CN106841135B - A method of a variety of miRNA are detected by fluorescence method simultaneously - Google Patents
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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 the technical field of biosensing, and particularly relates to a method for simultaneously detecting multiple miRNAs by a fluorescence method.
Background
Lung cancer is the leading cause of cancer death worldwide, with over 100 million deaths per year due to lung cancer, with non-small cell lung cancer (NSCLC) accounting for over 80% of these, including adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Despite recent advances in imaging and targeted therapy of lung cancer, the 5-year overall survival rate of NSCLC is still less than 15%. The lack of effective early diagnosis is a major cause of poor prognosis in NSCLC. Most patients are diagnosed in the middle and late stage of clinical diagnosis, and the chance of radical operation is lost. The specificity of chest radiography, CT and other examinations is low, while the sensitivity of the existing blood tumor markers to lung cancer diagnosis is low, and the efficiency of early diagnosis is lacked. Therefore, the search for markers with high sensitivity and specificity is an urgent need for early diagnosis and treatment of lung cancer. Recent research shows that some specific microRNAs (miRNAs) are closely related to the occurrence and development of lung cancer, play a role in promoting or inhibiting cancer in the occurrence process of the lung cancer, and can be used as targets for diagnosis and treatment of the lung cancer.
The miRNA is a group of evolutionary conserved single-stranded non-coding RNAs, the length of the miRNA is more than 21-23 nucleotides, the miRNA can specifically recognize target mRNA and regulate expression of coding genes, and mRNA expression and expression of the mRNA at a level after transcription are influenced by promoting mRNA degradation or inhibiting gene transcription. mirnas are involved in regulating a variety of biological processes, such as cell differentiation, proliferation, metabolism, and apoptosis. Researches show that different tumors have different miRNA expression profiles, and the miRNA plays the role of oncogenes or cancer suppressor genes in tumor tissues and participates in gene regulation loops of various tumors including lung cancer and the generation and development processes of the tumors. In addition, various body fluids of the body, including plasma, sputum, thoracic fluid and the like, have a large number of detectable extracellular miRNAs which exist stably and are not easily degraded by RNA enzyme, so that the miRNAs can be used as biological markers for early diagnosis of lung cancer, can also be used as effective indexes for judging prognosis of patients, and even can become a new treatment means.
Currently, some techniques for qualitative and quantitative analysis of miRNA, including blot hybridization, in situ hybridization of microsphere array hybridization microarray hybridization, and reverse transcription polymerase chain reaction (RT-PCR), are established. However, the analysis and detection of miRNA still have challenges, because of their unique properties, including short size, high sequence similarity (homology), and easy degradation, it is difficult to apply miRNA in the field of nucleic acid hybridization-based biosensors, and therefore, there is no urgent need to develop a series of methods for rapid, sensitive, and multichannel detection of miRNA.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects of the prior art, the invention provides a method for simultaneously detecting multiple miRNAs by a fluorescence method, the method is based on single-wavelength excitation of a fluorescence resonance energy transfer effect, simultaneously emits multiple fluorescence with different wavelengths so as to correspondingly detect multiple over-expressed miRNAs in lung cancer tissues, carries out crosstalk correction on fluorescence signals, obtains more accurate concentration of the miRNAs, and has the advantages of simple principle, short experimental period, high accuracy and no need of any large instrument.
The technical scheme is as follows: a method for simultaneously detecting multiple miRNAs by a fluorescence method is characterized by comprising the following steps:
1) respectively marking nucleic acid probes corresponding to the multiple miRNAs by using multiple fluoresceins; selecting as a fluorescence donor a nucleic acid dye that can transfer emission energy to the plurality of luciferin by a fluorescence resonance energy transfer effect;
2) calculating fluorescence crosstalk correction factors of fluorescein corresponding to multiple miRNAs;
3) respectively taking the multiple miRNA solutions with different known concentrations, hybridizing the multiple miRNAs, the nucleic acid probes and the auxiliary probes to form a nucleic acid double-helix structure, embedding the fluorescence donor into the nucleic acid double-helix structure, exciting the fluorescence donor, detecting fluorescence signals of fluorescein corresponding to the multiple miRNAs, and correcting the fluorescence signals by using a fluorescence crosstalk correction factor to obtain a corresponding relation between the corrected fluorescence intensity of the fluorescein corresponding to the multiple miRNAs and the miRNA concentration;
4) taking a miRNA solution to be detected, hybridizing the miRNA to be detected, the nucleic acid probe and the auxiliary probe to form a nucleic acid double-helix structure, embedding the fluorescence donor into the nucleic acid double-helix structure, exciting the fluorescence donor, detecting fluorescence signals of fluorescein corresponding to multiple miRNAs, and calculating by using the corresponding relational expression obtained in the step 3) to obtain the concentration of the miRNA.
