CN103773877B

CN103773877B - MicroRNA detection kit and detection method thereof is organized based on AllGlo fluorescence probe quantitative PCR

Info

Publication number: CN103773877B
Application number: CN201410041141.8A
Authority: CN
Inventors: 唐晶; 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: 2015-08-26
Anticipated expiration: 2034-01-28
Also published as: CN103773877A

Abstract

Organize microRNA detection kit and detection method thereof based on AllGlo fluorescence probe quantitative PCR, relate to microRNA.Test kit is provided with box body, dividing plate, stem ring reverse transcription reagents bottle and real-time fluorescence quantitative PCR reagent bottle.Conventionally extract microRNA in tissue; MiRNA reverse transcription is cDNA by the described stem ring reverse transcription reagents organizing miRNA detection kit to provide based on AllGlo fluorescence probe quantitative PCR of application; CDNA is carried out real-time fluorescence quantitative PCR amplification by the described real-time fluorescence quantitative PCR reagent organizing miRNA detection kit to provide based on AllGlo fluorescence probe quantitative PCR of application; Every data that composite analyser provides, set rational threshold value and baseline, carry out interpretation of result.

Description

MicroRNA detection kit and detection method thereof is organized based on AllGlo fluorescence probe quantitative PCR

Technical field

The present invention relates to microRNA, especially relate to and a kind ofly organize microRNA detection kit and detection method thereof based on AllGlo fluorescence probe quantitative PCR.

Background technology

MicroRNA (miRNA) is the non-coding RNA molecule being about 22 Nucleotide that a class is extensively present in eukaryotic cell, take part in many pathological processes in organism, expressed by regulatory gene, at cell proliferation, apoptosis, grow, play a significant role in cytodifferentiation, the process such as metabolism.Be embodied in miRNA in nucleus through forming initial primary transcribe pri-miRNA to pre-miRNA, after transport out nucleus, ripe miRNA is formed by shearing, 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 plays key player under the generation of tumour, development, Index for diagnosis and many other diseases states, its expression of accurate detection is significant for the effect of research miRNA, and the method detecting miRNA at present mainly contains northern blot technology, Real-Time Fluorescent Quantitative PCR Technique, hybridization in situ technique, microarray chip technology etc.Wherein northernblot technology is one of early stage means that RNA detects, but its specificity and susceptibility low, if sample rna content is low or there is degraded, may can't detect; Hybridization in situ technique, for detecting the distribution situation of tissue and cell levels miRNA, carries out half-quantitative detection to miRNA simultaneously, and its weak point is the miRNA that low expression accurately can not be detected; Microarray chip technology is a kind of high-throughout detection technique, can detect a large amount of miRNA of one or more samples simultaneously, but there is the problems such as chip manufacturing is time-consuming, effort, high, the detection sensitivity of mark cost and specificity are low.

Real time fluorescent quantitative technology (RT-qPCR) is commonly used to most at present one of technology that genetic expression detects, have easy fast, the feature such as the high and specificity of susceptibility is good.Comprise the quantitative fluorescent PCR two portions in RNA reverse transcription and downstream at present for the real time fluorescence quantifying PCR method detecting 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, and the probe wherein detecting miRNA mostly at present is Taqman-MGB probe (a kind of fluorescent probe being suitable for amplification short-movie section).It is short and small that miRNA due to maturation has fragment, and special detection, probe method advantageously, Sensitivity and Specificity is better than dye method.Based on the reverse transcriptase primer energy specific recognition of loop-stem structure, the miRNA sequence that amplification is ripe, and the precursor of miRNA is not amplified, the shortcoming of Taqman-MGB probe in detecting miRNA is to synthesize price, is unfavorable for promoting.

Current 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 YMDDmutant 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 ofdiagnostic accuracy of AllGlo (TM) probe-based techniques for invasive aspergillosis [J] .Zhong huaNei Ke Za Zhi, 2010,49 (2): 142-145) detections such as, K-ras transgenation, ankylosing spondylitis.

Summary of the invention

Organize the above-mentioned defect of test kit and the detection method existence used in miRNA method for existing detection, first object of the present invention is to provide detection two kinds to organize the special primer of miRNA.

