A kit for extracting RNA and use thereof

Abstract

The application provides a kit for extracting RNA and application thereof. In the application, deoxyribonuclease (DNase) is used for DNA digestion in the process of magnetic bead extraction. The reaction environment can meet the optimal reaction environment requirement of the deoxyribonuclease (DNase), and at the same time, a hydrophobic environment is provided when the magnetic beads are adsorbed, so as to ensure the yield of nucleic acid. In addition, an emulsion of an oil is added to increase the total volume during the extraction operation, so as to facilitate the convenience of automatic extraction operation of the instrument. The extracted RNA has high yield and high purity, and the method can easily realize automatic extraction of the instrument. The requirement of extracting high-purity RNA quickly and conveniently in the process of clinical practical application is solved, the difficulty and cost of extracting high-purity RNA are greatly reduced, and the extracted RNA can be well adapted to subsequent RT-PCR and RNA-seq application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological sample purification, and particularly relates to a DNA-contaminated magnetic bead method for extracting RNA and application thereof. BACKGROUND

[0002] RNA, as a genetic information carrier existing in biological cells and part of viruses and viroids, its extraction and purification are the basis for various clinical detection applications. In the process of RNA purification, removing DNA is a very important link. Incomplete removal of DNA may affect the downstream experiment. The current common method for extracting RNA is mainly through denaturing agent to break the cells or tissues, and then extracting RNA through chloroform and other organic solvents. This method can obtain RNA with high purity, but chloroform and other chemicals with high toxicity are used in the process of extracting RNA, and the experimental operation is complex and cannot be automated.

[0003] As an important method for nucleic acid extraction, the magnetic bead method is widely used in the extraction and detection of viral RNA because it is suitable for instrument automation. However, this method extracts RNA and DNA at the same time, so the contamination of DNA limits the application of downstream experiments. For example, in clinical practice, the detection of high-risk HPV E6 / E7 mRNA transcription is applied to the early diagnosis, early treatment and prognosis monitoring of cervical cancer, and the detection target is mRNA. The interference of DNA will affect the detection results.

[0004] The invention patent with the application publication number CN 114350650 A discloses a DNA-free residual blood RNA extraction kit and nucleic acid extraction method, which includes lysis solution, first rinsing solution, second rinsing solution, eluent, proteinase K buffer solution, and RNAase-free double distilled water. It also includes red blood cell lysis solution and DNA enzyme buffer solution. After cell lysis, the first rinsing solution is used for washing, then DNA enzyme buffer solution and DNA enzyme are added to the adsorption column for digestion, so as to remove DNA. Then the first rinsing solution and the second rinsing solution are used for washing, and finally RNA is obtained by elution. Although this method can obtain RNA with high purity, it uses adsorption column extraction, which is not conducive to instrument automation.

[0005] The invention patent with application publication number CN 110607395 A discloses a method and kit for detecting high-risk human papilloma virus mRNA across introns. The extraction and purification method uses conventional magnetic bead method. The characteristics further include using magnetic bead method nucleic acid extraction and purification reagent, selecting silicon-based magnetic beads with better RNA affinity than DNA, and adding DNA enzyme digestion step in extraction and purification. The patent proposes adding DNA enzyme for DNA digestion during proteinase K digestion. Based on previous knowledge, DNA enzyme as a protein should be inactivated by proteinase K digestion. Common reducing agents such as SDS, DTT, mercaptoethanol, and salt concentrations above 50-100 mM all have significant inhibitory effects on DNase I. Therefore, this environment cannot provide a favorable environment for DNA enzyme reaction, and the patent does not provide detailed example data and result display. The effect of DNA enzyme digestion is unknown.

[0006] The invention patent with application publication number CN 108949747 A discloses a kit and method for extracting ribonucleic acid (RNA) by magnetic bead method. The kit includes reagents: tissue digestion solution, lysis solution, proteinase K, DNase I, DNase I buffer, nucleic acid, extraction magnetic beads, washing solution I, washing solution II, and elution solution. The scheme performs DNase I digestion step after washing solution I treatment step. The DNase I concentration is 1-3 U / μl. The DNase I buffer includes: final concentration of 5-20 mmol / L Tris-HCl, final concentration of 10-50 mmol / L MgCl2, final concentration of 0.1-1 mmol / L CaCl2, reaction volume of 50 ul, and reaction environment of DNase I conventional reaction conditions, which can effectively remove DNA. However, the disadvantages are: first, the solution is in aqueous environment, and nucleic acid is easily dissolved in water phase, resulting in reduced recovery efficiency and yield during magnetic bead adsorption; second, the reaction volume is too small, which is not conducive to the realization of automatic extraction by magnetic bead method.