Wherein, the multiple miRNAs are three miRNAs of miRNA-155, miRNA-182 and miRNA-197 respectively; the multiple fluorescein is three fluorescein of Cy3, Cy3.5 and Cy 5; the fluorescence donor is nucleic acid dye TOTO-1.
The specific method for calculating the fluorescence crosstalk correction factors of the fluorescein corresponding to the multiple miRNAs in the step 2) comprises the following steps: and respectively comparing the fluorescence signal intensity of each fluorescein at the maximum emission wavelength with the fluorescence intensity of all other fluorescein at the wavelength to obtain a fluorescence crosstalk correction factor M.
The corrected fluorescence intensity P of fluorescein corresponding to multiple miRNA obtained in step 3)1、P2、P3With miRNA concentration CmiRNA-155、CmiRNA-182、CmiRNA-197The corresponding relation is as follows:
P1＝1297.7lnCmiRNA-155+2758.9；
P2＝1453.7lnCmiRNA-182+2748.1；
P3＝788.8lnCmiRNA-197+1240.7；
where, the concentration units are nM.
In the step 3), the concentration of various miRNA solutions with different known concentrations is in the range of 0-50 nM, and the concentration of the fluorescence donor is in the range of 100-300 nM.
In the step 3) and the step 4), the hybridization is carried out in a DNAse/RNAase-free phosphate buffer solution at the temperature of 20-37 ℃ for 2-4 hours; the method for intercalating a fluorescent donor into a nucleic acid duplex is: adding the fluorescence donor into a nucleic acid double-helix structure formed by hybridization, and reacting for 1-2 hours at 20-37 ℃; in the step 3) and the step 4), the wavelength of the excitation light for exciting the fluorescence donor is 440 nm.
The working principle of the invention is as follows: nucleic acid probes labeled with nucleic acid dye (TOTO-1) and fluorescein (Cy3, Cy3.5, Cy5) did not fluoresce in the free state. When the target miRNA, the auxiliary probe and the fluorescein-labeled nucleic acid probe exist simultaneously, a nucleic acid double helix structure is formed through a hybridization reaction. TOTO-1 embedded in the nucleic acid duplex structure emits intense fluorescence when excited with 440nm excitation light. The fluorescence emission spectrum of TOTO-1 overlaps with the excitation spectrum of Cy3, Cy3.5, Cy5, and its emission energy can be transferred to three fluorescein species by Fluorescence Resonance Energy Transfer (FRET). The maximum emission wavelengths of Cy3, Cy3.5 and Cy5 are 570nm, 596nm and 670nm respectively, so that corresponding fluorescent signals can be effectively distinguished. When miRNA-155, miRNA-182 or miRNA-197 are contained in the solution, a fluorescent signal corresponding to fluorescein can be detected. After the fluorescence crosstalk correction is carried out on the detection signal, the accurate concentration of any miRNA in the solution can be obtained.
Has the advantages that: compared with the prior art, the invention has the following characteristics and advantages:
(1) the invention realizes the simultaneous detection of multiple miRNAs by using single wavelength excitation, simplifies the detection method and improves the accuracy of lung cancer diagnosis;
(2) the invention effectively reduces the interference between the background signal and the fluorescein signal by using the fluorescence crosstalk correction method, and can accurately measure the concentration of any miRNA in the solution;
(3) the invention can realize the fluorescence detection of miRNA in human body fluid, does not need to use a precise instrument, simplifies the detection method, greatly reduces the detection cost, and has the advantages of low cost, rapidness, simplicity, convenience, sensitivity and good specificity.
Drawings
FIG. 1 is a flow chart of single-wavelength fluorescence excitation method for detecting 3 kinds of miRNA based on fluorescence resonance energy transfer effect and spectral crosstalk correction technology; wherein,the expression TOTO-1 is used,the representation of P1 is shown as,the representation of P2 is shown as,the representation of P3 is shown as,represents an ancillary ligand;
FIG. 2A shows absorption/emission spectra of fluorescence donor TOTO-1 and fluorescence acceptors Cy3, Cy3.5 and Cy 5; FIG. 2B is a schematic diagram showing the overlap of the emission spectrum of the fluorescence donor TOTO-1 and the absorption spectra of the fluorescence acceptors Cy3, Cy3.5 and Cy 5; it can be seen from the figure that the emission spectrum of the fluorescence donor TOTO-1 and the absorption spectra of the fluorescence acceptors Cy3, Cy3.5, Cy5 partially overlap, and satisfy the basic condition of Fluorescence Resonance Energy Transfer (FRET).
