A method for detecting the stability of siRNA in biological matrix

Abstract

The present invention belongs to the field of SiRNA detection, and provides a method for detecting the stability of SiRNA in a biological matrix, comprising the following steps: extracting the biological matrix with phenol and deionized water, washing with dichloromethane, obtaining a pretreated biological matrix, and then separating and analyzing the positive and antisense strands of SiRNA in the pretreated biological matrix by liquid chromatography-mass spectrometry, selecting the chromatogram peak area of ​​a certain mass-to-charge ratio of the positive and antisense strands in the spectrum for relative comparison. The use of this method can improve the recovery rate of SiRNA drug extraction in plasma samples in biological matrices, and the interference of SiRNA drugs is solved by using the accuracy of high resolution. The method of the present invention does not need to optimize the mass spectrometry method, has little interference, is qualitative and quantitative in one step, is simple and fast to operate, and has high sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of SiRNA detection and analysis, and in particular relates to a method for high-precision qualitative and quantitative measurement of SiRNA stability in a biological matrix. Background Art

[0002] SiRNA is usually a double-stranded RNA fragment containing 19-23 base pairs, which can be covalently coupled with a carrier to specifically target the diseased tissue to exert a gene silencing effect. After SiRNA enters the cell, the AgO2 enzyme in the cytoplasm will cleave the sense strand of the siRNA, and the antisense strand will be loaded into the RNA-induced silencing complex (RISC), which specifically binds to the target mRNA to degrade it, thereby inhibiting protein expression. There are many papers on SiRNA research, and the determination of the stability of SiRNA in plasma is a basic prerequisite for its continued in-depth research. At present, the main analytical methods for SiRNA are ultraviolet chromatography and triple quadrupole mass spectrometry, but the above methods generally have the disadvantages of low resolution and large interference. Therefore, a highly sensitive, high-throughput and reliable analytical method has been developed, which is of great help to the subsequent research of SiRNA. Summary of the invention

[0003] The purpose of the present invention is to provide a method for determining the stability of siRNA in a biological matrix, which is simple and rapid to operate, has accurate measurement and high sensitivity.

[0004] The technical solution provided by the present invention is as follows:

[0005] A method for detecting the stability of SiRNA in a biological matrix comprises the following steps: extracting the biological matrix by liquid-liquid extraction with phenol and deionized water, washing with dichloromethane to obtain a pretreated biological matrix, and then separating and analyzing the sense strand and antisense strand of SiRNA in the pretreated biological matrix by liquid chromatography-mass spectrometry, and selecting the chromatogram peak area of ​​a certain mass-to-charge ratio of the sense strand and the antisense strand in the spectrum for relative comparison.

[0006] Furthermore, the biological matrix is ​​one of plasma, serum and whole blood.

[0007] Furthermore, the pretreatment step includes: adding the biological matrix to a water-saturated phenol solution and shaking vigorously, then adding deionized water to mix, aspirating the supernatant after the first centrifugation, adding an organic solvent for washing, mixing and performing a second centrifugation to obtain the pretreated biological matrix.

[0008] Furthermore, in the liquid chromatography-mass spectrometry method, the first mobile phase is water containing 0.210wt% hexafluoroisopropanol, 0.175wt% N,N-diisopropylethylamine and 10 µM ethylenediaminetetraacetic acid, and the second mobile phase is methanol containing 0.210wt% hexafluoroisopropanol, 0.175wt% N,N-diisopropylethylamine and 10 µM ethylenediaminetetraacetic acid, and the two mobile phases are driven by their respective pumps and merged into the injector in front of the chromatographic column.

[0009] Furthermore, the volume ratio of the biological matrix, the water-saturated phenol solution and the deionized water is 1:1~3:3~5.

[0010] Furthermore, the volume ratio of the biological matrix, the water-saturated phenol solution and the deionized water is 1:2:4.

[0011] Furthermore, the rotation speeds of the first centrifugation and the second centrifugation are both 12000-14000 rpm, and the centrifugation time is both 8-12 min.

[0012] Furthermore, the organic solvent is dichloromethane.

[0013] Furthermore, the volume ratio of the supernatant after the first centrifugation to the organic solvent is 1:2-4.