[0007] Based on the above-mentioned patents, the products for RNA extraction and purification still have the following defects: 1. Most of them use guanidine salt-organic solvent method to extract high-purity RNA, but toxic organic solvents are used in the extraction process, and this method is not suitable for instrument extraction automation. 2. Some products do not need to use organic solvents to separate RNA and DNA, and they use Dnase enzyme to digest DNA during extraction, but this method is not suitable for instrument extraction automation. 3. The existing magnetic bead method for instrument automation has low RNA purity and is contaminated with a large amount of DNA. Some patents propose to use Dnase enzyme to digest DNA during the extraction process, but no detailed example data and results are provided. 4. Some patents use suitable Dnase enzyme reaction environment for digestion reaction, but the water phase environment will lead to low recovery efficiency of nucleic acid magnetic adsorption, and the small reaction volume is not suitable for magnetic bead automatic extraction operation. Therefore, it is necessary to find a suitable DNA digestion method for magnetic bead method to obtain high-purity RNA. This RNA extraction and purification method can meet the requirements of not using toxic organic solvents, remove DNA residual contamination, and easily realize instrument automation. SUMMARY

[0008] In clinical detection or scientific research, high-purity RNA needs to be extracted for detection, and DNA contamination will interfere with downstream detection. Previous studies and products use organic solvents such as chloroform to extract and separate RNA and DNA. Although the purity of the extracted RNA is high, this method uses toxic organic reagents and the operation is complex. Alternatively, Dnase enzyme is used for digestion treatment on the adsorption column to remove DNA contamination, which is not conducive to instrument automation extraction.

[0009] The purpose of the present application is to provide a method for extracting RNA by magnetic beads and effectively removing DNA contamination. The present application uses Dnase enzyme (deoxyribonuclease) for DNA digestion during the extraction process by magnetic beads, which can meet the optimal reaction environment requirements of Dnase enzyme, and provide a hydrophobic environment for magnetic bead adsorption to ensure the yield of nucleic acid. An oil emulsion is added to increase the total volume during extraction operation to facilitate the convenience of instrument automation extraction. The extracted RNA has high yield and high purity, and this method can easily realize instrument automation extraction. It solves the demand for rapid and convenient extraction of high-purity RNA in clinical practice, greatly reduces the difficulty and cost of high-purity RNA extraction, and the extracted RNA can be well adapted to subsequent RT-PCR and RNA-seq applications.

[0010] The present application adopts the following technical solutions:

[0011] A kit for extracting RNA, comprising the following components: proteinase K, lysis binding solution, magnetic beads, DNase reaction solution, washing solution, elution solution;

[0012] The DNase reaction solution comprises Dnase I, Dnase I buffer, the Dnase I buffer comprises an aqueous phase, an oil phase and an emulsifier, the oil phase accounts for 70%-90% of the total volume of the two phases, preferably 73-77%, most preferably 75%, and the emulsifier accounts for 0.5%-5% of the total volume of the two phases, preferably 0.5-2%, most preferably 1%.

[0013] The kit for extracting RNA,

[0014] The oil phase comprises one or more of the following components: silicone oil and its derivatives (such as dimethyl silicone oil, vinyl silicone oil, benzyl silicone oil, vinyl-terminated polymethyl silicone oil, polymethyl hydrogen silicone), fluorine oil and its derivatives (such as HFE-7100, HFE-7200, HFE-7500, FC-40); the emulsifier comprises one or more of the following components: non-ionic surfactants (such as Span / Triton / Tween), anionic surfactants (such as sodium dodecyl benzene sulfonate, sodium lauryl sulfonate), cationic surfactants (dodecyl dimethyl benzyl ammonium chloride, hexadecyl trimethyl ammonium chloride, octadecyl trimethyl ammonium chloride).

[0015] The kit for extracting RNA, the aqueous phase comprises PEG.

[0016] The kit for extracting RNA, the aqueous phase comprises 2.5%-10% PEG, preferably 4-6%, most preferably 5%.