FIG. 3A shows fluorescence emission spectra of fluorescence donor TOTO-1 without resonance energy transfer and fluorescence emission spectra of fluorescence acceptors Cy3, Cy3.5, Cy5 with resonance energy transfer induced by the presence of a corresponding one of the miRNAs in solution; from the figure, it can be seen that the fluorescence donor TOTO-1 emits a strong fluorescence signal when no fluorescence energy resonance transfer occurs, when miRNA-155 is contained in the solution, the miRNA-155 and the corresponding probe undergo a hybridization reaction, the energy of the fluorescence donor TOTO-1 embedded in the nucleic acid duplex structure is transferred to the fluorescence acceptor Cy3, and the fluorescence emission signal of Cy3 in the solution can be detected; similarly, when miRNA-182 or miRNA-197 is contained in the solution, the fluorescence emission signal of Cy3.5 or Cy5 in the solution can be detected. FIG. 3B shows fluorescence emission spectra of two or three miRNAs in solution when present in the corresponding solution; it can be seen from the figure that the fluorescence donor TOTO-1 can transfer its energy to two or three fluorescein at the same time, thereby realizing the simultaneous detection of three miRNAs in the solution.
FIGS. 4A, 4B and 4C are graphs of fluorescent signals before correction for fluorescent crosstalk as a function of miRNA concentration; fig. 4A ', 4B ', and 4C ' are graphs of fluorescence signals as a function of miRNA concentration after correction for fluorescence crosstalk using equation 1.
Detailed Description
Reagents and apparatus used in examples 1-3:
the nucleotide sequences of the probe P1' labeled with fluorescein Cy3, the fluorescent probe P1 labeled with fluorescein Cy3, the fluorescent probe P2 labeled with Cy3.5 and the fluorescent probe P3 labeled with Cy3.5, the auxiliary probes L1, L2, L3 and miRNA-155, miRNA-182 and miRNA-197 (Sanghai Sangon Biological Engineering Technology & Services Co. Ltd., Shanghai, China) are shown in Table 2. TOTO-1(Thermo Fisher Scientific, Massachusetts, USA), 1 XPBS without DNA/RNase (Sunshine Biotechnology, Nanjing, China), UV spectrophotometer (Shimadzu UV-2450, Kyoto, Japan), fluorometer (Fluoromax-4, Horiba Jobin Yvon, Japan), centrifuge (Eppendorf, German), quartz cuvette.
The inventors measured the excitation spectrum and emission spectrum of TOTO-1, Cy3, Cy3.5, and Cy5, respectively, using a fluorescence spectrometer, and normalized the obtained spectra to obtain FIG. 2.
As can be seen from FIG. 2, the emission spectrum of the fluorescence donor TOTO-1 and the absorption spectra of the fluorescence acceptors Cy3, Cy3.5, and Cy5 partially overlap each other, and satisfy the basic condition of Fluorescence Resonance Energy Transfer (FRET).
Example 1
(1) Preparation of probes and selection of fluorescence donors
The fluorescein Cy3 labeled nucleic acid probe corresponding to miRNA-155 is P1, the fluorescein Cy3.5 labeled nucleic acid probe corresponding to miRNA-182 is P2, the fluorescein Cy5 labeled nucleic acid probe corresponding to miRNA-197 is P3, the auxiliary probe corresponding to miRNA-155 is L1, the auxiliary probe corresponding to miRNA-182 is L2, the auxiliary probe corresponding to miRNA-197 is L3, and the nucleic acid dye TOTO-1 is selected as a fluorescence donor. Wherein, the nucleic acid sequences of miRNA-155, miRNA-182, miRNA-197, fluorescein labeled nucleic acid probes P1, P2, P3 and auxiliary probes L1, L2 and L3 are shown in Table 1.
TABLE 1
(2) Principle verification
Taking 8 EP tubes, adding 10nM L1, P1' (nucleic acid probe of unlabeled fluorescein Cy 3), miRNA-155 to the first tube, adding 10nM L1, P1 and miRNA-155 to the second tube, adding 10nM L2, P2 and miRNA-182 to the third tube, adding 10nM L3, P3 and miRNA-197 to the fourth tube, adding 10nM L1, P1, miRNA-155 and L2, P2 and miRNA-182 to the fifth tube, adding 10nM L1, P1, miRNA-155 and L3, P3 and miRNA-197 to the sixth tube, adding 10nM L2, P2, miRNA-182 and L3, P3 and miRNA-197 to the seventh tube, and adding 10nM L1, P1, miRNA-155, L2, P2, miRNA-182, L3, miRNA-3 and P-197 to the eighth tube.
Eight tubes of samples were hybridized in 1 XPBS without DNAse/RNAase for 2 hours at 25 ℃. 100nmTOTO-1 is added into the reaction solution respectively, the total reaction volume is 500 mu L, after the reaction is carried out for 1 hour at 25 ℃, a fluorescence emission spectrogram of the solution is detected by exciting at 440nm of a fluorescence instrument.
