGGGTGTAAAT-3＇；

SEQ?ID?NO.85＇-CTCGCTTCGGCAGCACA-3＇。

7. the hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR as claimed in claim 5, is characterized in that the nucleotides sequence of described general reverse primer is classified as:

SEQ?ID?NO.95＇-CAGTGCGTGTCGTGGAGT-3＇。

8. the hsa-miR-134 detection kit based on AllGlo fluorescence probe quantitative PCR as claimed in claim 5, is characterized in that described AllGlo probe is comprised of with reference to the specific probe of U6 and exogenous specific probe with reference to cel-miR-39 the specific probe of hsa-miR-134, endogenous;

SEQ?ID?NO.10MAR-TTGCACTGGATACGACCCCCTCTG-MAR；

SEQ?ID?NO.11JUP-CGATACAGAGAAGATTAGCATGGCCCC-JUP；

SEQ?ID?NO.12JUP-TGCACTGGATACGACCAAGCT-JUP。

9. the detection method of the hsa-miR-134 based on AllGlo fluorescence probe quantitative PCR, is characterized in that its step is as follows:

4) every data that composite analyser provides, set rational threshold value and baseline, carry out interpretation of result.

10. the detection method of the hsa-miR-134 based on AllGlo fluorescence probe quantitative PCR as claimed in claim 9, is characterized in that described AllGlo probe adopts the fluorescent quantitation probe of U.S. A1leLogic Biosciences Corporation company.