RNA extraction kit based on paramagnetic particle method and automatic extraction method

Abstract

The invention discloses an RNA extraction kit based on a paramagnetic particle method and an automatic extraction method, and belongs to the technical field of RNA extraction. According to the present invention, after the auxiliary agent is added to the DNase I buffer solution in the RNA extraction kit, the activity of the DNase I can be effectively maintained so as to completely degrade the gDNA; meanwhile, the auxiliary agent can enable the RNA to be always in a combined state with the magnetic beads in the process of digesting the DNA by the DNase I, other reagents are not needed to be added to enable the RNA to be combined with the magnetic beads again, and the RNA and the magnetic beads can directly enter subsequent steps together, so that the full-automatic extraction process of the RNA is completed; in addition, the auxiliary agent can also significantly improve the extraction yield of RNA of a low nucleic acid sample, so that the RNA extraction kit has a good application prospect in automatic extraction of RNA.

Description

Technical Field

[0001] The present invention belongs to the technical field of RNA extraction, and particularly relates to an RNA extraction kit based on magnetic bead method and an automated extraction method. Background Art

[0002] RNA is a kind of biological macromolecule existing in biological cells, as well as in some viruses and viroids. Similar to the function of DNA, RNA is also an important carrier of biological genetic and developmental information. In life science research, gene expression analysis, genetic information research, transcriptome sequencing, etc. can be carried out through RNA molecules to further deepen the understanding of the relevant mechanisms of organism inheritance, development, growth, etc.; in medical diagnosis, the pathogenic information can be accurately located by detecting or sequencing specific RNA molecular sequences, so as to achieve early detection, control or precise treatment of related diseases. And obtaining high-quality RNA molecules from various samples is an essential step for the above research. Along with the development of life science technology and the expansion of the diagnostic market, requirements for high-throughput, automation, simple and fast operation, and safe and non-toxic reagents are also put forward for RNA extraction.

[0003] Currently, the main RNA extraction techniques include: solution extraction methods represented by the TRIZOL method and the CTAB method, and kit methods using silica membrane columns and magnetic beads as purification carriers. Among them, the Trizol method and the CTAB method are both classic RNA extraction methods. They use phenol, guanidine isothiocyanate or strong surfactants to lyse samples to release nucleic acids, and after phase separation with chloroform, alcohol precipitation and centrifugation are used to obtain purified RNA. This method has the advantages of high sample adaptability, wide application, low cost, etc., but at the same time, there are also problems such as toxic reagents, difficulty in high-throughput automated extraction, relatively high risk of genomic DNA residue, and large influence of repeatability by operators. And although the existing magnetic bead method kit can be combined with an automatic extractor and special consumables to achieve high-throughput nucleic acid extraction, in general, RNA extraction kits not based on the Trizol method obtain total nucleic acid first and then use DNase I to digest DNA to obtain purified RNA. Because the currently widely used DNase I buffer is in an aqueous environment, nucleic acids will dissolve in it after contacting with it, which leads to the need to pause the extraction procedure after complete DNA digestion in the current market kits, and the experimenter needs to manually add reagents to make RNA re-bind to magnetic beads before entering the subsequent reaction steps. This operation brings a certain risk of environmental and sample contamination on the one hand, and reduces the simplicity of the RNA extraction process on the other hand.

[0004] Therefore, it is necessary to provide an RNA extraction kit based on magnetic bead method and an automated extraction method to solve the deficiencies in the prior art. Summary of the Invention

[0005] The object of the present invention is to provide an RNA extraction kit based on the magnetic bead method and an automated extraction method. In the present invention, by optimizing the components of the DNase I buffer in the RNA extraction kit, when using the RNA extraction kit to extract sample RNA, the RNA remains bound to the magnetic beads throughout the process of DNase I digesting DNA, without the need to add other reagents to re-bind the RNA to the magnetic beads. The RNA can directly enter the subsequent steps together with the magnetic beads, thereby completing the full-automatic extraction process of RNA. Moreover, the RNA extracted using this RNA extraction kit has good concentration, purity, and integrity.

[0006] In a first aspect, the present invention provides an RNA extraction kit based on the magnetic bead method, comprising a lysis solution, a binding solution, a magnetic bead suspension, a washing solution, a DNase I buffer, DNase I, and an elution solution; wherein, the pH value of the DNase I buffer is 5-9, and it comprises the following components: 5-50 mM of tris(hydroxymethyl)aminomethane, 0.5-5 mM of calcium chloride, 10-100 mM of magnesium chloride, and 25-200 mg / mL of an auxiliary agent, and the auxiliary agent comprises at least one of PEG, mannitol, and trehalose.