[0014] Furthermore, the volume ratio of the supernatant after the first centrifugation to the organic solvent is 1:3.

[0015] Beneficial Effects

[0016] This method uses a simple and rapid liquid-liquid extraction for pretreatment to remove proteins that interfere with SiRNA determination, removes phenol that interferes with SiRNA analysis through washing, and uses a chromatographic column to separate the positive and antisense strands of SiRNA, making the measurement results more accurate.

[0017] The present invention is a new method for determining the stability of siRNA in a biological matrix. The method does not require the optimization of a mass spectrometry method, has little interference, can be qualitatively and quantitatively determined in one step, is simple and fast to operate, and has high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the TIC mass spectrum of SiRNA in the biological matrix determined in Example 1 (based on high-resolution quadrupole time-of-flight mass spectrometer analysis, RT 2.06: internal standard RBD7022 sense strand; RT 2.16: SiRNA drug antisense strand; RT 2.29: SiRNASiRNA sense strand; RT 2.38: internal standard RBD7022 antisense strand);

[0019] Figure 2is the primary mass spectrum of the positive strand of siRNA in the biological matrix determined in Example 1 (based on high-resolution quadrupole time-of-flight mass spectrometer analysis);

[0020] Figure 3 is the primary mass spectrum of the antisense strand of siRNA in the biological matrix determined in Example 1 (based on high-resolution quadrupole time-of-flight mass spectrometer analysis);

[0021] Figure 4 is a chromatogram of the positive strand of siRNA in the biological matrix determined in Example 1 (based on high-resolution quadrupole time-of-flight mass spectrometer analysis);

[0022] Figure 5 4 is a chromatogram of the antisense strand of SiRNA in the biological matrix determined in Example 1 (based on high-resolution quadrupole time-of-flight mass spectrometry analysis). DETAILED DESCRIPTION

[0023] During the exploration process, the high-resolution characteristics were studied emphatically, and the resolution was increased to one hundred thousandth, which completely solved the interference problem, identified SiRNA in one step, and quantified it at the same time. The present invention is further described below in conjunction with the accompanying drawings. The following examples are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the scope of protection of the present invention.

[0024] The reagents and instruments used in the present invention are all common in the market and can be purchased from the market. Some of the reagents used are shown in Table 1.

[0025] Table 1 Information on some reagents used

[0026]

[0027] Example 1

[0028] Liquid-liquid extraction method to extract the sample

[0029] First, 200 µL of water-saturated phenol solution (containing 2 μg / mL RBD7022 as an internal standard solution) was added to 100 μL of plasma sample, and the mixture was shaken vigorously for 30 s. 400 µL of deionized water was added, mixed, and centrifuged at 13,000 rpm for 10 min. 300 µL of supernatant was quantitatively aspirated, and 900 µL of dichloromethane was added for washing to remove phenol that interfered with SiRNA analysis. The mixture was mixed, centrifuged at 13,000 rpm for 10 min, and 200 µL was quantitatively aspirated and transferred to the LC-MS instrument for qualitative and quantitative analysis.

[0030] Example 2

[0031] UPLC-HRMS quantitative analysis

[0032] The sample obtained after pretreatment was transferred to the chromatographic column, and the chromatographic column was used to separate the positive and antisense strands of SiRNA at high temperature, and finally entered the mass spectrometer for analysis (injection volume 10 μL). The first mobile phase was water containing 0.210% hexafluoroisopropanol + 0.175% N,N-diisopropylethylamine + 10 µM ethylenediaminetetraacetic acid, and the second mobile phase was methanol containing 0.210% hexafluoroisopropanol + 0.175% N,N-diisopropylethylamine + 10 µM ethylenediaminetetraacetic acid, and the two mobile phases were driven by their respective pumps to flow into the chromatographic column injector, and finally injected into the high-resolution quadrupole time-of-flight mass spectrometer. The model of the instrument used in this qualitative and quantitative analysis is ACQUITY PREMER ultra-high performance liquid phase system + Xevo G3 mass spectrometer.