[0017] The kit for extracting RNA, the aqueous phase further comprises a pH buffer, 1-5 mM MgCl2, preferably 2-3 mM, most preferably 2.5 mM, 0.25-0.75 mM CaCl2, preferably 0.4-0.6 mM, most preferably 0.5 mM; the pH buffer comprises one or more of the following components: 10 mM-50 mM NaAc-HAc buffer at pH 7.5, 10 mM-50 mM Tris-HCl buffer at pH 7.5.

[0018] The kit for extracting RNA, the Dnase I in the DNase reaction solution is 40 U / mL-120 U / mL, preferably 60-100 U / mL, most preferably 75 U / mL.

[0019] The kit for extracting RNA, the object for extracting RNA comprises cells, tissues, blood, urine, cervical swab, oral swab, nasopharyngeal swab samples, etc.

[0020] For cultured cells (concentration 10 5 For cultured cells (concentration 10

[0021] For blood, urine, cervical swab, buccal swab, nasopharyngeal swab samples, only proteinase K is needed, mixed well, no need to react for 30-60 min at 56°C.

[0022] Proteinase K (Tianguen RT403, concentration 20 mg / ml).

[0023] The proteinase K reaction buffer comprises the following components: 10-50 mM Tris-Cl, preferably 25-35 mM, most preferably 30 mM, 0.2%-2% SDS, further preferably 0.5%-1%, most preferably 1%, 5-20 mM urea, further preferably 10-15 mM, most preferably 12.5 mM;

[0024] The kit for extracting RNA, the lysis binding liquid comprises the following components: chaotropic salt, nucleic acid precipitant, nuclease inhibitor, reducing agent, surfactant, isopropyl alcohol, pH buffer; the chaotropic salt comprises one or more of the following components: guanidine hydrochloride, guanidine isothiocyanate, urea and sodium perchlorate, the concentration is 3-6M, further preferably 4-6M, most preferably 5M; the nucleic acid precipitant comprises one or more of the following components: 5%-20% PEG, further preferably 6%-12%, most preferably 10%, 2-4M LiCl, further preferably 2.5-4M, most preferably 3.2M, 0.1-0.5mg / mL Glycogen, further preferably 0.25-0.5mg / mL, most preferably 0.4mg / mL, 2-10μg / μL Carrier RNA, further preferably 3-8μg / μL, most preferably 6μg / μL; the nuclease inhibitor comprises one or more of the following components: 5mM-50mM EDTA, further preferably 10mM-40mM, most preferably 20mM, 5mM-50mM EGT A, further preferably 10mM-40mM, most preferably 20mM, 0.5-10mM ATA, further preferably 2mM-8mM, most preferably 5mM, 50mM-200mM glycerol, further preferably 75mM-150mM, most preferably 125mM, 1mg / ml-5mg / ml NaF, further preferably 2mg / ml-4mg / ml, most preferably 3mg / ml; the reducing agent comprises one or more of the following components: 20mM-200mM tris (2-carboxyethyl) phosphine hydrochloride, further preferably 50mM-150mM, most preferably 125mM, 2%-10% β-mercaptoethanol, further preferably 3%-8%, most preferably 5%, 50-400mM DTT, further preferably 100mM-250mM, most preferably 200mM; the surfactant comprises one or more of the following components: Tween 20, Triton X-100, Nonidet P40, Brij35, the content is 1%-10%, further preferably 1%-5%, most preferably 2%, the content of isopropyl alcohol is 30-60%, further preferably 35%-55%, most preferably 40%; the pH buffer comprises one or more of the following components: 50mM-250mM pH6.6NaAc-HAc buffer, 50mM-250mM pH6.6Tris-HCl buffer, further preferably 100mM-200mM, most preferably 200mM; the solvent is RNase-free water;

[0025] The magnetic beads comprise at least one of hydroxyl magnetic beads and carboxyl magnetic beads;

[0026] The washing liquid is divided into washing liquid 1 and washing liquid 2;