[0007] In the present invention, the inventors have found through research that after adding an auxiliary agent to the DNase I buffer, it can effectively maintain the activity of DNase I, enabling the complete degradation of gDNA; at the same time, the auxiliary agent can also keep the RNA bound to the magnetic beads throughout the process of DNase I digesting DNA, without the need to add other reagents to re-bind the RNA to the magnetic beads. The RNA can directly enter the subsequent steps together with the magnetic beads, thereby completing the full-automatic extraction process of RNA; in addition, the auxiliary agent can also significantly improve the extraction yield of RNA from low-nucleic acid samples.

[0008] In some preferred embodiments, the pH value of the DNase I buffer is 7-9, and it comprises the following components: 25 mM of tris(hydroxymethyl)aminomethane, 1 mM of calcium chloride, 20 mM of magnesium chloride, 100 mg / mL of an auxiliary agent, and the auxiliary agent comprises at least one of PEG, mannitol, and trehalose.

[0009] In some embodiments, the molecular weight of PEG is 800-8000.

[0010] In the present invention, the inventors have further found through research that when the molecular weight of PEG is within a specific range, RNA with higher purity and concentration can be extracted.

[0011] In some preferred embodiments, the molecular weight of PEG is 8000.

[0012] In some embodiments, the lysis solution comprises the following components: 10 - 100 mM tris(hydroxymethyl)aminomethane, 1 - 5 M guanidine isothiocyanate, 1 - 10 mg / mL sodium dodecyl sulfate, 5 - 20 mg / mL sodium lauroyl sarcosinate, 10 - 100 mM sodium citrate, 5 - 20 mM disodium ethylenediaminetetraacetate, 10 - 200 mM thiourea, and 1 - 3% (v / v) Triton-X-100.

[0013] It is understandable that the lysis solution can also be a conventional lysis solution in the prior art according to actual usage requirements, as long as it can lyse the sample to be extracted and release nucleic acids.

[0014] In some preferred embodiments, the lysis solution comprises the following components: 100 mM tris(hydroxymethyl)aminomethane, 4 M guanidine isothiocyanate, 2 mg / mL sodium dodecyl sulfate, 10 mg / mL sodium lauroyl sarcosinate, 50 mM sodium citrate, 10 mM disodium ethylenediaminetetraacetate, 100 mM thiourea, and 1% (v / v) Triton-X-100.

[0015] In some embodiments, the binding solution comprises the following components: an alcohol substance at 60 - 100% (v / v); wherein, the alcohol substance includes at least one of isopropanol and absolute ethanol.

[0016] It is understandable that the binding solution can also be a conventional binding solution in the prior art according to actual usage requirements, as long as it can promote the binding of nucleic acids to magnetic beads.

[0017] In some preferred embodiments, the binding solution comprises the following components: 90% (v / v) isopropanol.

[0018] In some embodiments, the concentration of magnetic beads in the magnetic bead suspension is 30 - 100 mg / mL, the magnetic beads include silica magnetic beads, and the particle size of the magnetic beads is 10 - 1000 nm.

[0019] It is understandable that the concentration and particle size of the magnetic beads in the magnetic bead suspension can also be conventionally adjusted according to actual usage requirements, as long as good binding of nucleic acids to magnetic beads can be achieved.

[0020] In some embodiments, the washing solution includes a first washing solution, a second washing solution, and a third washing solution; wherein, the first washing solution includes the following components: 1-4M guanidine isothiocyanate, 1-20mM EDTA, a surfactant with a volume percentage of 0.5-10%, ethanol with a volume percentage of 30-80%, and the surfactant includes at least one of Triton-X-100, NP-40, and Tween-20; the second washing solution includes the following components: an alcohol substance with a volume percentage of 60-90%, wherein the alcohol substance includes at least one of isopropanol and ethanol; the third washing solution includes the following components: ethanol with a volume percentage of 60-90%; the elution solution includes the following components: DEPC water with a volume percentage of 0.05-0.2%.

