CN106841135B - A method of a variety of miRNA are detected by fluorescence method simultaneously - Google Patents
A method of a variety of miRNA are detected by fluorescence method simultaneously Download PDFInfo
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Abstract
The invention discloses a kind of methods for detecting a variety of miRNA concentrations simultaneously by fluorescence method, comprising: the selection of miRNA；Prepare fluorescein-labeled nucleic acid probe；Select fluorogenic donor；Calculate the fluorescence crosstalk correction factor of the corresponding fluorescein of a variety of miRNA；The fluorescence intensity of the corresponding fluorescein of a variety of miRNA after being corrected and the corresponding relationship formula of miRNA concentration；The fluorescence signal for detecting the corresponding fluorescein of a variety of miRNA in miRNA solution to be measured, with after correction the fluorescence intensity of the corresponding fluorescein of a variety of miRNA and the corresponding relationship formula of miRNA concentration the concentration of miRNA is calculated.The present invention detects while realizing a variety of miRNA using single wavelength excitation, simplify detection method, the interference between background signal and fluorescein signal is effectively reduced, can accurately measure the concentration of any miRNA in solution, and at low cost, quick, easy, sensitive and specific good.
Description
Technical field
The invention belongs to biosensor technique fields, and in particular to a kind of to detect a variety of miRNA's simultaneously by fluorescence method
Method.
Background technique
Lung cancer is to lead to the main reason of global cancer mortality, every year because the number of lung cancer death is more than 1,000,000, wherein
Non-small cell lung cancer (no-small cell lung cancer, NSCLC) accounts for 80% or more, including gland cancer, squamous carcinoma and maxicell
Cancer.Although in recent years Imaging of Lung Cancer inspection and in terms of achieve greater advance, total existence in NSCLC 5 years
Rate is still less than 15%.Lacking effective early diagnosis is the main reason for NSCLC prognosis is bad.Most of patients is in clinical definite
Belong to middle and advanced stage, loses the chance of radical surgery.Rabat and CT etc. check that specificity is low, and existing neoplastic hematologic disorder mark
Object is lower for the susceptibility of pulmonary cancer diagnosis, lacks the efficiency of early diagnosis.Therefore, the mark of high sensitive and high specificity is sought
Will object becomes the urgent need that lung cancer is early diagnosed and treated.Recently research have indicated that some specific microRNA (miRNA) with
The occurrence and development of lung cancer are closely related, rise in the generating process of lung cancer and promote cancer or cancer suppressing action, can be used as pulmonary cancer diagnosis and control
The target spot for the treatment of.
MiRNA is the single-stranded non-coding RNA of one group of evolution conservative, and length, can specificity knowledge mostly in 21~23 nucleotide
Other said target mrna and the expression for regulating and controlling encoding gene are being transcribed by promoting mRNA degradation or suppressor transcription to influence mRNA
With the expression of post-transcriptional level.MiRNA participates in adjusting various biological process, such as cell differentiation, proliferation, metabolism and apoptosis.
The study found that different tumours has different miRNA express spectras, miRNA plays oncogene or suppression cancer in tumor tissues
The effect of gene takes part in the gene regulation loop of the kinds of tumors including lung cancer and generation, the development process of tumour.This
Outside, in the various body fluid of body, including blood plasma, sputum, chest fluid etc., there are a large amount of detectable extracellular miRNA, stabilization is deposited
And be not easy to be degraded by RNA enzyme so that miRNA can not only be used for lung cancer early diagnosis biological markers, also can be used as judgement
The efficiency index of patient's prognosis, it might even be possible to as a kind of new treatment means.
Currently, the technology of some qualitative and quantitative analysis miRNA include blot hybridization, in situ hybridization micro-sphere array hybridization it is micro-
Hybridization array and RT-polymerase chain reaction (RT-PCR) etc. have been set up at present.However, the analysis detection of miRNA
Still challenging, this is because its unique property, including size is short, sequence similarity high (homology), appearance are degraded etc.,
So that miRNA it is more difficult be applied to by nucleic acid hybridize based on field of biosensors in therefore, future there is an urgent need to develop
Serial of methods to miRNA carries out quick, sensitive and multichannel detection.
Summary of the invention
Goal of the invention: for overcome the deficiencies in the prior art, the present invention provides one kind, and by fluorescence method, detection is more simultaneously
The method of kind miRNA, this method is excited based on fluorescence resonance energy transfer effect Single wavelength, while emitting a variety of different wave lengths
A variety of miRNAs of the fluorescence to be overexpressed in corresponding detection cancerous lung tissue, and crosstalk correction is carried out to fluorescence signal, it obtains more
The concentration of accurate miRNA, principle is simple, experimental period is short, accuracy is high, and without any large-scale instrument.