The results of the experiment are shown in FIG. 3. The first tube sample is made by inserting TOTO-1 into nucleic acid double helix structure and emitting strong fluorescence signal, corresponding to the black curve labeled by TOTO-1 in FIG. 3A; the fluorescence emission spectrum of the second tube sample corresponds to the green curve labeled by Cy3 in FIG. 3A; the fluorescence emission spectrum of the third tube sample corresponds to the orange curve labeled by Cy3.5 in FIG. 3A; the fluorescence emission spectrum of the fourth tube sample corresponds to the red curve labeled by Cy5 in FIG. 3A; the fluorescence emission spectrum of the fifth tube sample corresponds to the black curve in fig. 3B; the fluorescence emission spectrum of the sixth tube sample corresponds to the green curve in fig. 3B; the fluorescence emission spectrum of the seventh tube sample corresponds to the blue curve in fig. 3B; the fluorescence emission spectrum of the eighth tube sample corresponds to the red curve in fig. 3B. As can be seen from fig. 3, when one (corresponding to the four curves in fig. 3A) and two or three mirnas (corresponding to the four curves in fig. 3B) are present in the solution, corresponding fluorescein signals can be detected, and the results indicate that a fluorescence method based on Fluorescence Resonance Energy Transfer (FRET) is feasible for detecting the three mirnas.
(3) Calculating a fluorescence crosstalk correction factor
According to fig. 3A, it is determined that the fluorescence maximum emission wavelengths of fluorescein Cy3, Cy3.5 and Cy5 are the output channels of the respective mirnas, the fluorescence maximum emission signal of the channel corresponding to fluorescein is the target signal, and the signals of the other two fluorescein in the respective channels are the fluorescence crosstalk signals. Then, the target signals in each channel are normalized, and the ratio of the fluorescence crosstalk signal to the target signal in each channel is calculated to obtain a corresponding fluorescence crosstalk correction factor M, where M is a 3 × 3 spectral crosstalk correction matrix, as shown in table 2. Table 2 shows the ratio of the spectral crosstalk intensity of different fluorescein in each detection channel under single wavelength excitation.
TABLE 2
*The bold section is the 3 x 3 spectral crosstalk correction matrix M in equation 1.
(4) Corresponding relation between fluorescent signal and miRNA concentration
Three sets of EP tubes were taken, 9 EP tubes per set. The first set of each EP tube was loaded with 10nM L1, P1, L2, P2, L3 and P3, followed by 0, 0.2, 0.5, 1, 2, 4, 6, 8 or 10nMmiRNA-155 to 9 EP tubes, respectively. The second set of each EP tube was loaded with 10nM L1, P1, L2, P2, L3 and P3, followed by 0, 0.2, 0.5, 1, 2, 4, 6, 8 or 10nMmiRNA-182 to 9 EP tubes, respectively. The third group had 10nM L1, P1, L2, P2, L3 and P3 added to each EP tube, followed by 0, 0.2, 0.5, 1, 2, 4, 6, 8 or 10nM miRNA-197 added to 9 EP tubes, respectively. Three sets of samples were hybridized in 1 XPBS without DNAse/RNAase for 2 hours at 25 ℃. 100nM TOTO-1 was added to the above solutions, respectively, and the total volume of the reaction solution was 500. mu.L, and after reaction at 25 ℃ for 1 hour, the fluorescence emission spectra of the above solutions were measured by excitation at 440nM with a fluorometer. And then, correcting the originally measured fluorescence intensity (I) by using a formula 1, and deducting fluorescence crosstalk background signals of the two fluorescein at the maximum emission wavelength of the third fluorescein to obtain the corrected fluorescence intensity (P) of each fluorescein at the maximum emission wavelength of each fluorescein, so as to obtain the true concentration of the corresponding miRNA.
The results of the experiment are shown in FIG. 4. 4A, 4B and 4C show the change of fluorescence signals before the fluorescence crosstalk correction with the miRNA concentration; from the figure, it can be seen that signal interference between fluorescein is significant; the A ' B ' C ' plot shows fluorescence intensity (P) as a function of miRNA concentration (C) after correction for fluorescence crosstalk using equation 1miRNA) A logarithmic change of (d); it can be seen from the figure that the signal interference between fluorescein is obviously eliminated, the logarithm of the concentration of miRNA between 0.2nM and 10nM and the corresponding fluorescence signal present a good linear relationship, and the corresponding relationship between the corrected fluorescence intensity and the concentrations of three miRNAs is as follows:
P1＝1297.7lnCmiRNA-155+2758.9；
P2＝1453.7lnCmiRNA-182+2748.1；
P3＝788.8lnCmiRNA-197+1240.7。
(5) simultaneous detection of three miRNAs
Three EP tubes were taken and noted as first, second and third tubes. The first tube was added with 10nM of L1, P1, L2, P2, L3, P3 and 0.5nM miRNA-155, 10nM miRNA-182, 10nM miRNA-197; adding 10nM L1, P1, L2, P2, L3, P3, 1nM miRNA-155, 7nM miRNA-182 and 10nM miRNA-197 into the second tube; the third tube was added with 10nM of L1, P1, L2, P2, L3, P3 and 3nM miRNA-155, 3nM miRNA-182, 3nM miRNA-197.