[0021] It can be understood that the first washing solution, the second washing solution, and the third washing solution can also select conventional washing solutions in the prior art according to actual usage needs, as long as they can better remove impurities from the sample to be extracted; the elution solution can also select conventional elution solutions in the prior art according to actual usage needs, as long as they can better elute RNA.

[0022] In some preferred embodiments, the first washing solution includes the following components: 3M guanidine isothiocyanate, 15mM EDTA, Triton-X-100 with a volume percentage of 3%, ethanol with a volume percentage of 50%; the second washing solution includes the following components: ethanol with a volume percentage of 80%; the third washing solution includes the following components: ethanol with a volume percentage of 70%; the elution solution includes the following components: DEPC water with a volume percentage of 0.1%.

[0023] In a second aspect, the present invention provides a method for automatically extracting RNA using any one of the above RNA extraction kits, including the following steps: S1. Mix the sample to be extracted with the lysis solution, add the binding solution and the magnetic bead suspension after shaking and mixing evenly, and after mixing and standing, perform separation treatment to obtain a nucleic acid-magnetic bead complex; S2. Treat the nucleic acid-magnetic bead complex with the first washing solution and the second washing solution respectively to obtain a treated nucleic acid-magnetic bead complex, continue to add DNase I buffer and DNase I to the treated nucleic acid-magnetic bead complex, mix evenly at room temperature, and then perform centrifugation treatment to obtain a digested nucleic acid-magnetic bead complex; S3. Treat the digested nucleic acid-magnetic bead complex with the third washing solution to obtain a purified nucleic acid-magnetic bead complex, and continue to elute the purified nucleic acid-magnetic bead complex with the elution solution to obtain the sample RNA.

[0024] It is understandable that the RNA extraction kit in the present invention can be used to manually extract RNA or automate the RNA extraction process. Preferably, the RNA extraction kit is used to automate the RNA extraction process in the present invention. The entire RNA extraction process is completed on a fully automatic nucleic acid extractor according to the above extraction steps. This method of automating the RNA extraction process solves the problem that most current nucleic acid extraction reagents using magnetic beads cannot achieve true fully automatic RNA extraction, and while reducing the risk of contamination and RNA degradation caused by suspension steps, it also makes the RNA extraction process faster and more convenient, saving the labor and time costs of the RNA extraction process.

[0025] In some embodiments, in step S1, the sample to be extracted includes at least one of cells, animal tissues, blood, and plant tissues; the addition amount of the lysis solution is 400 - 600 μL, the addition amount of the binding solution is 300 - 500 μL, and the addition amount of the magnetic bead suspension is 5 - 20 μL.

[0026] In some embodiments, in step S2, the addition amount of the first washing solution is 700 - 900 μL, the addition amount of the second washing solution is 700 - 900 μL, the addition amount of the DNase I buffer solution and DNase I is 50 - 150 μL, and the mixing time at room temperature is 15 - 20 min; wherein, the enzyme activity of DNase I in the DNase I buffer solution is 5 - 20 U.

[0027] In some embodiments, in step S3, the addition amount of the third washing solution is 700 - 900 μL, and the addition amount of the elution solution is 35 - 100 μL.

[0028] It is understandable that for different samples to be extracted, the addition amounts of the lysis solution, binding solution, magnetic bead suspension, first washing solution, second washing solution, DNase I buffer solution and DNase I, third washing solution and elution solution can also be routinely adjusted according to actual usage needs. For example, when the sample to be extracted is relatively large, the addition amounts of the lysis solution, binding solution, magnetic bead suspension, first washing solution, second washing solution, DNase I buffer solution and DNase I, third washing solution and elution solution can be appropriately increased; when the sample to be extracted is relatively small, the addition amounts of the lysis solution, binding solution, magnetic bead suspension, first washing solution, second washing solution, DNase I buffer solution and DNase I, third washing solution and elution solution can be appropriately decreased.