Technical solution: a method of a variety of miRNA being detected by fluorescence method simultaneously, which is characterized in that including following step
It is rapid:
1) nucleic acid probe corresponding with a variety of miRNA is marked respectively with a variety of fluoresceins；Selection can pass through fluorescence resonance energy
It measures transfer effect and emitted energy is transferred to the nucleic acid dye of a variety of fluoresceins as fluorogenic donor；
2) the fluorescence crosstalk correction factor of the corresponding fluorescein of a variety of miRNA is calculated；
3) a variety of miRNA solution for taking different known concentrations respectively visit a variety of miRNA, nucleic acid probe and auxiliary
Needle hybridizes to form nucleic acid duplex structure, is embedded into fluorogenic donor in nucleic acid duplex structure, excites fluorogenic donor, detects more
The fluorescence signal of the corresponding fluorescein of kind miRNA, is corrected, after being corrected with fluorescence crosstalk correction factor pair fluorescence signal
The corresponding fluorescein of a variety of miRNA fluorescence intensity and miRNA concentration corresponding relationship formula；
4) miRNA solution to be measured is taken, miRNA, nucleic acid probe and assist probes to be measured is made to hybridize to form nucleic acid duplex knot
Structure is embedded into fluorogenic donor in nucleic acid duplex structure, excites fluorogenic donor, detects the corresponding fluorescein of a variety of miRNA
The concentration of miRNA is calculated with the corresponding relationship formula that step 3) obtains for fluorescence signal.
Wherein, a variety of miRNA are respectively tri- kinds of miRNA of miRNA-155, miRNA-182 and miRNA-197；A variety of fluorescence
Element is respectively tri- kinds of fluoresceins of Cy3, Cy3.5 and Cy5；Fluorogenic donor is nucleic acid dye TOTO-1.
Step 2) calculates the fluorescence crosstalk correction factor of the corresponding fluorescein of a variety of miRNA method particularly includes: respectively will
Fluorescence signal intensity of the every kind of fluorescein at maximum emission wavelength and fluorescence intensity of all other fluorescein at this wavelength
It compares, obtains fluorescence crosstalk correction factor M.
The fluorescence intensity P of the corresponding fluorescein of a variety of miRNA after the correction that step 3) obtains1、P2、P3With miRNA concentration
CmiRNA-155、CmiRNA-182、CmiRNA-197Corresponding relationship formula are as follows:
P1=1297.7lnCmiRNA-155+2758.9；
P2=1453.7lnCmiRNA-182+2748.1；
P3=788.8lnCmiRNA-197+1240.7；
Wherein, concentration unit nM.
In step 3), a variety of miRNA solution concentrations of different known concentrations within the scope of 0~50nM, fluorogenic donor it is dense
Degree is within the scope of 100~300nM.
In step 3) and step 4), hybridization is in the phosphate buffer solution of no DNAse/RNAase, at 20~37 DEG C
Reaction 2~4 hours；Fluorogenic donor is set to be embedded into the method in nucleic acid duplex structure are as follows: the nucleic acid duplex formed to hybridization
The fluorogenic donor is added in structure, is reacted 1~2 hour at 20~37 DEG C；In step 3) and step 4), fluorogenic donor is excited
The a length of 440nm of excitation light wave.
The working principle of the invention: the nucleic acid of nucleic acid dye (TOTO-1) and fluorescein (Cy3, Cy3.5, Cy5) label is visited
Needle does not fluoresce under free state.As target miRNA, when assist probes and fluorescein-labeled nucleic acid probe exist simultaneously,
Nucleic acid duplex structure can be formed by hybridization reaction.When with the excitation of 440nm, it is embedded into nucleic acid duplex structure
In TOTO-1 issue strong fluorescence.The fluorescence emission spectrum and Cy3 of TOTO-1, the excitation spectrum of Cy3.5, Cy5 overlap,
Its emitted energy can be transferred to three kinds of fluoresceins by fluorescence resonance energy transfer effect (FRET).Cy3, Cy3.5 and Cy5's
Maximum emission wavelength is respectively 570nm, 596nm and 670nm, can the corresponding fluorescence signal of effective district point.When containing in solution
When miRNA-155, miRNA-182 or miRNA-197, the fluorescence signal of corresponding fluorescein can detecte.By believing detection
Number carry out fluorescence crosstalk correction after, the actual concentrations of any one miRNA in available solution.
The utility model has the advantages that compared with prior art, the present invention have the advantages that following characteristic and:
(1) it is detected while the present invention realizes a variety of miRNA using single wavelength excitation, simplifies detection method, improved
The accuracy of pulmonary cancer diagnosis；
(2) present invention is effectively reduced dry between background signal and fluorescein signal using the method for fluorescence crosstalk correction
It disturbs, can accurately measure the concentration of any miRNA in solution；
(3) fluorescence detection to miRNA in human body fluid can be achieved in the present invention, does not need to simplify by precision instrument
Detection method, significantly reduces testing cost, and the present invention has at low cost, quick, easy, sensitive and specific good excellent
Point.
Detailed description of the invention
Fig. 1 is to excite Fluorometric assay 3 based on fluorescence resonance energy transfer effect and spectra overlap alignment technique Single wavelength
The flow chart of kind miRNA；Wherein,Indicate TOTO-1,Indicate P1,Indicate P2,Indicate P3,Indicate assistant ligand；
Fig. 2A is absorption/emission spectrum of fluorogenic donor TOTO-1 and fluorescent receptor Cy3, Cy3.5, Cy5；Fig. 2 B is fluorescence
The overlapping cases schematic diagram of the absorption spectrum of the emission spectrum and fluorescent receptor Cy3, Cy3.5, Cy5 of donor TOTO-1；From figure
It can be seen that the emission spectrum of fluorogenic donor TOTO-1 and the absorption spectrum of fluorescent receptor Cy3, Cy3.5, Cy5 overlap,
Meet the primary condition of fluorescence resonance energy transfer (FRET).