The three tubes of samples were hybridized in 1 XPBS without DNAse/RNAase for 2 hours at 25 ℃. 100nM TOTO-1 was added to the above solutions, respectively, in a total volume of 500. mu.L, and after 1 hour at 25 ℃ the fluorescence emission spectra of the above solutions was detected by excitation at 440nM with a fluorometer. And calculating the original measured fluorescence intensity (I) by using a formula 1 to obtain corrected fluorescence intensity (P), substituting the corrected fluorescence intensity into a corresponding relation between the corrected fluorescence intensity and the concentrations of the three miRNAs to obtain the measured values of the three miRNAs in each tube sample, and comparing the measured values with known addition values to obtain the recovery rates of the three miRNAs in the three tube samples.
Example 2
(1) Preparation of probes and selection of fluorescence donors
See example 1, section (1).
(2) Principle verification
Taking 8 EP tubes, adding 5nM L1, P1' (nucleic acid probe of unlabeled fluorescein Cy 3), miRNA-155 to a first tube, adding 5nM L1, P1 and miRNA-155 to a second tube, adding 5nM L2, P2 and miRNA-182 to a third tube, adding 5nM L3, P3 and miRNA-197 to a fourth tube, adding 5nM L1, P1, miRNA-155 and L2, P2 and miRNA-182 to a fifth tube, adding 5nM L1, P1, miRNA-155 and L3, P3 and miRNA-197 to a sixth tube, adding 5nM L2, P2, miRNA-182 and L3, P3 and miRNA-197 to a seventh tube, and adding 5nM L1, P1, miRNA-155, L2, P2, miRNA-182, L3, miRNA-3 and P-197 to an eighth tube.
Eight tubes of samples were hybridized in 1 XPBS without DNAse/RNAase for 4 hours at 20 ℃. 100nmTOTO-1 is added into the reaction solution respectively, the total reaction volume is 500 mu L, after the reaction is carried out for 2 hours at the temperature of 20 ℃, a fluorescence emission spectrogram of the solution is detected by exciting at the position of 440nm by a fluorescence instrument.
The experimental results show that the fluorescence emission spectrum obtained in this example is the same as that obtained in example 1, and the experimental results are shown in FIG. 3. The first tube sample is made by inserting TOTO-1 into nucleic acid double helix structure and emitting strong fluorescence signal, corresponding to the black curve labeled by TOTO-1 in FIG. 3A; the fluorescence emission spectrum of the second tube sample corresponds to the green curve labeled by Cy3 in FIG. 3A; the fluorescence emission spectrum of the third tube sample corresponds to the orange curve labeled by Cy3.5 in FIG. 3A; the fluorescence emission spectrum of the fourth tube sample corresponds to the red curve labeled by Cy5 in FIG. 3A; the fluorescence emission spectrum of the fifth tube sample corresponds to the black curve in fig. 3B; the fluorescence emission spectrum of the sixth tube sample corresponds to the green curve in fig. 3B; the fluorescence emission spectrum of the seventh tube sample corresponds to the blue curve in fig. 3B; the fluorescence emission spectrum of the eighth tube sample corresponds to the red curve in fig. 3B. As can be seen from fig. 3, when one (corresponding to the four curves in fig. 3A) and two or three mirnas (corresponding to the four curves in fig. 3B) are present in the solution, corresponding fluorescein signals can be detected, and the results indicate that a fluorescence method based on Fluorescence Resonance Energy Transfer (FRET) is feasible for detecting the three mirnas.
(3) Calculating a fluorescence crosstalk correction factor
According to fig. 3A, it is determined that the fluorescence maximum emission wavelengths of fluorescein Cy3, Cy3.5 and Cy5 are the output channels of the respective mirnas, the fluorescence maximum emission signal of the channel corresponding to fluorescein is the target signal, and the signals of the other two fluorescein in the respective channels are the fluorescence crosstalk signals. Then, the target signals in each channel are normalized, and the ratio of the fluorescence crosstalk signal to the target signal in each channel is calculated to obtain a corresponding fluorescence crosstalk correction factor M, where M is a 3 × 3 spectral crosstalk correction matrix, as shown in table 2. Table 2 shows the ratio of the spectral crosstalk intensity of different fluorescein in each detection channel under single wavelength excitation.