[0029] The beneficial effects of the present invention are as follows: Different from the prior art, after adding an auxiliary agent to the DNase I buffer in the RNA extraction kit, the present invention can effectively maintain the activity of DNase I, enabling the complete degradation of gDNA. At the same time, the auxiliary agent can also keep the RNA in a bound state with the magnetic beads during the process of DNase I digesting DNA. Without adding other reagents to re-bind the RNA to the magnetic beads, the RNA can directly enter the subsequent steps together with the magnetic beads, thus completing the fully automated RNA extraction process. In addition, the auxiliary agent can significantly improve the extraction yield of RNA from low-nucleic acid samples. Therefore, this RNA extraction kit has good application prospects in the automated extraction of RNA. Description of the Drawings

[0030] Figure 1 It is the agarose gel electrophoresis results of the RNA products extracted in Examples 1-3 and Comparative Example 1 of the present invention; Figure 2 It is the agarose gel electrophoresis results of the RNA products extracted in Example 1 and Comparative Example 2 of the present invention; Figure 3 It is the agarose gel electrophoresis results of the RNA products extracted in Example 1 and Comparative Example 3 of the present invention; Figure 4 It is the agarose gel electrophoresis results of the RNA products extracted in Example 4 and Comparative Example 4 of the present invention; Figure 5 It is the agarose gel electrophoresis results of the RNA products extracted in Example 4 and Comparative Example 5 of the present invention; Figure 6 It is the agarose gel electrophoresis results of the RNA products extracted in Example 5 of the present invention. Detailed Embodiments

[0031] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0032] For the experimental methods without specifying specific conditions in the embodiments, they are usually carried out according to the conventional conditions and the conditions described in the manuals, or according to the conditions recommended by the manufacturers. For the general equipment, materials, reagents, etc., without special instructions, they can all be obtained from commercial channels.

[0033] Example 1 In this embodiment, the RNA extraction kit based on the magnetic bead method includes a lysis solution, a binding solution, a magnetic bead suspension, a first washing solution, a second washing solution, a DNase I buffer solution, DNase I, a third washing solution, and an elution solution; Among them, the lysis solution includes the following components: 100 mM tris(hydroxymethyl)aminomethane, 4 M guanidine isothiocyanate, 2 mg / mL sodium dodecyl sulfate, 10 mg / mL sodium lauroyl sarcosinate, 50 mM sodium citrate, 10 mM disodium ethylenediaminetetraacetate, 100 mM thiourea, and 1% (v / v) Triton-X-100; The binding solution includes the following component: 90% (v / v) isopropanol; The concentration of magnetic beads in the magnetic bead suspension is 100 mg / mL. The magnetic beads include silica magnetic beads, and the particle size of the magnetic beads is 200 - 400 nm; The first washing solution includes the following components: 3 M guanidine isothiocyanate, 15 mM EDTA, 3% (v / v) Triton-X-100, and 50% (v / v) ethanol; The second washing solution includes the following component: 80% (v / v) ethanol; The DNase I buffer solution includes the following components: 25 mM tris(hydroxymethyl)aminomethane, 1 mM calcium chloride, 20 mM magnesium chloride, 100 mg / mL PEG (molecular weight 8000), and the pH value is 7 - 9; The third washing solution includes the following component: 70% (v / v) ethanol; The elution solution includes the following component: 0.1% (v / v) DEPC water.

[0034] Using the kit in this embodiment, the total RNA of 293F cells (concentrations are 5×10 5 cells / T and 1×10 5 cells / T respectively, where "T" represents each reaction) stored in 1.5 mL EP tubes is extracted by a NanoMagBio S-48 automatic nucleic acid extractor. Each sample is detected in triplicate, and the instrument parameters are set as shown in Table 1 below.

[0035] Table 1 Program for extracting total RNA of cells by NanoMagBio S-48 automatic nucleic acid extractor

[0036] Take out the corresponding number of deep well plates (one deep well plate for every 16 samples), and add the respective components (be sure to shake well before use) to the corresponding wells of the deep well plate in sequence as shown in Table 2 below.

[0037] Table 2 Aliquot volume of RNA extraction reagent in the kit and corresponding positions in the deep well plate

[0038] Extract RNA according to the following steps specifically: 1) Add 500 μL of lysis buffer to the 293F cells frozen in a 1.5 mL EP tube. After mixing well, transfer it to the corresponding wells in the 1st / 7th column of the deep well plate; 2) Place the deep well plate into the card slot of the instrument, place the magnetic rod sleeve, close the chamber door, and start running the program; 3) After the program ends, the instrument stops automatically. The nucleic acid sample at the 5th station in the deep well plate is transferred to a clean RNase-free centrifuge tube, which is the RNA of the extracted 293F cells.