Fig. 3 A shows fluorescence emission spectrum of fluorogenic donor TOTO-1 when there is no resonance energy transfer and molten
There are the correspondence fluorescence hairs of fluorescent receptor Cy3, Cy3.5, Cy5 when a kind of corresponding miRNA initiation resonance energy transfer in liquid
Penetrate spectrum；As can be seen from the figure fluorogenic donor TOTO-1 issues strong fluorescence when there is no fluorescence resonance energy transfer
Signal, when containing miRNA-155 in solution, with corresponding probe hybridization reaction occurs for miRNA-155, is embedded into the double spiral shells of nucleic acid
The energy of fluorogenic donor TOTO-1 in rotation structure can be transferred to fluorescent receptor Cy3, can detecte the fluorescence hair of Cy3 in solution
Penetrate signal；Similarly, when containing miRNA-182 or miRNA-197 in solution, it can detecte the glimmering of Cy3.5 or Cy5 in solution
Light emission signal.Fig. 3 B show when in solution there are when two or three of miRNA, the fluorescence emission spectrum of corresponding solution；From figure
In it can be seen that fluorogenic donor TOTO-1 can by its energy simultaneously be transferred to two or three of fluorescein, thus realize at the same detect
Three kinds of miRNA in solution.
Fig. 4 A, 4B and 4C are the fluorescence signal before fluorescence crosstalk correction with the variation of miRNA concentration；Fig. 4 A ', 4B ' and 4C '
To use the fluorescence signal after 1 fluorescence crosstalk correction of formula with the variation of miRNA concentration.
Specific embodiment
The reagent and instrument used in Examples 1 to 3:
The fluorescence probe of fluorescence probe P1, the Cy3.5 label of nucleic acid probe P1 ', the Cy3 label of unmarked fluorescein Cy3
The fluorescence probe P3, assist probes L1, L2, L3 and miRNA-155, miRNA-182, miRNA-197 of P2 and Cy3.5 label
(Shanghai Sangon Biological Engineering Technology&Services Co.Ltd., Shanghai,
China), specific base sequence is shown in Table 2.TOTO-1 (Thermo Fisher Scientific, Massachusetts,
USA), 1 × PBS (Sunshine Biotechnology, Nanjing, China) of no DNA/RNA enzyme, ultraviolet specrophotometer
(Shimadzu UV-2450, Kyoto, Japan), luminoscope (Fluoromax-4, Horiba Jobin Yvon, Japan), from
Scheming (Eppendorf, German), quartz colorimetric utensil.
Inventor measures the excitation spectrum and transmitting light of TOTO-1, Cy3, Cy3.5 and Cy5 using Fluorescence Spectrometer respectively
Spectrum, is normalized obtained map, obtains Fig. 2.
As can be seen from Figure 2 the absorption of the emission spectrum of fluorogenic donor TOTO-1 and fluorescent receptor Cy3, Cy3.5, Cy5
Spectrum overlaps, and meets the primary condition of fluorescence resonance energy transfer (FRET).
Embodiment 1
(1) selection of the preparation of probe and fluorogenic donor
The nucleic acid probe that fluorescein Cy3 corresponding to miRNA-155 is marked is P1, the fluorescence of corresponding miRNA-182 preparation
The nucleic acid probe of plain Cy3.5 label is P2, and the nucleic acid probe of the fluorescein Cy5 label corresponding to miRNA-197 preparation is P3, right
The assist probes that should be prepared in miRNA-155 are L1, and the assist probes corresponding to miRNA-182 preparation are L2, are corresponded to
The assist probes of miRNA-197 preparation are L3, select nucleic acid dye TOTO-1 as fluorogenic donor.Wherein, miRNA-155,
MiRNA-182, miRNA-197, the nucleic acid sequence of fluorescein-labeled nucleic acid probe P1, P2, P3 and assist probes L1, L2, L3
Column are as shown in table 1.
Table 1
(2) Proof-Of Principle
8 EP pipes are taken, L1, the P1 ' (nucleic acid probe of unmarked fluorescein Cy3), miRNA- of 10nM is added in the first pipe
155, L1, P1, miRNA-155 of 10nM is added in the second pipe, and L2, P2, miRNA-182 of 10nM is added in third pipe, and the 4th pipe adds
Entering L3, P3, miRNA-197 of 10nM, the 5th pipe is added L1, P1, miRNA-155 and L2, P2, miRNA-182 of 10nM, and the 6th
L1, P1, miRNA-155 and L3, P3, miRNA-197 of 10nM is added in pipe, and L2, P2, miRNA-182 of 10nM is added in the 7th pipe
With L3, P3, miRNA-197, the 8th pipe be added L1, P1, miRNA-155, L2, P2, miRNA-182 and L3 of 10nM, P3,
miRNA-197。
Eight pipe samples are hybridized 2 hours for 25 DEG C in 1 × PBS of no DNAse/RNAase.Add respectively into reaction solution
Enter 100nMTOTO-1, reaction total volume is 500 μ L, after 25 DEG C are reacted 1 hour, excites, detects above-mentioned molten at luminoscope 440nm
The fluorescent emission spectrogram of liquid.