(4) Corresponding relation between fluorescent signal and miRNA concentration
Three sets of EP tubes were taken, 9 EP tubes per set. The first set of each EP tube was charged with 5nM L1, P1, L2, P2, L3 and P3, followed by 0, 0.1, 0.2, 0.5, 1, 2, 3, 4 or 5nMmiRNA-155 to 9 EP tubes, respectively. The second set of EP tubes each had 10nM L1, P1, L2, P2, L3 and P3 added to them, followed by 0, 0.1, 0.2, 0.5, 1, 2, 3, 4 or 5nMmiRNA-182 added to 9 EP tubes, respectively. The third group was added 10nM L1, P1, L2, P2, L3 and P3 to each EP tube, followed by 0, 0.1, 0.2, 0.5, 1, 2, 3, 4 or 5nMmiRNA-197 to 9 EP tubes, respectively. Three sets of samples were hybridized in 1 XPBS without DNAse/RNAase for 4 hours at 20 ℃. 100nM TOTO-1 was added to the above solutions, respectively, and the total volume of the reaction solution was 500. mu.L, and after reaction at 20 ℃ for 2 hours, the fluorescence emission spectra of the above solutions were measured by excitation at 440nM with a fluorometer. And then, correcting the originally measured fluorescence intensity (I) by using a formula 1, and deducting fluorescence crosstalk background signals of the two fluorescein at the maximum emission wavelength of the third fluorescein to obtain the corrected fluorescence intensity (P) of each fluorescein at the maximum emission wavelength of each fluorescein, so as to obtain the true concentration of the corresponding miRNA.
The results of the experiment are shown in FIG. 4. 4A, 4B and 4C show the change of fluorescence signals before the fluorescence crosstalk correction with the miRNA concentration; from the figure, it can be seen that signal interference between fluorescein is significant; the A ' B ' C ' plot shows fluorescence intensity (P) as a function of miRNA concentration (C) after correction for fluorescence crosstalk using equation 1miRNA) A logarithmic change of (d); it can be seen from the figure that the signal interference between fluorescein is obviously eliminated, the logarithm of the concentration of miRNA between 0.1nM and 5nM and the corresponding fluorescence signal present a good linear relationship, and the corresponding relationship between the corrected fluorescence intensity and the concentrations of three miRNAs is as follows:
P1＝1297.7lnCmiRNA-155+2758.9；
P2＝1453.7lnCmiRNA-182+2748.1；
P3＝788.8lnCmiRNA-197+1240.7。
(5) simultaneous detection of three miRNAs
And taking 3 EP tubes, and marking as a fourth tube, a fifth tube and a sixth tube respectively. The fourth tube was added with 10nM L1, P1, L2, P2, L3, P3 and 7nM mirAN-155, 1nM miRNA-182, 10nM miRNA-197; the fifth tube was added with 10nM L1, P1, L2, P2, L3, P3 and 10nM miRAN-155, 0.5nM miRNA-182, 10nM miRNA-197; the sixth tube was loaded with 10nM of L1, P1, L2, P2, L3, P3, and 10nM mirAN-155, 1nM miRNA-182, 7nM miRNA-197.
The three tubes of samples were hybridized in 1 XPBS without DNAse/RNAase for 4 hours at 20 ℃. 100nM TOTO-1 was added to the above solutions, respectively, in a total volume of 500. mu.L, and after 2 hours at 20 ℃ the fluorescence emission spectra of the above solutions was detected by excitation at 440nM with a fluorometer. And calculating the original measured fluorescence intensity (I) by using a formula 1 to obtain corrected fluorescence intensity (P), substituting the corrected fluorescence intensity into a corresponding relation between the corrected fluorescence intensity and the concentrations of the three miRNAs to obtain the measured values of the three miRNAs in each tube sample, and comparing the measured values with known addition values to obtain the recovery rates of the three miRNAs in the three tube samples.
Example 3
(1) Preparation of probes and selection of fluorescence donors
See example 1, section (1).
(2) Principle verification procedure
Taking 8 EP tubes, adding 10nM L1, P1' (nucleic acid probe of unlabeled fluorescein Cy 3), miRNA-155 to the first tube, adding 10nM L1, P1 and miRNA-155 to the second tube, adding 10nM L2, P2 and miRNA-182 to the third tube, adding 10nM L3, P3 and miRNA-197 to the fourth tube, adding 10nM L1, P1, miRNA-155 and L2, P2 and miRNA-182 to the fifth tube, adding 10nM L1, P1, miRNA-155 and L3, P3 and miRNA-197 to the sixth tube, adding 10nM L2, P2, miRNA-182 and L3, P3 and miRNA-197 to the seventh tube, and adding 10nM L1, P1, miRNA-155, L2, P2, miRNA-182, L3, miRNA-3 and P-197 to the eighth tube.
Eight tubes of samples were hybridized in 1 XPBS without DNAse/RNAase for 3 hours at 37 ℃. 100nmTOTO-1 is added into the reaction solution respectively, the total reaction volume is 500 mu L, after the reaction is carried out for 1 hour at 37 ℃, a fluorescence emission spectrogram of the solution is detected by exciting at 440nm of a fluorescence instrument.