[0039] Example 2 In this example, the components of the RNA extraction kit based on the magnetic bead method are basically the same as those in Example 1. The difference is that the DNase I buffer includes the following components: 5 mM tris(hydroxymethyl)aminomethane, 5 mM calcium chloride, 80 mM magnesium chloride, 200 mg / mL mannitol, and the pH value is 7 - 9.

[0040] Use the kit in this example to extract the total RNA of 293F cells with a NanoMagBio S-48 automatic nucleic acid extractor. The extraction method is the same as that in Example 1, and the RNA of 293F cells is obtained.

[0041] Example 3 In this example, the components of the RNA extraction kit based on the magnetic bead method are basically the same as those in Example 1. The difference is that the DNase I buffer includes the following components: 50 mM tris(hydroxymethyl)aminomethane, 0.5 mM calcium chloride, 10 mM magnesium chloride, 25 mg / mL trehalose, and the pH value is 7 - 9.

[0042] Use the kit in this example to extract the total RNA of 293F cells with a NanoMagBio S-48 automatic nucleic acid extractor. The extraction method is the same as that in Example 1, and the RNA of 293F cells is obtained.

[0043] Example 4 In this example, the components of the RNA extraction kit based on the magnetic bead method are the same as those in Example 1. The difference is that use the kit in this example to extract the total RNA of animal tissues with a NanoMagBio S-48 automatic nucleic acid extractor. Among them, the animal tissues need to be pretreated as follows: Weigh the required weight of tissue blocks (30 mg of rabbit liver, 10 mg of rabbit brain, 20 mg of rabbit heart, 20 mg of rabbit muscle), place them in a 2 mL centrifuge tube, add 3 steel beads with a diameter of 5 mm, and immediately cool them in liquid nitrogen. Grind them at 60 Hz for 30 s (with a metal module pre-cooled by liquid nitrogen) until the tissue becomes powdery. Then, directly add 500 μL of lysis buffer to the centrifuge tube (if the temperature is too low, the liquid will freeze. You can shake the centrifuge tube to quickly melt it). Immediately shake and mix well for 2 min until there are no obvious lumps / flocculent tissue particles in the tube (for heart and muscle, 10 μL of protease K solution with a concentration of 20 mg / mL needs to be added after lysis, and mix well and let it stand for 2 - 3 min). Subsequently, centrifuge at 4°C and 16,000×g for 5 min, take the supernatant for subsequent extraction, and the subsequent extraction method is the same as that in Example 1, and animal tissue RNA is obtained by extraction.

[0044] Example 5 In this example, the components of the RNA extraction kit based on the magnetic bead method are the same as those in Example 1. The difference is that the total RNA of the blood sample is extracted using the NanoMagBio S-48 automatic nucleic acid extractor with the kit in this example. Among them, the blood sample needs to be pretreated as follows, which specifically includes the following steps: 1) Red blood cell lysis: Take 1 - 1.5 mL of fresh whole blood (fresh anticoagulated whole blood sample without freezing and thawing), add 3 times the volume (3 - 4.5 mL) of red blood cell lysis buffer (150 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM disodium ethylenediaminetetraacetate), gently vortex or invert to mix well, and let it stand on ice for 15 min. During this period, gently vortex and mix well twice (after red blood cell lysis, the solution should be clear and transparent); centrifuge at 4°C and 450×g for 10 min, and carefully aspirate and discard the supernatant; 2) White blood cell collection: Then add 2 times the volume (2 - 3 mL) of red blood cell lysis buffer, and gently pipette to resuspend the white blood cells; centrifuge at 4°C and 450×g for 10 min to precipitate the white blood cells, and carefully and thoroughly aspirate and discard the supernatant to obtain the white blood cell pellet; 3) White blood cell lysis: Add 500 μL of lysis buffer, vortex to mix well, and then add 60 μL of protease K, and vortex to mix well for 1 - 2 min until there are no obvious cell clumps in the liquid; 1 mL and 1.5 mL of human peripheral blood whole blood samples anticoagulated with EDTA are obtained respectively. The above samples are subjected to subsequent extraction, and the subsequent extraction method is the same as that in Example 1 (in the mixing step, the mixing time is 10 min), and blood sample RNA is obtained by extraction.

[0045] Comparative Example 1 In this comparative example, the components of the RNA extraction kit based on the magnetic bead method are basically the same as those in Example 1, except that the third washing solution is not included, and the DNase I buffer solution includes the following components: 25 mM tris(hydroxymethyl)aminomethane, 1 mM calcium chloride, 20 mM magnesium chloride, and the pH value is 7 - 9.