Experimental result is shown in Fig. 3.First pipe sample be TOTO-1 be embedded into launch in nucleic acid duplex structure it is strong glimmering
Optical signal, the black curve of TOTO-1 mark in corresponding diagram 3A；Second pipe fluorescent emits Cy3 mark in spectrogram corresponding diagram 3A
Green curve；Third pipe fluorescent emits the orange curve of Cy3.5 mark in spectrogram corresponding diagram 3A；4th pipe fluorescent
Emit the red curve of Cy5 mark in spectrogram corresponding diagram 3A；It is bent that 5th pipe fluorescent emits the black in spectrogram corresponding diagram 3B
Line；6th pipe fluorescent emits the green curve in spectrogram corresponding diagram 3B；7th pipe fluorescent emits spectrogram corresponding diagram 3B
In blue curve；8th pipe fluorescent emits the red curve in spectrogram corresponding diagram 3B.As seen from Figure 3, work as solution
It is middle to there is a kind of (four curves in corresponding diagram 3A), two or three of miRNA (four curves in corresponding diagram 3B) Shi Junke
Detect corresponding fluorescein signal, the results showed that be based on three kinds of Fluorometric assay of fluorescence resonance energy transfer (FRET)
The method of miRNA is feasible.
(3) the fluorescence crosstalk correction factor is calculated
According to Fig. 3 A, establish at the fluorescence maximum emission wavelength of fluorescein Cy3, Cy3.5 and Cy5 as respectively corresponding miRNA
Output channel, channel correspond to fluorescein fluorescence emission maximum signal be echo signal, other two kinds in respective channel
The signal of fluorescein is fluorescence crosstalk signal.Then the echo signal in each channel is normalized, calculates fluorescence string in each channel
The ratio for disturbing signal and echo signal, obtains corresponding fluorescence crosstalk correction factor M, and M is 3 × 3 spectra overlap correction matrixs, ginseng
It is shown in Table 2.Table 2 is different fluoresceins spectra overlap intensity rate in each sense channel under Single wavelength excitation.
Table 2
*Thickened portion is 3 × 3 spectra overlap correction matrix M in equation 1.
(4) corresponding relationship of fluorescence signal and miRNA concentration
Take three groups of EP pipes, every group of 9 EP pipe.10nM L1, P1, L2, P2, L3 and P3 is added in first group of each EP pipe,
Then 0,0.2,0.5,1,2,4,6,8 or 10nMmiRNA-155 is added into 9 EP pipes respectively.Second group of each EP Guan Zhongjun
10nM L1, P1, L2, P2, L3 and P3 is added, 0,0.2,0.5,1,2,4,6,8 or 10nM is then added into 9 EP pipes respectively
miRNA-182.10nM L1, P1, L2, P2, L3 and P3 is added in each EP pipe of third group, then respectively to 9 EP Guan Zhongjia
Enter 0,0.2,0.5,1,2,4,6,8 or 10nM miRNA-197.Three groups of samples are 25 DEG C in 1 × PBS of no DNAse/RNAase
Hybridization 2 hours.100nM TOTO-1 is separately added into above-mentioned solution, reaction solution total volume is 500 μ L, and 25 DEG C are reacted 1 hour
Afterwards, it is excited at luminoscope 440nm, detects the fluorescent emission spectrogram of above-mentioned solution.Then strong to original measurement fluorescence using formula 1
Degree (I) is corrected, and is deducted fluorescence crosstalk background signal of two kinds of fluoresceins at the third fluorescein maximum emission wavelength, is obtained
To correction of fluorescence intensity (P) of each fluorescein at respective maximum emission wavelength, to obtain the true dense of corresponding miRNA
Degree.
Experimental result is shown in Fig. 4.4A, 4B and 4C figure show the fluorescence signal before fluorescence crosstalk correction with the change of miRNA concentration
Change；As can be seen from the figure the signal interference between fluorescein is apparent；A ' B ' C ' figure is shown using 1 fluorescence crosstalk school of formula
Fluorescence intensity (P) after just is with miRNA concentration (CmiRNA) logarithm variation；As can be seen from the figure the signal between fluorescein is dry
It disturbs and is obviously eliminated, good linear pass is presented in the miRNA logarithm of its concentration and corresponding fluorescence signal between 0.2nM to 10nM
System, the corresponding relationship formula of fluorescence intensity and three kinds of miRNA concentrations after correction are as follows:
P1=1297.7lnCmiRNA-155+2758.9；
P2=1453.7lnCmiRNA-182+2748.1；
P3=788.8lnCmiRNA-197+1240.7。
(5) three kinds of miRNA are detected simultaneously
Three EP pipes are taken, the first pipe, the second pipe and third pipe are denoted as.First pipe be added L1, P1 of 10nM, L2, P2, L3,
P3 and 0.5nM miRNA-155,10nM miRNA-182,10nM miRNA-197；Second pipe be added L1, P1 of 10nM, L2,
P2, L3, P3 and 1nM miRNA-155,7nM miRNA-182,10nM miRNA-197；L1, P1 of third pipe addition 10nM,
L2, P2, L3, P3 and 3nM miRNA-155,3nM miRNA-182,3nM miRNA-197.