[0027] The washing liquid 1 comprises the following components: chaotropic salt, ethanol, pH buffer, surfactant; the chaotropic salt comprises one or more of the following components: guanidine hydrochloride, guanidine isothiocyanate, urea and sodium perchlorate, with a concentration of 1-4M, further preferably 2-3M, most preferably 2.5M; the ethanol content is 30-60%, further preferably 40-55%, most preferably 50%; the pH buffer comprises one or more of the following components: 50mM-250mM pH 6.6 NaAc-HAc buffer, 50mM-250mM pH 6.6 Tris-HCl buffer, further preferably 50mM-100mM, most preferably 50mM; the surfactant comprises one or more of the following components: Tween 20, Triton X-100, Nonidet P40, Brij35, with a content of 1%-10%, further preferably 1%-5%, most preferably 1%;

[0028] The washing liquid 2 comprises the following components: 70%-80% ethanol, most preferably 80%, pH buffer, 400-800mM NaCl, most preferably 500mM; the pH buffer comprises one or more of the following components: 10mM-50mM pH 7.5 NaAc-HAc buffer, 10mM-50mM pH 7.5 Tris-HCl buffer, most preferably 20mM;

[0029] The elution liquid comprises the following components: 0.1% DEPC-treated Water (DNase, RNase free).

[0030] The application also provides the use of the kit in extracting RNA.

[0031] Specifically comprising the following steps:

[0032] 1) Put the collected sample into a centrifugal tube, add 150-250ul of sample buffer, then add 18-22ul of proteinase K, vortex to mix,

[0033] The sample buffer of the application includes but is not limited to proteinase K reaction buffer. If it is a cell or tissue sample, after adding the proteinase K reaction buffer and proteinase K, react at 56℃ for 30-60min; for blood samples, urine samples, cervical swabs containing preservative, oral swabs, nasopharyngeal swabs, directly take 200ul of sample, without supplementing the proteinase K reaction buffer, then add 20ul of proteinase K for reaction;

[0034] 2) After low-speed centrifugation at 500-2000rpm for at least 5s, add 250-350ul of lysis binding liquid and 18-22ul of magnetic beads, shake to mix, lysis and binding at 56℃ for 8-12min, during which time shake to mix;

[0035] 3) After centrifuging at a low speed of 500-2000 rpm for at least 5 seconds, place the centrifuge tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear. Then carefully aspirate the liquid.

[0036] 4) Add 550-650 μl of washing solution 1 and shake to mix for at least 1 min;

[0037] 5) After centrifuging at a low speed of 500-2000 rpm for at least 5 seconds, place the centrifuge tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear. Then carefully aspirate the liquid.

[0038] 6) Gently premix DNase I and DNase I Buffer in the specified ratio. Add 250-700 μl of the premix and gently mix. React at room temperature for 10-20 min. Centrifuge at 500-2000 rpm for at least 5 s. Let the centrifuge tube stand until the magnetic beads are completely adsorbed. Carefully remove the liquid. Add 550-650 μl of Washing Buffer 1 and vortex to mix for at least 1 min. Centrifuge at 500-2000 rpm for at least 5 s. Place the centrifuge tube on a magnetic rack and let it stand for 2-5 min until the solution is completely clear. Carefully remove the liquid.

[0039] 7) Add 550-650 μl of washing solution 1, shake to mix for at least 1 min, centrifuge at a low speed of 500-2000 rpm for at least 5 s, place the centrifuge tube on a magnetic rack and let it stand for 2-5 min until the solution is completely clear, then carefully aspirate the liquid.

[0040] 8) Add 550-650 μl of washing solution 2, shake to mix; centrifuge at low speed of 500-2000 rpm for at least 5 seconds, then place the centrifuge tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear, then carefully aspirate the liquid.

[0041] 9) After centrifuging at a low speed of 500-2000 rpm for at least 5 seconds, place the centrifuge tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear. Then carefully remove all the liquid.

[0042] 10) Allow the centrifuge tubes to air dry at room temperature for 3-5 minutes;

[0043] 11) Add 50-100 μl of elution buffer to the centrifuge tube, vortex to mix, and incubate at room temperature for at least 5 min, inverting the tube to mix during the incubation period; 12) Place the centrifuge tube on a magnetic rack and let it stand for 2-5 min until the solution is completely clear. Then carefully transfer the supernatant to a new centrifuge tube for storage.

[0044] The applications described in this invention involve extracting RNA from the following samples: cells, tissues, blood, urine, cervical swabs, oral swabs, and nasopharyngeal swabs.

[0045] The method of this invention can remove DNA contamination, obtain high-purity RNA, and easily automate the process. The effectiveness of this extraction method for RNA extraction was comprehensively evaluated through various validation methods, including RNA concentration, RNA purity, and downstream applications.