Second order of the present invention is to provide organizes miRNA detection kit based on AllGlo fluorescence probe quantitative PCR.

3rd object of the present invention is to provide a kind of based on AllGlo fluorescence probe quantitative PCR two kinds to organize miRNA detection method.

Organize miRNA to refer to hsa-miR-504 and hsa-miR-133b respectively for described two kinds, both are humanized miRNA.

Described detection is organized the special primer of miRNA to comprise and is organized the specific reverse transcriptase primer of miRNA and organize the specific forward primer of miRNA.

The specific reverse transcriptase primer of the described miRNA of organizing is made up of the specific reverse transcriptase primer of hsa-miR-504 and the specific reverse transcriptase primer of hsa-miR-133b.

The nucleotides sequence of the specific reverse transcriptase primer of described hsa-miR-504 is classified as SEQ ID NO.15 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACGATAGAGT-3 ＇;

The nucleotides sequence of the specific reverse transcriptase primer of described hsa-miR-133b is classified as SEQ ID NO.25 ＇-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTAGCTGGT-3 ＇;

The specific forward primer of the described miRNA of organizing is made up of the specific forward primer of hsa-miR-504 and the specific forward primer of hsa-miR-133b:

The nucleotides sequence of the specific forward primer of described hsa-miR-504 is classified as SEQ ID NO.35 ＇-GCTGGTTAAGACCCTGGT-3 ＇;

The nucleotides sequence of the specific forward primer of described hsa-miR-133b is classified as SEQ ID NO.45 ＇-TAGCGTCTTTGGTCCCCT-3 ＇.

The specificity AllGlo probe of miRNA is organized to be made up of the specificity AllGlo probe of hsa-miR-504 and the specificity AllGlo probe of hsa-miR-133b.

The nucleotides sequence of the specificity AllGlo probe of described hsa-miR-504 is classified as SEQ ID NO.5MAR-CTGGATACGACGATAGAGTGC-MAR;

The nucleotides sequence of the specificity AllGlo probe of described hsa-miR-133b is classified as SEQ ID NO.6JUP-CTGGATACGACTAGCTGGTTGA-JUP.

The nucleotides sequence of the general reverse primer sequences of miRNA is organized to be classified as SEQ ID NO.75 ＇-CAGTGCGTGTCGTGGAGT-3 ＇ for described two kinds.

The described microRNA detection kit of organizing based on AllGlo fluorescence probe quantitative PCR is provided with box body, dividing plate, stem ring reverse transcription reagents bottle and real-time fluorescence quantitative PCR reagent bottle; Dividing plate is located in box body, and stem ring ring reverse transcription reagents bottle and real-time fluorescence quantitative PCR reagent bottle are inserted on dividing plate, and stem ring ring reverse transcription reagents bottle is built with stem ring reverse transcription reagents, and real-time fluorescence quantitative PCR reagent bottle is built with real-time fluorescence quantitative PCR reagent.

Described stem ring reverse transcription reagents 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 ₂, 50m molDTT forms), the RNA enzyme inhibitors of 40 units/μ L, the MgCl of 10m mol ₂, 1 μm of ol reverse transcriptase primer, the miRNA standard substance (miRNA standard substance here refer to the miRNA of synthetic, and concentration is 100n mol) of special miRNA.

Described real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid (10 × Taq damping fluid comprises 100mmol Tris-HCl, 500m mol KCl), 10m mol ₂, the Taq polysaccharase of 5 units/μ L, 10mM dNTP mixed solution, 100ml nuclease free water, 10 μm of ol specific forward primer, 10 μm of general reverse primers of ol and AllGlo probes.