Three pipe samples respectively in 1 × PBS of no DNAse/RNAase 25 DEG C hybridize 2 hours.Respectively into above-mentioned solution
100nM TOTO-1 is added, reaction total volume is 500 μ L, after 25 DEG C are reacted 1 hour, excites, detects above-mentioned at luminoscope 440nm
The fluorescent emission spectrogram of solution.Original measurement fluorescence intensity (I) is calculated with formula 1 to obtain correction of fluorescence intensity (P), is substituted into
The corresponding relationship formula of fluorescence intensity and three kinds of miRNA concentrations after correction, obtains the measured value of three kinds of miRNA in each pipe sample,
Measured value is compared to obtain the rate of recovery of three kinds of miRNA in three pipe samples with known addition value.
Embodiment 2
(1) selection of the preparation of probe and fluorogenic donor
Referring to 1 (1) part of embodiment.
(2) Proof-Of Principle
8 EP pipes are taken, L1, the P1 ' (nucleic acid probe of unmarked fluorescein Cy3), miRNA-155 of 5nM is added in the first pipe,
L1, P1, miRNA-155 of 5nM is added in second pipe, and L2, P2, miRNA-182 of 5nM is added in third pipe, and the 4th pipe is added 5nM's
L1, P1, miRNA-155 and L2, P2, miRNA-182 of 5nM is added in L3, P3, miRNA-197, the 5th pipe, and 5nM is added in the 6th pipe
L1, P1, miRNA-155 and L3, P3, miRNA-197, the 7th pipe be added L2, P2, miRNA-182 and L3 of 5nM, P3,
L1, P1, miRNA-155, L2, P2, miRNA-182 and L3, P3, miRNA-197 of 5nM is added in miRNA-197, the 8th pipe.
Eight pipe samples are hybridized 4 hours for 20 DEG C in 1 × PBS of no DNAse/RNAase.Add respectively into reaction solution
Enter 100nMTOTO-1, reaction total volume is 500 μ L, after 20 DEG C are reacted 2 hours, excites, detects above-mentioned molten at luminoscope 440nm
The fluorescent emission spectrogram of liquid.
The experimental results showed that the fluorescent emission spectrogram that the present embodiment obtains is same as Example 1, experimental result is shown in Fig. 3.The
One pipe sample is embedded into nucleic acid duplex structure for TOTO-1 launches strong fluorescence signal, and TOTO-1 is marked in corresponding diagram 3A
The black curve of note；Second pipe fluorescent emits the green curve of Cy3 mark in spectrogram corresponding diagram 3A；Third pipe fluorescent
Emit the orange curve of Cy3.5 mark in spectrogram corresponding diagram 3A；4th pipe fluorescent emits Cy5 mark in spectrogram corresponding diagram 3A
Red curve；5th pipe fluorescent emits the black curve in spectrogram corresponding diagram 3B；6th pipe fluorescent emits spectrogram
Green curve in corresponding diagram 3B；7th pipe fluorescent emits the blue curve in spectrogram corresponding diagram 3B；8th pipe sample is glimmering
Red curve in light emitting spectrogram corresponding diagram 3B.As seen from Figure 3, when in solution exist one kind (four in corresponding diagram 3A
Curve), corresponding fluorescein signal can be detected in when two or three of miRNA (four curve in corresponding diagram 3B), ties
Fruit shows that the method for three kinds of miRNA of Fluorometric assay based on fluorescence resonance energy transfer (FRET) is feasible.
(3) the fluorescence crosstalk correction factor is calculated
According to Fig. 3 A, establish at the fluorescence maximum emission wavelength of fluorescein Cy3, Cy3.5 and Cy5 as respectively corresponding miRNA
Output channel, channel correspond to fluorescein fluorescence emission maximum signal be echo signal, other two kinds in respective channel
The signal of fluorescein is fluorescence crosstalk signal.Then the echo signal in each channel is normalized, calculates fluorescence string in each channel
The ratio for disturbing signal and echo signal, obtains corresponding fluorescence crosstalk correction factor M, and M is 3 × 3 spectra overlap correction matrixs, ginseng
It is shown in Table 2.Table 2 is different fluoresceins spectra overlap intensity rate in each sense channel under Single wavelength excitation.