The experimental results show that the fluorescence emission spectrum obtained in this example is the same as that of example 1, as shown in FIG. 3. The first tube sample is made by inserting TOTO-1 into nucleic acid double helix structure and emitting strong fluorescence signal, corresponding to the black curve labeled by TOTO-1 in FIG. 3A; the fluorescence emission spectrum of the second tube sample corresponds to the green curve labeled by Cy3 in FIG. 3A; the fluorescence emission spectrum of the third tube sample corresponds to the orange curve labeled by Cy3.5 in FIG. 3A; the fluorescence emission spectrum of the fourth tube sample corresponds to the red curve labeled by Cy5 in FIG. 3A; the fluorescence emission spectrum of the fifth tube sample corresponds to the black curve in fig. 3B; the fluorescence emission spectrum of the sixth tube sample corresponds to the green curve in fig. 3B; the fluorescence emission spectrum of the seventh tube sample corresponds to the blue curve in fig. 3B; the fluorescence emission spectrum of the eighth tube sample corresponds to the red curve in fig. 3B. As can be seen from fig. 3, when one (corresponding to the four curves in fig. 3A) and two or three mirnas (corresponding to the four curves in fig. 3B) are present in the solution, corresponding fluorescein signals can be detected, and the results indicate that a fluorescence method based on Fluorescence Resonance Energy Transfer (FRET) is feasible for detecting the three mirnas.
(3) Calculating a fluorescence crosstalk correction factor
According to fig. 3A, it is determined that the fluorescence maximum emission wavelengths of fluorescein Cy3, Cy3.5 and Cy5 are the output channels of the respective mirnas, the fluorescence maximum emission signal of the channel corresponding to fluorescein is the target signal, and the signals of the other two fluorescein in the respective channels are the fluorescence crosstalk signals. Then, the target signals in each channel are normalized, and the ratio of the fluorescence crosstalk signal to the target signal in each channel is calculated to obtain a corresponding fluorescence crosstalk correction factor M, where M is a 3 × 3 spectral crosstalk correction matrix, as shown in table 2. Table 2 shows the ratio of the spectral crosstalk intensity of different fluorescein in each detection channel under single wavelength excitation.
(4) Corresponding relation between fluorescent signal and miRNA concentration
Three sets of EP tubes were taken, 9 EP tubes per set. The first set of each EP tube was loaded with 10nM L1, P1, L2, P2, L3 and P3, followed by 0, 0.2, 0.5, 1, 2, 4, 6, 8 or 10nMmiRNA-155 to 9 EP tubes, respectively. The second set of each EP tube was loaded with 10nM L1, P1, L2, P2, L3 and P3, followed by 0, 0.2, 0.5, 1, 2, 4, 6, 8 or 10nMmiRNA-182 to 9 EP tubes, respectively. The third group had 10nM L1, P1, L2, P2, L3 and P3 added to each EP tube, followed by 0, 0.2, 0.5, 1, 2, 4, 6, 8 or 10nMmiRNA-197 added to 9 EP tubes, respectively. Three sets of samples were hybridized in 1 XPBS without DNAse/RNAase for 3 hours at 37 ℃. 200nM TOTO-1 was added to the above solutions, respectively, in a total volume of 500. mu.L, and after reaction at 37 ℃ for 2 hours, the fluorescence emission spectra of the above solutions were detected by excitation at 440nM with a fluorometer. And then, correcting the originally measured fluorescence intensity (I) by using a formula 1, and deducting fluorescence crosstalk background signals of the two fluorescein at the maximum emission wavelength of the third fluorescein to obtain the corrected fluorescence intensity (P) of each fluorescein at the maximum emission wavelength of each fluorescein, so as to obtain the true concentration of the corresponding miRNA.
The results of the experiment are shown in FIG. 4. 4A, 4B and 4C show the change of fluorescence signals before the fluorescence crosstalk correction with the miRNA concentration; from the figure, it can be seen that signal interference between fluorescein is significant; the A ' B ' C ' plot shows fluorescence intensity (P) as a function of miRNA concentration (C) after correction for fluorescence crosstalk using equation 1miRNA) A logarithmic change of (d); from the figure canSo as to show that the signal interference between fluorescein is obviously eliminated, the logarithm of the concentration of miRNA between 0.2nM and 10nM and the corresponding fluorescence signal present a good linear relationship, and the corresponding relationship between the corrected fluorescence intensity and the concentrations of three miRNAs is as follows:
P1＝1297.7lnCmiRNA-155+2758.9；
P2＝1453.7lnCmiRNA-182+2748.1；
P3＝788.8lnCmiRNA-197+1240.7。
(5) simultaneous detection of three miRNAs
Three EP tubes were taken and were designated as the seventh, eighth and ninth tubes, respectively. The seventh tube was added with 10nM of L1, P1, L2, P2, L3, P3 and 10nM mirAN-155, 3nM miRNA-182, 3nM miRNA-197. The eighth tube was charged with 10nM of L1, P1, L2, P2, L3, P3 and 10nM mirAN-155, 7nM miRNA-182, 1nM miRNA-197. The ninth tube was added with 10nM of L1, P1, L2, P2, L3, P3 and 10nM mirAN-155, 10nM miRNA-182, 0.5nM miRNA-197.