[0046] Use the kit in this comparative example to extract the total RNA of 293F cells with a NanoMagBio S-48 fully automatic nucleic acid extractor, and each sample is detected in triplicate. The instrument parameters are set as shown in Table 3 below.

[0047] Table 3 Program for extracting total cellular RNA with a NanoMagBio S-48 fully automatic nucleic acid extractor

[0048] Take out the corresponding number of deep well plates (one deep well plate for every 16 samples), and add the respective components (be sure to shake well before use) to the corresponding wells in the deep well plate according to Table 4 below in sequence.

[0049] Table 4 Dispensing volume of RNA extraction reagents in the kit and corresponding positions on the deep well plate

[0050] Extract RNA specifically according to the following steps: 1) Add 500 μL of lysis solution to the 293F cells frozen in a 1.5 mL EP tube, mix well, and transfer them to the corresponding wells in the 1st / 7th column of the deep well plate; 2) Place the deep well plate in the card slot of the instrument, place the magnetic rod sleeve, close the chamber door, and start running the program; 3) During the operation of the instrument, after the DNA digestion is completed, the instrument will pause and beep. Open the instrument chamber door, take out the deep well plate, add 800 μL of the second washing solution to the wells corresponding to the samples in the 4th / 10th column of the 96-well deep well plate, then put the deep well plate back into the card slot, close the chamber door, click Resume on the lower right corner of the screen to continue the extraction program; 3) After the program ends, the instrument stops automatically. The nucleic acid sample at the 5th station in the deep well plate is transferred to a clean RNase-free centrifuge tube, which is the RNA of the extracted 293F cells.

[0051] Comparative Example 2 In this comparative example, the components of the RNA extraction kit based on the magnetic bead method are basically the same as those in Example 1, except that in the DNase I buffer solution, the molecular weights of PEG are 200, 400, and 20000 respectively.

[0052] Using the kit in this comparative example, the total RNA of 293F cells was extracted with a NanoMagBio S-48 automatic nucleic acid extractor. The extraction method was the same as that in Example 1, and the 293F cell RNA was obtained.

[0053] Comparative Example 3 In this comparative example, the components of the RNA extraction kit based on the magnetic bead method were basically the same as those in Example 1, except that in the DNase I buffer, the concentrations of PEG were 10 mg / mL and 250 mg / mL, respectively.

[0054] Using the kit in this comparative example, the total RNA of 293F cells was extracted with a NanoMagBio S-48 automatic nucleic acid extractor. The extraction method was the same as that in Example 1, and the 293F cell RNA was obtained.

[0055] Comparative Example 4 In this comparative example, the components of the RNA extraction kit based on the magnetic bead method were basically the same as those in Example 4, except that in the DNase I buffer, the additives were isopropanol and ethanol.

[0056] Using the kit in this comparative example, the total RNA of rabbit liver in Example 4 was extracted with a NanoMagBio S-48 automatic nucleic acid extractor. The extraction method was the same as that in Example 4, and the rabbit liver RNA was obtained.

[0057] Comparative Example 5 In this comparative example, the components of the RNA extraction kit based on the magnetic bead method were the same as those in Comparative Example 1, except that using the kit in this comparative example, the total RNA of animal tissues in Example 4 was extracted with a NanoMagBio S-48 automatic nucleic acid extractor. The extraction method was the same as that in Comparative Example 1, and the animal tissue RNA was obtained.

[0058] Performance Detection The concentrations and purities of the RNA of 293F cells extracted in Examples 1-3 and Comparative Example 1 were tested (using Thermo Fisher Nanodrop One), and the results are shown in Table 5 below.

[0059] Table 5 Results of the concentration and purity of the RNA of 293F cells extracted

[0060] Furthermore, the RNA products extracted in Examples 1-3 and Comparative Example 1 above were subjected to agarose gel electrophoresis, and the results are as Figure 1 shown.

[0061] From Table 5 and Figure 1As can be seen, in Examples 1-3 of the present invention, after adding PEG to the DNase I buffer, not only can the automatic extraction of cellular RNA be achieved, and for samples with low nucleic acid content (1×10 5 cells / T), after adding the auxiliary agent, the extraction yield and purity of the kit for samples with low nucleic acid content can be significantly improved.