(4) corresponding relationship of fluorescence signal and miRNA concentration
Take three groups of EP pipes, every group of 9 EP pipe.5nM L1, P1, L2, P2, L3 and P3 is added in first group of each EP pipe,
Then 0,0.1,0.2,0.5,1,2,3,4 or 5nMmiRNA-155 is added into 9 EP pipes respectively.Second group of each EP Guan Zhongjun
Be added 10nM L1, P1, L2, P2, L3 and P3, then respectively into 9 EP pipes be added 0,0.1,0.2,0.5,1,2,3,4 or
5nMmiRNA-182.10nM L1, P1, L2, P2, L3 and P3 is added in each EP pipe of third group, is then managed respectively to 9 EP
0,0.1,0.2,0.5,1,2,3,4 or 5nMmiRNA-197 of middle addition.Three groups of samples are in 1 × PBS of no DNAse/RNAase
20 DEG C hybridize 4 hours.100nM TOTO-1 is separately added into above-mentioned solution, reaction solution total volume is 500 μ L, 20 DEG C of reactions 2
After hour, is excited at luminoscope 440nm, detect the fluorescent emission spectrogram of above-mentioned solution.Then glimmering to original measurement using formula 1
Luminous intensity (I) is corrected, and deducts fluorescence crosstalk background letter of two kinds of fluoresceins at the third fluorescein maximum emission wavelength
Number, correction of fluorescence intensity (P) of each fluorescein at respective maximum emission wavelength is obtained, to obtain the true of corresponding miRNA
Real concentration.
Experimental result is shown in Fig. 4.4A, 4B and 4C figure show the fluorescence signal before fluorescence crosstalk correction with the change of miRNA concentration
Change；As can be seen from the figure the signal interference between fluorescein is apparent；A ' B ' C ' figure is shown using 1 fluorescence crosstalk school of formula
Fluorescence intensity (P) after just is with miRNA concentration (CmiRNA) logarithm variation；As can be seen from the figure the signal between fluorescein is dry
It disturbs and is obviously eliminated, good linear pass is presented in the miRNA logarithm of its concentration and corresponding fluorescence signal between 0.1nM to 5nM
System, the corresponding relationship formula of fluorescence intensity and three kinds of miRNA concentrations after correction are as follows:
P1=1297.7lnCmiRNA-155+2758.9；
P2=1453.7lnCmiRNA-182+2748.1；
P3=788.8lnCmiRNA-197+1240.7。
(5) three kinds of miRNA are detected simultaneously
3 EP pipes are taken, are denoted as the 4th pipe, the 5th pipe and the 6th pipe respectively.4th pipe be added L1, P1 of 10nM, L2, P2,
L3, P3 and 7nM miRAN-155,1nM miRNA-182,10nM miRNA-197；5th pipe be added L1, P1 of 10nM, L2,
P2, L3, P3 and 10nM miRAN-155,0.5nM miRNA-182,10nM miRNA-197；The L1 of 6th pipe addition 10nM,
P1, L2, P2, L3, P3 and 10nM miRAN-155,1nM miRNA-182,7nM miRNA-197.
Three pipe samples respectively in 1 × PBS of no DNAse/RNAase 20 DEG C hybridize 4 hours.Respectively into above-mentioned solution
100nM TOTO-1 is added, reaction total volume is 500 μ L, after 20 DEG C are reacted 2 hours, excites, detects above-mentioned at luminoscope 440nm
The fluorescent emission spectrogram of solution.Original measurement fluorescence intensity (I) is calculated with formula 1 to obtain correction of fluorescence intensity (P), is substituted into
The corresponding relationship formula of fluorescence intensity and three kinds of miRNA concentrations after correction, obtains the measured value of three kinds of miRNA in each pipe sample,
Measured value is compared to obtain the rate of recovery of three kinds of miRNA in three pipe samples with known addition value.
Embodiment 3
(1) selection of the preparation of probe and fluorogenic donor
Referring to 1 (1) part of embodiment.
(2) Proof-Of Principle step
8 EP pipes are taken, L1, the P1 ' (nucleic acid probe of unmarked fluorescein Cy3), miRNA- of 10nM is added in the first pipe
155, L1, P1, miRNA-155 of 10nM is added in the second pipe, and L2, P2, miRNA-182 of 10nM is added in third pipe, and the 4th pipe adds
Entering L3, P3, miRNA-197 of 10nM, the 5th pipe is added L1, P1, miRNA-155 and L2, P2, miRNA-182 of 10nM, and the 6th
L1, P1, miRNA-155 and L3, P3, miRNA-197 of 10nM is added in pipe, and L2, P2, miRNA-182 of 10nM is added in the 7th pipe
With L3, P3, miRNA-197, the 8th pipe be added L1, P1, miRNA-155, L2, P2, miRNA-182 and L3 of 10nM, P3,
miRNA-197。
Eight pipe samples are hybridized 3 hours for 37 DEG C in 1 × PBS of no DNAse/RNAase.Add respectively into reaction solution
Enter 100nMTOTO-1, reaction total volume is 500 μ L, after 37 DEG C are reacted 1 hour, excites, detects above-mentioned molten at luminoscope 440nm
The fluorescent emission spectrogram of liquid.