[0033] More specifically, the liquid phase conditions are:

[0034] Column: ACQUITY UPLC® BEH C18 1.7 μm 2.1 mm × 50 mm;

[0035] Table 2 Elution gradient

[0036]

[0037] Table 3 Mass spectrometry parameters (different instrument conditions have appropriate changes)

[0038]

[0039] The experimental results are shown in Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 .

[0040] Figure 1 The TIC mass spectrum of the sample obtained by the liquid-liquid extraction treatment method in Example 1, Figure 2 , Figure 3 is the primary mass spectrum of the sense chain and the antisense chain, Figure 4 The chromatogram is in the range of 829.2465-829.2537 for the mass-to-charge ratio of the positive chain. Figure 5 The chromatogram of the positive chain mass-to-charge ratio range is 776.7667-776.7686. Figure 1 It can be seen that the sense strand and the antisense strand can be completely separated. Figure 2 , Figure 3 It can be seen that there is no interference between the positive and antisense chains, and the resolution can reach one in 100,000, which can accurately determine the charge number and molecular weight. Figure 4 , Figure 5The chromatograms of a certain mass-to-charge ratio range of the sense chain and the antisense chain can be seen, and the high sensitivity and high resolution provide a high guarantee for the stability detection of plasma samples. This method uses a variety of volatile reagents during the experiment. From the perspective of personnel health, the present invention has stronger practicality.

[0041] In summary, the present invention is a new method for determining the stability of siRNA in a biological matrix. The method does not require optimization of the mass spectrometry method, has little interference, and can be qualitatively and quantitatively determined in one step. The operation is simple, fast, and highly sensitive.

[0042] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting the stability of siRNA in a biological matrix, characterized in that: The following steps are involved: The biological matrix was extracted with phenol and deionized water and washed with dichloromethane to obtain the pretreated biological matrix. Then, liquid chromatography-mass spectrometry was used to separate and analyze the sense chain and antisense chain of SiRNA in the pretreated biological matrix, and the chromatogram peak area of ​​a certain mass-to-charge ratio of the sense chain and the antisense chain in the spectrum was selected for relative comparison.

2. The method for detecting the stability of siRNA in a biological matrix according to claim 1, characterized in that: The biological matrix is ​​one of plasma, serum and whole blood.

3. The method for detecting the stability of siRNA in a biological matrix according to claim 1, characterized in that: The pretreatment step includes: adding the biological matrix to a water-saturated phenol solution and shaking vigorously, then adding deionized water to mix, aspirating the supernatant after the first centrifugation, adding an organic solvent for washing, mixing and performing a second centrifugation to obtain the pretreated biological matrix.

4. The method for detecting the stability of siRNA in a biological matrix according to claim 1, characterized in that: In the liquid chromatography-mass spectrometry method, the first mobile phase is water containing 0.210wt% hexafluoroisopropanol, 0.175wt% N,N-diisopropylethylamine and 10 μM ethylenediaminetetraacetic acid, and the second mobile phase is methanol containing 0.210wt% hexafluoroisopropanol, 0.175wt% N,N-diisopropylethylamine and 10 μM ethylenediaminetetraacetic acid, and the two mobile phases are driven by their respective pumps to flow into the injector in front of the chromatographic column.

5. The method for detecting the stability of siRNA in a biological matrix according to claim 3, characterized in that: The volume ratio of the biological matrix, water-saturated phenol solution and deionized water is 1:1~3:3~5.

6. The method for detecting the stability of siRNA in a biological matrix according to claim 3, characterized in that: The volume ratio of biological matrix, water-saturated phenol solution and deionized water was 1:2:

4.

7. The method for detecting the stability of siRNA in a biological matrix according to claim 3, characterized in that: The rotation speeds of the first centrifugation and the second centrifugation are both 12000-14000 rpm, and the centrifugation time is both 8-12 min.

8. The method for detecting the stability of siRNA in a biological matrix according to claim 3, characterized in that: The organic solvent is dichloromethane.

9. The method for detecting the stability of siRNA in a biological matrix according to claim 3, characterized in that: The volume ratio of the supernatant and the organic solvent after the first centrifugation is 1:2~4.

10. The method for detecting the stability of siRNA in a biological matrix according to claim 3, characterized in that: The volume ratio of the supernatant after the first centrifugation to the organic solvent was 1:3.

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