[0062] The concentrations and purities of the RNA extracted from 293F cells in Example 1 and Comparative Example 2 were tested (measured using Thermo Fisher Nanodrop One), and the results are shown in Table 6 below.

[0063] Table 6 Results of the concentrations and purities of the RNA extracted from 293F cells

[0064] Furthermore, the RNA products extracted in the above Example 1 and Comparative Example 2 were subjected to agarose gel electrophoresis, and the results are as Figure 2 shown.

[0065] As can be seen from Table 6 and Figure 2 in Comparative Example 2, if the molecular weight of PEG is too low or too high, both will cause a decrease in the concentration and purity of the extracted RNA. Therefore, in the present invention, using PEG with a specific molecular weight as the auxiliary agent can significantly improve the concentration and purity of the extracted RNA.

[0066] The concentrations and purities of the RNA extracted from 293F cells in Example 1 and Comparative Example 3 were tested (measured using Thermo Fisher Nanodrop One), and the results are shown in Table 7 below.

[0067] Table 7 Results of the concentrations and purities of the RNA extracted from 293F cells

[0068] Furthermore, the RNA products extracted in the above Example 1 and Comparative Example 3 were subjected to agarose gel electrophoresis, and the results are as Figure 3 shown.

[0069] As can be seen from Table 7 and Figure 3 in Comparative Example 3, if the concentration of PEG is too low, nucleic acid loss will occur, resulting in a decrease in the product concentration; if the concentration of PEG is too high, incomplete digestion of gDNA will occur, resulting in the appearance of heterobands and a falsely high nucleic acid measurement value, affecting the result interpretation. Therefore, in the present invention, using PEG with a specific concentration as the auxiliary agent can significantly improve the concentration and purity of the extracted RNA.

[0070] The concentration and purity of the RNA extracted from the rabbit liver in Example 4 and Comparative Example 4 were tested (using ThermoFisher Nanodrop One), and the results are shown in Table 8 below.

[0071] Table 8 Results of the concentration and purity of the RNA extracted from the rabbit liver

[0072] Furthermore, agarose gel electrophoresis was performed on the RNA products extracted in Example 4 and Comparative Example 4 above, and the results are as Figure 4 shown.

[0073] As can be seen from Table 8 and Figure 4 In Comparative Example 4, when the auxiliary agent was replaced with isopropanol and ethanol, both the concentration and purity of the extracted RNA decreased. Therefore, in the present invention, the auxiliary agent used can significantly improve the concentration and purity of the extracted RNA.

[0074] The concentration and purity of the animal tissue RNA extracted in Example 4 and Comparative Example 5 were tested (using Thermo Fisher Nanodrop One), and the results are shown in Table 9 below.

[0075] Table 9 Results of the concentration and purity of the RNA extracted from the animal tissue

[0076] Furthermore, agarose gel electrophoresis was performed on the RNA products extracted in Example 4 and Comparative Example 5 above, and the results are as Figure 5 shown.

[0077] As can be seen from Table 9 and Figure 5 In Example 4 of the present invention, after adding PEG to the DNase I buffer, not only can the automatic extraction of animal tissue RNA be achieved, but also the concentration and purity of the RNA extracted from the animal tissue are significantly higher than those in Comparative Example 5.

[0078] The concentration and purity of the blood sample RNA extracted in Example 5 were tested (using ThermoFisher Nanodrop One), and the results are shown in Table 10 below.

[0079] Table 10 Results of the concentration and purity of the extracted blood sample

[0080] Furthermore, agarose gel electrophoresis was performed on the RNA products extracted in Example 5 above, and the results are as Figure 6 shown.

[0081] As can be seen from Table 10 and Figure 6 it can be seen that the RNA extracted using the kit of the present invention has good concentration and purity, and the main band of the blood sample is bright and clear, without other miscellaneous bands and incomplete DNA digestion.

[0082] In summary, after adding an auxiliary agent to the DNase I buffer in the RNA extraction kit of the present invention, it can effectively maintain the activity of DNase I, so that gDNA can be completely degraded; at the same time, the auxiliary agent can also make RNA always in a bound state with magnetic beads during the process of DNase I digesting DNA. Without adding other reagents to make RNA bind to magnetic beads again, RNA can directly enter the subsequent steps together with magnetic beads, thus completing the automatic extraction process of RNA.