The experimental results showed that the fluorescent emission spectrogram that the present embodiment obtains is same as Example 1, Fig. 3 is seen.First pipe sample
It is embedded into nucleic acid duplex structure for TOTO-1 and launches strong fluorescence signal, the black that TOTO-1 is marked in corresponding diagram 3A
Curve；Second pipe fluorescent emits the green curve of Cy3 mark in spectrogram corresponding diagram 3A；Third pipe fluorescent emits spectrogram
The orange curve that Cy3.5 is marked in corresponding diagram 3A；The red that 4th pipe fluorescent emits Cy5 mark in spectrogram corresponding diagram 3A is bent
Line；5th pipe fluorescent emits the black curve in spectrogram corresponding diagram 3B；6th pipe fluorescent emits spectrogram corresponding diagram 3B
In green curve；7th pipe fluorescent emits the blue curve in spectrogram corresponding diagram 3B；8th pipe fluorescent emission spectra
Red curve in figure corresponding diagram 3B.As seen from Figure 3, when there are a kind of (four curves in corresponding diagram 3A) in solution,
Corresponding fluorescein signal can be detected when two or three of miRNA (four curve in corresponding diagram 3B), the results showed that base
In the method for three kinds of miRNA of Fluorometric assay of fluorescence resonance energy transfer (FRET) be feasible.
(3) the fluorescence crosstalk correction factor is calculated
According to Fig. 3 A, establish at the fluorescence maximum emission wavelength of fluorescein Cy3, Cy3.5 and Cy5 as respectively corresponding miRNA
Output channel, channel correspond to fluorescein fluorescence emission maximum signal be echo signal, other two kinds in respective channel
The signal of fluorescein is fluorescence crosstalk signal.Then the echo signal in each channel is normalized, calculates fluorescence string in each channel
The ratio for disturbing signal and echo signal, obtains corresponding fluorescence crosstalk correction factor M, and M is 3 × 3 spectra overlap correction matrixs, ginseng
It is shown in Table 2.Table 2 is different fluoresceins spectra overlap intensity rate in each sense channel under Single wavelength excitation.
(4) corresponding relationship of fluorescence signal and miRNA concentration
Take three groups of EP pipes, every group of 9 EP pipe.10nM L1, P1, L2, P2, L3 and P3 is added in first group of each EP pipe,
Then 0,0.2,0.5,1,2,4,6,8 or 10nMmiRNA-155 is added into 9 EP pipes respectively.Second group of each EP Guan Zhongjun
Be added 10nM L1, P1, L2, P2, L3 and P3, then respectively into 9 EP pipes be added 0,0.2,0.5,1,2,4,6,8 or
10nMmiRNA-182.10nM L1, P1, L2, P2, L3 and P3 is added in each EP pipe of third group, is then managed respectively to 9 EP
0,0.2,0.5,1,2,4,6,8 or 10nMmiRNA-197 of middle addition.Three groups of samples are 37 in 1 × PBS of no DNAse/RNAase
DEG C hybridization 3 hours.200nM TOTO-1 is separately added into above-mentioned solution, reaction solution total volume is 500 μ L, and 37 DEG C of reactions 2 are small
It is excited at Shi Hou, luminoscope 440nm, detects the fluorescent emission spectrogram of above-mentioned solution.Then using formula 1 to original measurement fluorescence
Intensity (I) is corrected, and deducts fluorescence crosstalk background signal of two kinds of fluoresceins at the third fluorescein maximum emission wavelength,
Correction of fluorescence intensity (P) of each fluorescein at respective maximum emission wavelength is obtained, to obtain the true dense of corresponding miRNA
Degree.
Experimental result is shown in Fig. 4.4A, 4B and 4C figure show the fluorescence signal before fluorescence crosstalk correction with the change of miRNA concentration
Change；As can be seen from the figure the signal interference between fluorescein is apparent；A ' B ' C ' figure is shown using 1 fluorescence crosstalk school of formula
Fluorescence intensity (P) after just is with miRNA concentration (CmiRNA) logarithm variation；As can be seen from the figure the signal between fluorescein is dry
It disturbs and is obviously eliminated, good linear pass is presented in the miRNA logarithm of its concentration and corresponding fluorescence signal between 0.2nM to 10nM
System, the corresponding relationship formula of fluorescence intensity and three kinds of miRNA concentrations after correction are as follows:
P1=1297.7lnCmiRNA-155+2758.9；
P2=1453.7lnCmiRNA-182+2748.1；
P3=788.8lnCmiRNA-197+1240.7。
(5) three kinds of miRNA are detected simultaneously
Three EP pipes are taken, are denoted as the 7th pipe, the 8th pipe and the 9th pipe respectively.L1, P1, L2, the P2 of 10nM is added in 7th pipe,
L3, P3 and 10nM miRAN-155,3nM miRNA-182,3nM miRNA-197.L1, P1, the L2 of 10nM is added in 8th pipe,
P2, L3, P3 and 10nM miRAN-155,7nM miRNA-182,1nM miRNA-197.L1, the P1 of 10nM is added in 9th pipe,
L2, P2, L3, P3 and 10nM miRAN-155,10nM miRNA-182,0.5nM miRNA-197.
