CN111665351B

CN111665351B - Method for quickly and specifically determining RNA content

Info

Publication number: CN111665351B
Application number: CN202010569437.2A
Authority: CN
Inventors: 刘延峰; 李江华; 伍业旭; 卢川川
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-06-20
Filing date: 2020-06-20
Publication date: 2021-12-03
Anticipated expiration: 2040-06-20
Also published as: CN111665351A

Abstract

The invention discloses a method for quickly and specifically determining RNA content. The method uses the specific dye SYTO^TMRNASelect^TMStaining of RNAThe fluorescence intensity was then measured. The results show that SYTO^TMRNASelect^TMThe fluorescence intensity observed after the RNA is combined has a good linear relation with the RNA concentration, and the content of the low-concentration RNA can be accurately detected; and the requirement on the purity of RNA is low, so that the interference of other impurities mixed in the RNA extraction process, such as DNA, nucleic acid, phosphate and protein, can be avoided, and the RNA content can be accurately determined.

Description

Method for quickly and specifically determining RNA content

Technical Field

The invention relates to a method for quickly and specifically determining RNA content, belonging to the technical field of biology.

Background

RNA is an important biological molecule responsible for many functions, including physical transmission, interpretation of genetic information, structural support of molecular machinery, and regulation of gene expression. Quantification of RNA in biological samples and living cells has a very important role. The existing RNA detection method comprises an ultraviolet absorption spectrometry and a orcinol colorimetry, wherein the former utilizes the maximum absorption peak of RNA at 260nm to quantify nucleic acid, while the latter utilizes the degradation of ribose into furfural in an acidic environment and utilizes the coloration of orcinol and furfural to quantify furfural, thereby indirectly quantifying RNA. In addition, the phosphorus determination method is also frequently used for detection of nucleic acid substances, and quantification of nucleic acid substances is mainly achieved by a color reaction between phosphorus in nucleic acids and molybdic acid. However, these methods cannot avoid the interference of other substances in the biological sample, such as protein and DNA substances which have large interference on the determination of RNA by ultraviolet absorption method, ribose substances which have interference on the detection by the lichen phenol method and other phosphorus-containing substances such as phosphate on the phosphorus determination method, and the like.

In recent years, fluorescence measurement of nucleic acids has been widely developed due to the advantages of high sensitivity, rapid detection, and high throughput of fluorescence. The most commonly used fluorescent dye for nucleic acid detection is Hochest 33258, but Hochest 33258 is a non-specific dye capable of simultaneously binding DNA and RNA, so when a sample to be detected contains DNA, RNA is degraded by RNase, the DNA in the sample is quantified, and then the DNA content in the sample is subtracted during RNA quantification, so the method has the defects of complicated and time-consuming operation process and increased error in experiments.

Therefore, it is necessary to develop a method for rapidly and specifically determining the RNA content, which eliminates the interference of impurities in the RNA quantification process, improves the RNA quantification accuracy, simplifies the operation and improves the efficiency.

Disclosure of Invention

In view of the above, the present invention is to overcome the shortcomings of the prior art and provide a method for rapidly and specifically determining the content of RNA.

In order to solve the technical problem, the invention adopts the following scheme:

the invention provides a method for determining RNA content, which comprises the steps of mixing and incubating an RNA specific dye and an RNA sample, and calculating the RNA content by determining fluorescence intensity.

In one embodiment of the present invention, the RNA sample further contains DNA, phenols, alcohols, and proteins.

In one embodiment of the invention, the RNA content in the RNA sample is at least 2. mu.g/mL

In one embodiment of the invention, the RNA specific dye is SYTO^TMRNASelect^TM Green Fluorescent cell Stain。

In one embodiment of the present invention, the concentration of the RNA specific dye is 5 to 10. mu. mol/L.

In one embodiment of the invention, fluorescence intensity is measured by using a microplate reader under the conditions of excitation wavelength of 400-500 nm and emission wavelength of 500-600 nm.

In one embodiment of the present invention, the incubation time is 2-7 min.

In one embodiment of the invention, the total time is determined to be not more than 10 min.

The invention provides a method for specifically determining RNA, which comprises the steps of adding an RNA specific dye into a sample to be determined, and calculating the content of RNA by using fluorescence intensity; detecting fluorescence under the conditions of excitation wavelength of 400-500 nm and emission wavelength of 500-600 nm; the detection result has fluorescence reaction, namely the sample contains RNA.