Describedly organize MicroRNA detection method based on AllGlo fluorescence probe quantitative PCR, comprise the following steps:

1) conventionally microRNA in tissue is extracted;

2) miRNA reverse transcription is cDNA by the described stem ring reverse transcription reagents organizing miRNA detection kit to provide based on AllGlo fluorescence probe quantitative PCR of application;

3) cDNA is carried out real-time fluorescence quantitative PCR amplification by the described real-time fluorescence quantitative PCR reagent organizing miRNA detection kit to provide based on AllGlo fluorescence probe quantitative PCR of application;

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

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

Described AllGlo probe has following advantage: 1. improve Tm value (can reach more than 10 DEG C), probe is shorter can reach 15 ~ 16 bases, can be suitable for A, the sequences Design probe that T comparision contents is high; 2. increase the selection of multiple fluorescence quantitative, because not only often kind of dyestuff is reporter group but also be quenching group, the Taqman probe that breaks traditions, because of wavelength reason Marker selection difficulty, does not limit by quenching group wavelength; 3. greatly improve signal to noise ratio, without background signal, space length is near, better quenching effects; 4. cost is lower, and price is only equivalent to the half of Taqman-MGB probe, has MGB probe and had superiority.

Present invention employs AllGlo probe technique, devise special forward primer and general reverse primer and probe, probe two ends only need mark a kind of special fluorophor MAR, the ability simultaneously detecting multiple miRNA can be reached, greatly save cost, and this technology reaction conditions is optimized, establish a kind of method simultaneously detecting multiple miRNA based on AllGlo probe technique associating Real-Time Fluorescent Quantitative PCR Technique, have a extensive future.

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

The invention provides the composition of a kind of method based on AllGlo probe in detecting microRNA and relevant test kit, by designing primer and the probe of microRNA, reach accurate quantification testing goal, compare current detection means, it is advantageous that: 1. comparatively Taqman-MGB is low for AllGlo probe synthesis cost and difficulty, is beneficial to popularization; 2. AllGlo probe itself has two identical fluorophors, both reporter group each other, again quenching group each other, and the fluorescent signal of generation is stronger, sensitive higher than other probes; 3. there is not mutual fluorescence interference between several groups that probe itself designs, be applicable to carrying out multi-PRC reaction; 4. increasing of concentration and probe concentration can't Fluorophotometry quantitative PCR; 5. detect multiple microRNA, the fluorescence quantitative PCR detection comparing the single microRNA of single tube general at present more can save sample simultaneously.

Beneficial effect of the present invention is as follows:

The invention provides a kind of miRNA that detects based on the detection method of AllGlo probe RT-qPCR and test kit, its specificity and susceptibility high, quick and precisely can detect the content of miRNA in sample, compared with prior art there is following advantage:

1. compare with northern blot, miRNA chip, the specificity of AllGlo probe in detecting and susceptibility all want high, and cost ratio microRNA chip wants cheap;

2. compared with taqman-MGB probe, AllGlo probe adopts identical fluorophor, each other reporter group and quenching group, and the fluorescent signal of release 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. the RT-qPCR that the present invention establishes based on AllGlo probe detects miRNA method, be suitable for as cDNA microarray miRNA later phase clinical sample checking, and research miRNA is at the differential expression etc. of different field, has promoted the further investigation of miRNA at relative disease.

Accompanying drawing explanation

Fig. 1 is the structure composition schematic diagram organizing microRNA detection kit embodiment based on AllGlo fluorescence probe quantitative PCR of the present invention.

Embodiment

Embodiment 1

See Fig. 1, the described microRNA detection kit embodiment of organizing based on AllGlo fluorescence probe quantitative PCR is provided with box body 1, dividing plate 2, stem ring reverse transcription reagents bottle 3 and real-time fluorescence quantitative PCR reagent bottle 4; Dividing plate 2 is located in box body 1, stem ring ring reverse transcription reagents bottle 3 and real-time fluorescence quantitative PCR reagent bottle 4 are inserted on dividing plate 2, stem ring ring reverse transcription reagents bottle 3 is built with stem ring reverse transcription reagents, and real-time fluorescence quantitative PCR reagent bottle 4 is built with real-time fluorescence quantitative PCR reagent.

1. described stem ring reverse transcription reagents 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 ₂, 50m molDTT forms), the RNA enzyme inhibitors of 40 units/μ L, the MgCl of 10m mol ₂, 1 μm of ol reverse transcriptase primer, the miRNA standard substance (miRNA standard substance here refer to the miRNA of synthetic, and concentration is 100n mol) of special miRNA.