The three tubes of samples were hybridized in 1 XPBS without DNAse/RNAase for 3 hours at 37 ℃. 100nM TOTO-1 was added to the above solutions, respectively, in a total volume of 500. mu.L, and after 1 hour of reaction at 37 ℃, the fluorescence emission spectra of the above solutions were detected by excitation at 440nM with a fluorometer. And calculating the original measured fluorescence intensity (I) by using a formula 1 to obtain corrected fluorescence intensity (P), substituting the corrected fluorescence intensity into a corresponding relation between the corrected fluorescence intensity and the concentrations of the three miRNAs to obtain the measured values of the three miRNAs in each tube sample, and comparing the measured values with known addition values to obtain the recovery rates of the three miRNAs in the three tube samples.
Referring to the data of the first tube to the ninth tube obtained in the (5) th section in examples 1-3, the recovery rate of the sample after the correction of the fluorescence crosstalk is 90% to 109%, and thus, the error of the concentration of the three miRNAs detected by the scheme of the invention is within +/-10%. The result shows that the method can be used for simultaneously detecting three miRNAs.
TABLE 3
The above description is only of the preferred embodiments of the present invention, and it should be noted that: for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications may be made, and the method of the present invention is not limited to the detection of the above three mirnas, but is also applicable to the detection of other mirnas, and these improvements and modifications should be considered as the protection scope of the present invention.
SEQUENCE LISTING
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Claims (7)
1. A method for simultaneously detecting multiple miRNAs by a fluorescence method is characterized by comprising the following steps:
1) respectively marking nucleic acid probes corresponding to the multiple miRNAs by using multiple fluoresceins; selecting as a fluorescence donor a nucleic acid dye that can transfer emission energy to the plurality of luciferin by a fluorescence resonance energy transfer effect;
2) calculating fluorescence crosstalk correction factors of fluorescein corresponding to the multiple miRNAs;
3) respectively taking the multiple miRNA solutions with different known concentrations, hybridizing the multiple miRNAs, the nucleic acid probe and the auxiliary probe to form a nucleic acid double-helix structure, embedding the fluorescence donor into the nucleic acid double-helix structure, exciting the fluorescence donor, detecting fluorescence signals of fluorescein corresponding to the multiple miRNAs, and correcting the fluorescence signals by using the fluorescence crosstalk correction factor to obtain a corresponding relation between the fluorescence intensity of the fluorescein corresponding to the multiple miRNAs and the miRNA concentration after correction;
4) taking a miRNA solution to be detected, hybridizing the miRNA to be detected, the nucleic acid probe and the auxiliary probe to form a nucleic acid double-helix structure, embedding the fluorescence donor into the nucleic acid double-helix structure, exciting the fluorescence donor, detecting fluorescence signals of fluorescein corresponding to the multiple miRNAs, and calculating by using the corresponding relational expression obtained in the step 3) to obtain the concentration of the miRNA;
the multiple miRNAs are miRNA-155, miRNA-182 and miRNA-197 respectively;
the fluorescein is Cy3, Cy3.5 or Cy 5;
the fluorescence donor is nucleic acid dye TOTO-1.
2. The method according to claim 1, wherein the specific method for calculating the fluorescence crosstalk correction factor of fluorescein corresponding to multiple miRNAs in step 2) is as follows: and respectively comparing the fluorescence signal intensity of each fluorescein at the maximum emission wavelength with the fluorescence intensity of all other fluorescein at the wavelength to obtain a fluorescence crosstalk correction factor M.
3. The method according to claim 1, wherein the corrected fluorescence intensity P of fluorescein corresponding to the multiple miRNAs obtained in step 3)1、P2、P3With miRNA concentration CmiRNA-155、CmiRNA-182、CmiRNA-197The corresponding relation is as follows:
P1＝1297.7lnCmiRNA-155+2758.9；
P2＝1453.7lnCmiRNA-182+2748.1；
P3＝788.8lnCmiRNA-197+1240.7；
where, the concentration units are nM.
4. The method according to any one of claims 1 to 3, wherein in step 3), the concentrations of the different known concentrations of the plurality of miRNA solutions are in the range of 0-50 nM, and the concentration of the fluorescence donor is in the range of 100-300 nM.
5. The method according to any one of claims 1 to 3, wherein in the step 3) and the step 4), the hybridization is carried out in a DNAse/RNAase-free phosphate buffer solution at 20 to 37 ℃ for 2 to 4 hours.
6. The method according to any one of claims 1 to 3, wherein the method for intercalating the fluorescence donor into the nucleic acid duplex structure in step 3) and step 4) comprises: and adding the fluorescence donor into a nucleic acid double-helix structure formed by hybridization, and reacting for 1-2 hours at 20-37 ℃.
7. The method according to any one of claims 1 to 3, wherein in step 3) and step 4), the wavelength of the excitation light for exciting the fluorescence donor is 440 nm.
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