In one embodiment of the invention, the RNA sample further comprises DNA, ribose, phosphate and/or protein.

The RNA sample also contains DNA, ribose, phosphate and/or protein.

In one embodiment of the present invention, the concentration of the DNA is not less than 2. mu.g/mL.

In one embodiment of the invention, the ribose concentration is not less than 0.6 μ g/mL; the concentration of the phosphate is not lower than 0.6 mu g/mL; the concentration of the protein is not lower than 0.6 mu g/mL.

The invention also protects the application of the method in the determination of RNA.

The invention has the advantages that: the method for detecting RNA provided by the invention can be realized by SYTO^TMRNASelect^TMAnd (3) mixing the dye with the sample, rapidly measuring the fluorescence intensity by using an enzyme-labeling instrument, and obtaining the corresponding RNA content according to a standard curve, wherein the method is rapid, simple and convenient. The requirement on the purity of RNA is low, the RNA can be specifically combined with the RNA in an environment containing various impurities, and the content of the RNA can be accurately determined; and the content of RNA can be accurately determined even at a low concentration (2. mu.g/mL). And the detection time is short, and the method has important significance for quickly and specifically determining the RNA content.

Drawings

FIG. 1 is a schematic flow chart of the detection method of the present invention.

SYTO in FIG. 2^TM RNASelect^TMAnd detecting the fluorescence intensity of the RNA at different concentrations.

FIG. 3 is a standard curve of fluorescence intensity versus RNA concentration.

SYTO in FIG. 4^TM RNASelect^TMAnd (4) performing fluorescence quantification on different RNA samples compared with Hoechst 33255.

FIG. 5 shows the result of RNA detection of samples containing other impurities.

FIG. 6 shows UV spectrophotometry, lichenin method and SYTO^TM RNASelect^TMThe method detects the comparative result of the RNA content.

Detailed Description

Materials and equipment

RNA standard (sigma): yeast single-stranded rRNA

DNA standard (sigma): salmon sperm DNA

PBS buffer (1L): NaCl 8g, KCl 0.2g, Na₂HPO₄ 1.44g,KH₂PO₄ 0.24g

RNA dye (ThermoFisher): SYTO^TM RNASelect^TM Green Fluorescent cell Stain

RNA dye (sigma) Hoechst33258

Enzyme-labeling instrument SynergyH4/Elx800(BioTek)

Black flat-bottom microplate (96 wells) (Corning 3603)

Second, standard curve making

1. Accurately weighing the RNA standard substance, diluting with ultrapure water to a proper concentration, and preparing the RNA standard solution with a specific concentration.

2. Respectively absorbing different volumes of RNA stock solutions, adding the RNA stock solutions into a microplate, and then complementing the RNA stock solutions to 100uL with deionized water, wherein each gradient is 2 in parallel.

3、SYTO^TM RNASelect^TMGreen Fluorescent Cell Stain is diluted by PBS solution until the concentration is proper, and a certain volume of staining solution is sucked into a pore plate to be mixed with the sample.

4. The reaction system was left to stand in the dark for 5min in the dark.

Example 1: SYTO^TMRNASelect^TMOptimum concentration exploration

1. SYTO buffer with PBS^TMRNASelect^TMDiluting by 500, 1000, 2000 and 5000 times respectively to prepare dye solutions with the concentration of 10 mu mol/L, 5 mu mol/L, 2.5 mu mol/L and 1 mu mol/L of stock solution respectively, and standing at 4 ℃ for later use.

2. Accurately weighing 10mg of RNA standard, and making the volume constant to 100mL to obtain 100mg/L RNA standard solution.

3.

Pipette

2, 5, 10, 15, 20, 25, 30, 35, 40, 50 μ L of RNA standard solution into a black 96 microwell plate, add deionized water, make up to 100 μ L, two replicates per gradient.

4. And (3) sucking 100 mu L of dye solutions with different concentrations prepared in the step 1, respectively mixing the dye solutions with the RNA samples, incubating the mixture for 5min in a dark place, placing the mixture in a microplate reader SynergyH4/Elx800(BioTek) and measuring fluorescence by using ultrapure water as a control, wherein the exciting light is 490nm, and the emitted light is 530 nm.

5. The blank was subtracted from the measured fluorescence intensity and corrected data was used to linearly fit the RNA concentration to the fluorescence intensity (fig. 2).