Three pipe samples respectively in 1 × PBS of no DNAse/RNAase 37 DEG C hybridize 3 hours.Respectively into above-mentioned solution
100nM TOTO-1 is added, reaction total volume is 500 μ L, after 37 DEG C are reacted 1 hour, excites, detects above-mentioned at luminoscope 440nm
The fluorescent emission spectrogram of solution.Original measurement fluorescence intensity (I) is calculated with formula 1 to obtain correction of fluorescence intensity (P), is substituted into
The corresponding relationship formula of fluorescence intensity and three kinds of miRNA concentrations after correction, obtains the measured value of three kinds of miRNA in each pipe sample,
Measured value is compared to obtain the rate of recovery of three kinds of miRNA in three pipe samples with known addition value.
The related data for the first pipe to the 9th pipe that (5) part obtains in Examples 1 to 3 is referring to table 3, by fluorescence string
Sample recovery rate is 90% to 109% after disturbing correction, it can be seen that, the solution of the present invention detects three kinds of miRNA concentration errors and exists
Within ± 10%.This method can be used for detecting three kinds of miRNA simultaneously as the result is shown.
Table 3
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, and method of the invention is not only
It is limited to the detection of three of the above miRNA, applies also for the detection of other a variety of miRNA, these improvements and modifications also should be regarded as this
The protection scope of invention.
SEQUENCE LISTING
<110>Southeast China University
<120>a kind of method that a variety of miRNA are detected simultaneously by fluorescence method
<130> SG20170105001
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<170> PatentIn version 3.3
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Claims (7)
1. a kind of method for detecting a variety of miRNA simultaneously by fluorescence method, which comprises the following steps:
1) nucleic acid probe corresponding with a variety of miRNA is marked respectively with a variety of fluoresceins；Selection can be turned by fluorescence resonance energy
It moves effect and emitted energy is transferred to the nucleic acid dye of a variety of fluoresceins as fluorogenic donor；
2) the fluorescence crosstalk correction factor of the corresponding fluorescein of a variety of miRNA is calculated；
3) a variety of miRNA solution for taking different known concentrations respectively make a variety of miRNA, the nucleic acid probe and auxiliary
It helps probe to hybridize to form nucleic acid duplex structure, is embedded into the fluorogenic donor in nucleic acid duplex structure, excite described glimmering
Light donor detects the fluorescence signal of the corresponding fluorescein of a variety of miRNA, the letter of the fluorescence described in the fluorescence crosstalk correction factor pair
It number is corrected, the fluorescence intensity of the corresponding fluorescein of a variety of miRNA after being corrected and the corresponding relationship of miRNA concentration
Formula；
4) miRNA solution to be measured is taken, the miRNA to be measured, the nucleic acid probe and assist probes is made to hybridize to form the double spiral shells of nucleic acid
Structure is revolved, is embedded into the fluorogenic donor in nucleic acid duplex structure, excites the fluorogenic donor, is detected described a variety of
The concentration of miRNA is calculated with the corresponding relationship formula that step 3) obtains for the fluorescence signal of the corresponding fluorescein of miRNA；
A variety of miRNA are respectively tri- kinds of miRNA of miRNA-155, miRNA-182 and miRNA-197；
A variety of fluoresceins are respectively tri- kinds of fluoresceins of Cy3, Cy3.5 and Cy5；
The fluorogenic donor is nucleic acid dye TOTO-1.
2. the method according to claim 1, wherein step 2) calculates the glimmering of the corresponding fluorescein of a variety of miRNA
Optical crosstalk correction factor method particularly includes: the fluorescence signal intensity by every kind of fluorescein at maximum emission wavelength and institute respectively
There is fluorescence intensity of other fluoresceins at this wavelength to compare, obtains fluorescence crosstalk correction factor M.
3. the method according to claim 1, wherein a variety of miRNA after the correction that step 3) obtains are corresponding
The fluorescence intensity P of fluorescein1、P2、P3With miRNA concentration CmiRNA-155、CmiRNA-182、CmiRNA-197Corresponding relationship formula are as follows:
P1=1297.7lnCmiRNA-155+2758.9；
P2=1453.7lnCmiRNA-182+2748.1；
P3=788.8lnCmiRNA-197+1240.7；
Wherein, concentration unit nM.
4. method according to any one of claims 1 to 3, which is characterized in that dense known to the difference in step 3)
A variety of miRNA solution concentrations of degree are within the scope of 0~50nM, and the concentration of the fluorogenic donor is in 100~300nM range
It is interior.
5. method according to any one of claims 1 to 3, which is characterized in that described miscellaneous in step 3) and step 4)
Friendship is reacted 2~4 hours at 20~37 DEG C in the phosphate buffer solution of no DNAse/RNAase.
6. method according to any one of claims 1 to 3, which is characterized in that in step 3) and step 4), make described
Fluorogenic donor is embedded into the method in nucleic acid duplex structure are as follows: is added in the nucleic acid duplex structure formed to hybridization described glimmering
Light donor reacts 1~2 hour at 20~37 DEG C.
7. method according to any one of claims 1 to 3, which is characterized in that in step 3) and step 4), excite institute
State a length of 440nm of excitation light wave of fluorogenic donor.
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| CN108486222B (en) * | 2018-03-20 | 2021-08-10 | 东南大学 | Method for detecting telomerase activity based on fluorescence enhancement of nucleic acid dye TOTO-1 on single-chain G sequence |
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