2. 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, the Taq polysaccharase of 5 units/μ L, 10mM dNTP mixed solution, 100mL nuclease free water, 10 μm of ol specific forward primer, 10 μm of general reverse primers of ol and AllGlo probes.

Embodiment 2

Concrete operation step of the present invention and reaction system as follows:

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

2. extract miRNA

The miRNA of the production of Tian Gen bio tech ltd is adopted to extract test kit, tissue samples miRNA extraction is carried out according to process specifications, finally dissolve miRNA with the stoning enzyme water elution of 50 μ L, concentration and purity is measured respectively on nucleic acid spectrophotometric instrument Nano Drop2000, get the reverse transcription of the miRNA extracted for downstream of 2 ~ 20ng, remaining miRNA is all frozen in-80 DEG C.

The reverse transcription of 3.miRNA:

Composition needed for reverse transcription dissolves by 3.1 under room temperature, and concussion mixing is placed on ice.

3.2 preparation reverse transcription reaction mixed solutions, prepare by table 1.

Table 1

Component	Volume/pipe (μ L)
		MMLV reversed transcriptive enzyme (200 units/μ L) (promega company)	0.2
MMLV5 times of reverse transcriptase buffer (promega company)	4
		Deoxynucleoside mixed solution (10m mol)	0.8
RNA enzyme inhibitors (40 units/μ L)	0.2
		Specificity stem ring reverse transcriptase primer (1 μm of ol)	1.2
Nuclease free water	11.6
		Cumulative volume	18

3.3 often pipe packing in the independent PCR pipe of 0.2 μ L

The 18 μ L mixed solutions prepared are sub-packed in the PCR pipe without RNA enzyme, after add the miRNA solution of 2 μ L Extraction and enrichment, with fully mixing without RNA enzyme rifle head (generation that bubble is avoided in this operation) as far as possible, be placed at the bottom of of short duration centrifugal drying to pipe on regular-PCR instrument (production of Biorad company), reverse transcription reaction condition is as table 2.

Table 2

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

Reaction end is placed on 4 DEG C.

The fluorescence quantitative PCR detection of 4.miRNA

Each component of quantitative fluorescent PCR is placed in room-temperature dissolution by 4.1, and mixing 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
MgCl ₂(25m mol)	2
		10 × Taq damping fluid is (without Mg ²⁺)	2
RTaq (5 units/μ L) (takara company)	0.2
		Forward primer (10 μm of ol)	0.4
Reverse primer (10 μm of ol)	0.4
		Probe (10 μm of ol)	0.2
Stoning enzyme water	10.8
		Cumulative volume	18

4.3 in 8 unions packing prepare PCR reaction mixture, often pipe adds the cDNA of 2 μ L, fully mixes, and 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

Detect carry out on real-time fluorescence quantitative PCR instrument, can ABI7300 be used, Applied Biosystems company of the 7500(U.S.) etc. multiple instrument.

5. data analysis and standardization: apply SPSS13.0 and Excel and carry out data analysis, the CT value of target miRNA and endogenous control U6 in the tissue can surveyed with aforesaid method, CT value level according to reference tries to achieve object miRNA relative content in the tissue, with 2 of relative quantification in qPCR ^{-Δ Ct}mode represents the level difference of the Ct value of miRNA and interior source reference (for the purpose of the Δ Ct) of object miRNA in tissue.

Embodiment 3

The foundation of double fluorescent quantitative PCR reaction system and typical curve is as table 5.

Table 5

Composition	Volume/often manage (μ L)
		dNTP Mixture(10m mol)	4
MgCl ₂(25m mol)	4
		10 × Taq damping fluid is (without Mg ²⁺)	4
RTaq (5 units/μ L) (takara company)	0.4
		Two align, reverse primer mixed solution (10 μ L)	3.2
Two kinds of probe mixed solutions (10 μ L)	0.8
		Stoning enzyme water	21.6
Cumulative volume	38

In advance 3 kinds of microRNA standard substance are diluted to 5 × 10 ⁷the microRNA initial concentration of copy/microlitre, after mixing, carries out continuous 10 times of dilutions, after carry out RT-qPCR detection.