As shown, with SYTO^TM RNASelect^TMThe linear relationship between the RNA concentration and the fluorescence intensity gradually increases when the concentration increases, when SYTO^TM RNASelect^TMAt a concentration of 10. mu. mol/L, the linear relationship is good, R²0.9965. The concentration of the dye is optimally 10 mu mol/L by combining the experimental cost and the linear relation result.

Example 2: standard curve of RNA concentration-fluorescence intensity

1. Accurately weighing 10mg of RNA standard, and making the volume constant to 100mL to obtain 100mg/L RNA standard solution.

2.

Pipetting

2, 5, 10, 15, 20, 25, 30, 35, 40 and 50 mu L of RNA standard solution into a black 96 micro-well plate respectively, adding deionized water to make up 100 mu L, and preparing into 2, 5, 10, 15, 20, 25, 30, 35, 40 and 50 mu g/mL of RNA solution respectively, wherein each gradient is parallel.

3. SYTO buffer with PBS^TM RNASelect^TMGreen Fluorescent Cell Stain was diluted to 10. mu. mol/L and 100. mu.L of diluted Stain was added to each well.

4. After incubating the 96-well plate for 5min in the dark, the plate was placed in a microplate reader SynergyH4/Elx800(BioTek) and fluorescence was measured with ultra pure water as a control, wherein excitation light was 490nm and emission light was 530 nm.

5. The blank was subtracted from the measured fluorescence intensity and a standard curve was calculated using the corrected fluorescence intensity and the corresponding RNA concentration (fig. 3).

As can be seen from the figure, the RNA concentration is in the range of 2-40 mug/mL, and the RNA concentration has good linear relation with the corresponding fluorescence intensity (R)²0.9965), this is illustrated by SYTO^TMRNASelect^TMCan more accurately quantify RNA.

Example 3: determination of RNA concentration in samples containing DNA interference and verification of SYTO^TMRNASelect^TMIs a specificity of

1. Preparation of RNA sample containing DNA interference:

the corresponding DNA samples were added to the RNA standard to prepare RNA samples containing 10mg/mL, 20mg/mL, 30mg/mL, 50mg/mL, and 100mg/mL of DNA, respectively, and the RNA samples were dissolved in deionized water to give 100mg/L of RNA sample concentration.

2. Preparation of DNA standard solution:

accurately weighing 10mg of DNA standard substance, and making the DNA standard substance into 100mL of deionized water to prepare 100mg/L of DNA standard solution.

3. The RNA sample is prepared into 2, 5, 10, 15, 20, 25, 30, 35, 40 and 50 mu g/mL RNA solution according to the method in the example 1;

configuring an RNA sample containing DNA interference into RNA solutions of 2, 5, 10, 15, 20, 25, 30, 35, 40 and 50 mu g/mL, wherein the concentrations of DNA in the solutions are respectively as follows:

TABLE 1 concentration of DNA in RNA samples at different concentrations

Preparing a DNA sample into a DNA solution of 2, 5, 10, 15, 20, 25, 30, 35, 40 and 50 mu g/mL;

the samples were mixed with 10. mu. mol/L SYTO^TMRNASelect^TMMixed and the corresponding fluorescence intensity was measured (fig. 4A).

As shown in FIG. 4A, to verify SYTO^TMRNASelect^TMWas mixed with the DNA sample, the maximum fluorescence intensity measured was only 2.9% of 40. mu.g/mL RNA, and the fluorescence intensity did not increase with increasing DNA concentration, indicating that SYTO^TMRNASelect^TMThe specificity is high.

In addition, DNA with different concentrations is mixed in the RNA sample, and then the fluorescence intensity and the RNA concentration are fitted, and the fitted curve is found to have extremely high similarity, which indicates that the method has little influence on the quantitative result of the RNA sample containing the DNA.

Comparative example 1

See example 3 for details, except that SYTO^TMRNASelect^TMReplacement was with Hoechst 33258.

Hoechst33258 was diluted to 15. mu. mol/L in PBS buffer according to SYTO^TMRNASelect^TMIn the same manner, the fluorescence value is measured at 350nm/461nm after incubation with RNA sample, RNA sample containing DNA interference, and DNA sample.

As shown in FIG. 4(B), when different RNA samples were quantified using Hoechst33258, it was found that the curve fit was greatly changed when RNA samples were mixed with DNA at different concentrations, and the curve difference between different samples was large, indicating that DNA had a large influence on the experimental results when RNA was quantified using Hoechst 33258.