[0046] The advantages or beneficial effects of this invention compared to the prior art.

[0047] Advantage 1: High-purity RNA is extracted using the magnetic bead method. The extraction process is short and simple, and it can be used for automated instrument extraction, which greatly expands the scope of clinical applications.

[0048] Advantage 2: The extraction process does not use toxic organic reagents, making it green and environmentally friendly.

[0049] Advantage 3: This invention effectively and thoroughly removes DNA contamination during the extraction process, resulting in high-yield and high-purity RNA, which is well-suited for subsequent RT-PCR and RNA-seq applications.

[0050] Advantage 4: This invention utilizes clinical cervical exfoliated cells as samples for extensive extraction and subsequent testing verification. This extraction method can be effectively applied to the detection of HPV E6 / E7 mRNA. Detailed Implementation

[0051] The following examples are intended to further illustrate the present invention, but not to limit it.

[0052] Example 1: Testing of oils and emulsifiers

[0053] The components and formulations of the kit of this invention are as follows: proteinase K (20 mg / mL), proteinase K reaction buffer (30 mM Tris-HCl, 1% SDS, 12.5 mM urea), lysis binding buffer (5 M guanidine isothiocyanate, 10% PEG, 3.2 M LiCl, 0.4 mg / mL Glycogen, 20 mM EDTA, 125 mM tris(2-carboxyethyl)phosphine hydrochloride, 2% Triton X-100, 40% isopropanol, 125 mM tris(2-carboxyethyl)phosphine hydrochloride, 200 mM pH 6.6 NaAc-HAc buffer, solvent is RNase-free water), carboxyl magnetic beads, DNase reaction solution (DNase reaction solution includes Dnase I and Dnase I buffer. The amount of Dnase I in the DNase reaction solution is 75 U / mL. Dnase I The buffer consists of an aqueous phase, an oil phase, and an emulsifier. The oil phase (dimethyl silicone oil) accounts for 75% of the total volume of the two phases, and the emulsifier (Tween 20) accounts for 1% of the total volume of the two phases. The aqueous phase contains 10 mM Tris-HCl buffer (pH 7.5), 2.5 mM MgCl2, 0.5 mM CaCl2, and 5% PEG. Washing buffer 1 (2.5 M guanidine isothiocyanate, 50% ethanol, 50 mM pH 6.6 NaAc-HAc buffer, and 1% Triton X-100), washing buffer 2 (80% ethanol, 500 mM NaCl, and 20 mM pH 7.5 NaAc-HAc buffer), and elution buffer (0.1% DEPC-treated water).

[0054] This embodiment sets up three groups: Group 1 is the Dnase I treatment group with oil and emulsifier of the present invention; Group 2 is the Dnase I treatment group without oil and emulsifier (only oil and emulsifier are not added, the volume of the aqueous phase is the same as the total volume of Dnase I buffer in Group 1, and the other components are the same as Group 1); Group 3 is the untreated group (only Dnase I and Dnase I buffer are not used, and the other components are the same as Group 1). Groups 1 and 2 perform step 6 (Dnase I treatment step) of the specific operation process described below, while Group 3 does not perform step 6. All other steps are the same.

[0055] The specific operation process of Example 1 is as follows:

[0056] 1) Take 200 μl of the suspension of cervical exfoliated cell samples (suspended in PBS) from each of the three groups, and dilute to 1.5 μL.

[0057] Add 20 μl of proteinase K to a centrifuge tube and vortex for 10 seconds to mix.

[0058] 2) After centrifuging at 2000 rpm for 5 seconds, add 300 μl of lysis binding solution and 20 μl of magnetic beads, shake to mix, and lyse the binding at 56℃ for 10 min, inverting and mixing 3 times during the process, 3-5 times each time.

[0059] 3) After instantaneous centrifugation at 2000 rpm for 5 seconds, place the centrifuge tube on a magnetic rack and let it stand for 2-3 minutes until the magnetic beads are completely attracted. Then carefully remove the liquid.

[0060] 4) Add 600 μl of washing solution 1 and shake to mix for 1 min.

[0061] 5) After centrifuging at 2000 rpm for 5 seconds, place the centrifuge tube on a magnetic rack and let it stand for 2-3 minutes until the magnetic beads are completely clear. Then carefully remove the liquid.