[0083] It should be noted that the above embodiments all belong to the same inventive concept. The descriptions of the embodiments have their own emphases. For the parts not described in detail in individual embodiments, reference can be made to the descriptions in other embodiments.

[0084] The above embodiments only express the implementation modes of the present invention. The descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A RNA extraction kit based on magnetic bead method, characterized in that: It includes lysis solution, binding solution, magnetic bead suspension, washing solution, DNase I buffer, DNase I and elution solution; The pH value of the DNase I buffer is 5-9, and it includes the following components: 5-50 mM tris(hydroxymethyl)aminomethane, 0.5-5 mM calcium chloride, 10-100 mM magnesium chloride and 25-200 mg / mL of an auxiliary agent, wherein the auxiliary agent includes at least one of PEG, mannitol and trehalose.

2. The RNA extraction kit based on the magnetic bead method according to claim 1, characterized in that The molecular weight of the PEG is 800-8000.

3. The RNA extraction kit based on the magnetic bead method according to claim 1, characterized in that The lysate comprises the following components: 10-100 mM tris(hydroxymethyl)aminomethane, 1-5 M guanidine isothiocyanate, 1-10 mg / mL sodium dodecyl sulfonate, 5-20 mg / mL sodium lauryl sarcosinate, 10-100 mM sodium citrate, 5-20 mM disodium ethylenediaminetetraacetic acid, 10-200 mM thiourea, and 1-3% by volume of Triton-X-100.

4. The RNA extraction kit based on the magnetic bead method according to claim 1, characterized in that The combination liquid includes the following components: 60-100% by volume of an alcohol substance; Wherein, the alcohol substance includes at least one of isopropanol and anhydrous ethanol.

5. The RNA extraction kit based on the magnetic bead method according to claim 1, characterized in that The concentration of the magnetic beads in the magnetic bead suspension is 30-100 mg / mL, the magnetic beads include silica magnetic beads, and the particle size of the magnetic beads is 10-1000 nm.

6. The RNA extraction kit based on the magnetic bead method according to claim 1, characterized in that The washing liquid comprises a first washing liquid, a second washing liquid and a third washing liquid; The first washing solution comprises the following components: 1-4M guanidine thiocyanate, 1-20mM EDTA, 0.5-10% by volume of a surfactant, and 30-80% by volume of ethanol, and the surfactant comprises at least one of Triton-X-100, NP-40, and Tween-20; The second washing liquid comprises the following components: 60-90% by volume of an alcohol substance, wherein the alcohol substance comprises at least one of isopropanol and ethanol; The third washing liquid comprises the following components: 60-90% by volume of ethanol; The eluent comprises the following components: DEPC water with a volume percentage of 0.05-0.2%.

7. A method for automatically extracting RNA using the RNA extraction kit according to any one of claims 1 to 6, characterized in that: The steps include: S1, mixing the sample to be extracted with the lysate, oscillating and mixing, adding the binding solution and the magnetic bead suspension, mixing and standing, and then performing separation treatment to obtain a nucleic acid-magnetic bead complex; S2, using the first washing solution and the second washing solution to treat the nucleic acid-magnetic bead complex respectively to obtain a treated nucleic acid-magnetic bead complex, and continuing to add DNase I buffer and DNase I to the treated nucleic acid-magnetic bead complex, mixing at room temperature, and centrifuging to obtain a digested nucleic acid-magnetic bead complex; S3. Using a third washing solution to treat the digested nucleic acid-magnetic bead complex to obtain a purified nucleic acid-magnetic bead complex, and continuing to use an elution solution to elute the purified nucleic acid-magnetic bead complex to obtain sample RNA.

8. The method according to claim 7, characterized in that In step S1, the sample to be extracted includes at least one of cells, animal tissues, blood, and plant tissues; The amount of the lysate added is 400-600 μL, the amount of the binding solution added is 300-500 μL, and the amount of the magnetic bead suspension added is 5-20 μL.

9. The method according to claim 7, characterized in that In step S2, the amount of the first washing solution added is 700-900 μL, the amount of the second washing solution added is 700-900 μL, the amount of the DNase I buffer and DNase I added is 50-150 μL, and the mixing time at room temperature is 15-20 min; Wherein, the enzyme activity of the DNase I in the DNase I buffer is 5-20U.

10. The method according to claim 7, characterized in that In step S3, the amount of the third washing solution added is 700-900 μL, and the amount of the elution solution added is 35-100 μL.

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