As can be seen from a comparison of the two graphs of FIG. 4A and FIG. 4B, SYTO^TMRNASelect^TMThe method has high specificity and sensitivity for RNA quantification, can quickly detect RNA without treating a sample with DNase or RNase, can finish the whole detection process within 10min, has high flux and greatly improves RNA quantification efficiency.

Example 4: determination of RNA concentration in samples containing multiple interferences and verification of SYTO^TM RNASelect^TMIs a specificity of

1. Preparing an RNA sample containing DNA, polysaccharide, protein, alcohol and phenolic substance interference:

ribose, protein, and phosphate were added to the RNA solution in the same manner as in example 2 to prepare 2, 5, 10, 15, 20, 25, 30, 35, 40, 50 μ g/mL RNA solutions, in which the protein concentration was 0.6, 1.5, 3, 4.5, 6, 7.5, 9, 10.5, 12, and 15 μ g/mL, the ribose concentration was 0.6, 1.5, 3, 4.5, 6, 7.5, 9, 10.5, 12, and 15 μ g/mL, and the potassium dihydrogen phosphate concentration was 0.6, 1.5, 3, 4.5, 6, 7.5, 9, 10.5, 12, and 15 μ g/mL, respectively.

2. Mixing the above sample with 10. mu. mol/L SYTO^TMRNASelect^TMMixed and the corresponding fluorescence intensity is measured.

The results are shown in FIG. 5 for the samplesAfter adding protein, ribose and phosphate, the measured curve is highly coincident with the curve of the pure RNA sample, which shows that the protein, ribose and phosphate are in pair SYTO^TMRNASelect^TMThe results of RNA quantification did not have much impact. However, ribose has larger interference on RNA determination of orcinol, and phosphate has larger interference on RNA determination by a phosphate method. SYTO can be seen from this^TMRNASelect^TMThe quantitative RNA has higher specificity.

Comparative example 2

Referring to example 4, the difference is that 2. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL of the LRNA sample was measured using a UV spectrophotometer nanodrop 2000, and the results are shown in FIG. 6. At an RNA concentration of 2. mu.g/mL, the UV spectrophotometer measurement is an absorbance of less than 0.1, making the calculated RNA concentration error larger, but SYTO^TMRNASelect^TMThe measured concentration does not differ much from the actual concentration. SYTO at RNA concentrations of 10. mu.g/mL and 40. mu.g/mL^TMRNASelect^TMThe concentration of the RNA is not greatly different from that of the RNA measured by an ultraviolet spectrophotometer.

Comparative example 3

Referring to example 4, except that the result of the lichenin method is shown in FIG. 6, the results are shown in FIG. 6, and the results are shown in FIG. 6 for 2. mu.g/mL, 20. mu.g/mL, and 40. mu.g/mL of the LRNA sample. The result of orcinol assay was more erratic at RNA concentrations of 2. mu.g/mL and SYTO at RNA concentrations of 20. mu.g/mL and 40. mu.g/mL^TMRNASelect^TMThe result is not very different from the result measured by orcinol.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for specifically determining RNA is characterized in that an RNA specific dye is added into a sample to be determined, the RNA specific dye and the sample to be determined are mixed and incubated, and the content of the RNA is calculated by determining the fluorescence intensity; detecting fluorescence under the conditions of excitation wavelength of 400-500 nm and emission wavelength of 500-600 nm; the detection result has fluorescence reaction, namely the sample contains RNA; the RNA sample also contains DNA, ribose, phosphate and/or protein; the RNA specific dye is SYTO (ribonucleic acid) cell Stain with a Green Fluorescent cell Stain; the RNA concentration in the RNA sample is 2-40 mug/mL; the concentration of the RNA specific dye was 10. mu. mol/L.

2. The method according to claim 1, wherein the incubation time is 2-7 min.

3. The method according to claim 1 or 2, characterized in that the total time determined does not exceed 10 min.

4. The method according to claim 3, wherein the concentration of the DNA is not less than 2. mu.g/mL.

5. The method of claim 4, wherein the concentration of ribose is not less than 0.6 μ g/mL; the concentration of the phosphate is not lower than 0.6 mu g/mL; the concentration of the protein is not lower than 0.6 mu g/mL.

6. Use of the method of any one of claims 1 to 5 for the determination of RNA.