Embodiment 4

Tissue samples detects:

Choose Colorectal Carcinoma 5 example, application AllGlo probe method detects hsa-miR-133b, compares with the method for the microRNA Taqman MGB of the detection hsa-miR-133b of the life technologies company of the U.S., does 2 multiple holes respectively.

Three kinds of methods detect the results contrast of hsa-miR-133b in 5 routine Colorectal Carcinoma samples respectively as table 6:

Table 6

Illustrate: detect same sample, CT value lower explanation susceptibility is higher, data presentation from upper table: compared with detecting 5 different samples with Taqman-MGB probe method, substance AllGlo probe method and the CT value that triple AllGlo probe method is detecting plasma sample has-miR-133b will lower than Taqman-MGB probe methods, and the susceptibility of both explanations will apparently higher than Taqman-MGB probe method.

Claims

1. detect the special primer that two kinds are organized miRNA, it is characterized in that comprising and organize the specific reverse transcriptase primer of miRNA, organize the specific forward primer of miRNA and organize the general reverse primer of miRNA, organize miRNA to refer to hsa-miR-504 and hsa-miR-133b respectively for described two kinds, both are humanized miRNA;

The specific reverse transcriptase primer of the described miRNA of organizing is made up of the specific reverse transcriptase primer of hsa-miR-504 and the specific reverse transcriptase primer of hsa-miR-133b;

The specific forward primer of the described miRNA of organizing is made up of the specific forward primer of hsa-miR-504 and the specific forward primer of hsa-miR-133b;

The nucleotides sequence of the specific forward primer of described hsa-miR-133b is classified as SEQ ID NO.45 ＇-TAGCGTCTTTGGTCCCCT-3 ＇;

The nucleotides sequence of the general reverse primer sequences of the described miRNA of organizing is classified as SEQ ID NO.75 ＇-CAGTGCGTGTCGTGGAGT-3 ＇.

2. organize the specificity AllGlo probe of miRNA, it is characterized in that being made up of the specificity AllGlo probe of hsa-miR-504 and the specificity AllGlo probe of hsa-miR-133b;

3. organize microRNA detection kit based on AllGlo fluorescence probe quantitative PCR, it is characterized in that being provided with box body, dividing plate, stem ring reverse transcription reagents bottle and real-time fluorescence quantitative PCR reagent bottle; Dividing plate is located in box body, and stem ring ring reverse transcription reagents bottle and real-time fluorescence quantitative PCR reagent bottle are inserted on dividing plate, and stem ring ring reverse transcription reagents bottle is built with stem ring reverse transcription reagents, and real-time fluorescence quantitative PCR reagent bottle is built with real-time fluorescence quantitative PCR reagent;

Described stem ring reverse transcription reagents 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 μm of ol 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, 375m mol KCl, 15m mol MgCl ₂, 50m mol DTT forms;

Described real-time fluorescence quantitative PCR reagent comprises following component: the MgCl of 10 × Taq damping fluid, 10m mol ₂, the Taq polysaccharase of 5 units/μ L, 10mM dNTP mixed solution, 100ml nuclease free water, 10 μm of ol specific forward primer, 10 μm of general reverse primers of ol and AllGlo probes; Described 10 × Taq damping fluid comprises 100m mol Tris-HCl, 500m mol KCl; Described specific forward primer is for organizing the specific forward primer of miRNA described in claim 1, described general reverse primer is for organizing the general reverse primer of miRNA described in claim 1, described AllGlo probe is for organizing the specificity AllGlo probe of miRNA described in claim 2.

4. based on the detection method organizing microRNA of AllGlo fluorescence probe quantitative PCR, this detection method is not used in the Diagnosis and Treat of disease, it is characterized in that comprising the following steps:

1) conventionally microRNA in tissue is extracted;

2) miRNA reverse transcription is cDNA based on the stem ring reverse transcription reagents organizing microRNA detection kit to provide of AllGlo fluorescence probe quantitative PCR by application as claimed in claim 3;

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

5. organize MicroRNA detection method as claimed in claim 4 based on AllGlo fluorescence probe quantitative PCR, it is characterized in that described AllGlo probe adopts the fluorescent quantitation probe of A1leLogic Biosciences Corporation company of the U.S..