[0062] 6) Gently premix DNase I and DNase I Buffer in the specified ratio. Add 400 μl of the premix and gently mix. React at room temperature for 15 min. Centrifuge briefly at 2000 rpm for 5 seconds, then place the centrifuge tube on a magnetic rack and let it stand for 2-3 min until the magnetic beads are completely adsorbed. Carefully remove the liquid. Add 600 μl of Wash Buffer 1 and vortex to mix for 1 min. Centrifuge briefly at 2000 rpm for 5 seconds, then place the centrifuge tube on a magnetic rack and let it stand for 2-3 min until the magnetic beads are completely adsorbed. Carefully remove the liquid.

[0063] 7) Add 600 μl of washing buffer 2 and vortex to mix. Centrifuge briefly at 2000 rpm for 5 seconds, then place the centrifuge tube on a magnetic rack and let it stand for 2 minutes until the magnetic beads are completely adsorbed. Carefully remove the liquid after this process.

[0064] 8) After instantaneous centrifugation at 2000 rpm for 5 seconds, place the centrifuge tube on a magnetic rack and let it stand for 2 minutes. After the magnetic beads are completely attracted, carefully remove all the liquid.

[0065] 9) Place the centrifuge tubes on a magnetic rack and let them air dry at room temperature for 5 minutes.

[0066] 10) Remove the centrifuge tube from the magnetic rack, add 80 μl of elution buffer, vortex to mix, and incubate at room temperature for 5 min, inverting the tube 3 times during the incubation period, 3-5 times each time.

[0067] 11) Place the centrifuge tube on a magnetic rack and let it stand for 2 minutes until the solution is completely clear. Then carefully transfer the supernatant to a new centrifuge tube and store it at -20±5℃. For long-term storage, it can be stored in a -80℃ refrigerator.

[0068] The obtained RNA was analyzed for concentration and purity using a microplate reader. RT-PCR was performed using primers specifically designed to amplify both ACTB RNA and ACTB DNA. The results are as follows:

[0069] Table 1

[0070]

[0071] Table 2

[0072]

[0073] Both DNase I treatment methods yielded high RNA nucleic acid concentrations and purity, and both DNase I-treated groups showed DNA removal effects compared to the untreated group. DNase I treatment with oil and emulsifiers resulted in slightly higher RNA yields than treatment without oil and emulsifiers, while maintaining consistent DNA removal effectiveness.

[0074] Example 2: Instrument Extraction Test

[0075] This embodiment uses the same setup as Example 1, with a total of three groups: Group 1 is the DNase I treatment group with added oil and emulsifier; Group 2 is the DNase I treatment group without added oil and emulsifier; and Group 3 is the untreated group. This embodiment uses a nucleic acid extractor (using the same reagents as in Example 1). Groups 1 and 2 have DNase I treatment steps, while Group 3 does not. The extraction process is the same as in Example 1.

[0076] The obtained RNA was analyzed for concentration and purity using a microplate reader. RT-PCR was performed using primers designed to amplify only ACTB RNA and primers designed to amplify only ACTB DNA. The results are as follows.

[0077] Table 3

[0078]

[0079] Table 4

[0080]

[0081] Both DNase I treatment methods yielded high RNA nucleic acid concentrations and purity, and both DNase I-treated groups showed DNA removal efficiency compared to the untreated group. DNase I treatment with oil and emulsifier yielded slightly higher RNA yields than treatment without oil and emulsifier, while DNase I treatment with oil and emulsifier showed better DNA removal efficiency. This is the innovation of this invention: by adding oil and emulsifier, without changing the effective concentration of the DNase I reactants, the volume of the DNase I reaction is increased, improving the ease of operation of the instrument extraction process and thus enhancing DNA digestion efficiency.

[0082] Example 3: Test of PEG content in Dnase I buffer

[0083] This embodiment sets up 6 groups, of which groups 1-5 are DNase I treatment groups, with DNase I in the DNA enzyme reaction solution at a concentration of 75 U / mL. Group 1's DNase I buffer does not contain PEG, group 2's DNase I buffer contains 2.5% PEG, group 3's DNase I buffer contains 5% PEG, group 4's DNase I buffer contains 10% PEG, group 5's DNase I buffer contains 20% PEG, and group 6 is the group without DNase I treatment. The formulations of the remaining components are the same as those of group 1 in Example 1. Groups 1-5 undergo step 6 (DNase I treatment step) of the specific operation process described below, while group 6 does not perform step 6. All other steps are the same.

[0084] The specific operation process is as follows:

[0085] 1. Take 200 μl of the suspension of cervical exfoliated cell samples from each of the 6 groups and put it into a 1.5 ml centrifuge tube. Add 20 μl of proteinase K and vortex for 10 seconds to mix.

[0086] 2. Subsequent operating steps are the same as steps 2-11 in Example 1.

[0087] The obtained RNA was detected by RT-PCR using primers designed to amplify only ACTB RNA and primers designed to amplify only ACTB DNA. The detection results are as follows:

[0088] Table 5

[0089]

[0090] Adding a certain amount of PEG can improve RNA yield. However, when the PEG concentration exceeds 10%, the high viscosity of the reaction system leads to a decrease in RNA yield and DNA digestion efficiency. The optimal concentration range determined by this invention is 2.5%-10%.

[0091] Example 4: Test of the effect of magnetic bead method on removing DNA contamination when extracting RNA from cervical exfoliated cell samples

[0092] This embodiment sets up two groups: an experimental group (treated with Dnase I) and a control group (not treated with Dnase I). The formulations of all other components are the same and identical to those of Group 1 in Example 1. That is, the experimental group performs step 6 of the specific operation process described below, while the control group does not perform step 6. Apart from this, all other steps are the same.

[0093] 1. Take 200 μl of the collected sample suspension and put it into a 1.5 ml centrifuge tube. Add 20 μl of proteinase K and vortex for 10 seconds to mix.

[0094] 2. Subsequent operating steps are the same as steps 2-11 in Example 1.

[0095] The obtained RNA was detected by RT-PCR using primers designed to amplify only ACTB RNA and primers designed to amplify only ACTB DNA. The detection results are as follows:

[0096] Table 6

[0097]

[0098]

[0099] Based on the RT-PCR results of this embodiment, the proportion of DNA in the experimental group (treated with Dnase I) was much lower than that in the control group (not treated with Dnase I), meaning that the proportion of DNA decreased after Dnase I treatment.

[0100] The obtained RNA was processed by Hieff The Fast RNA Library Prep Kit for Illumina (cot12304ES96) was used to perform whole-transcript sequencing after host (human) rRNA removal. The sequencing results are as follows:

[0101] Table 7

[0102]

[0103] Based on the RNA-seq results of this embodiment, the ratio of introns in the experimental group (treated with Dnase I) was much lower than that in the control group (untreated with Dnase I), indicating that the proportion of DNA decreased after Dnase I treatment.

[0104] Example 5: Test of the effect of magnetic bead method on removing DNA contamination during RNA extraction from cell lines

[0105] This embodiment sets up two groups: an experimental group (treated with Dnase I) and a control group (not treated with Dnase I). The formulations of all other components are the same and identical to those of Group 1 in Example 1. That is, the experimental group performs step 6 of the specific operation process described below, while the control group does not perform step 6. Apart from this, all other steps are the same.

[0106] The specific operation process is as follows:

[0107] 1. Take two aliquots of cells from each of the two groups, add PBS to a final volume of 200 μl, and transfer to a 1.5 ml centrifuge tube. Add 20 μl of proteinase K and 200 μl of proteinase K reaction solution, and vortex to mix.

[0108] 2. Subsequent operating steps are the same as steps 2-11 in Example 1.

[0109] The obtained RNA was detected by RT-PCR using primers designed to amplify only ACTB RNA and primers designed to amplify only ACTB DNA. The detection results are as follows:

[0110] Table 8

[0111]

[0112] Based on the RT-PCR results of this embodiment, the proportion of DNA in the experimental group (treated with Dnase I) was much lower than that in the control group (not treated with Dnase I), meaning that the proportion of DNA decreased after Dnase I treatment.

[0113] Example 6: Test of the effect of magnetic bead method on removing DNA contamination during blood RNA extraction

[0114] This embodiment sets up two groups: an experimental group (treated with Dnase I) and a control group (not treated with Dnase I). The formulations of all other components are the same and identical to those of Group 1 in Example 1. That is, the experimental group performs step 6 of the specific operation process described below, while the control group does not perform step 6. Apart from this, all other steps are the same.

[0115] 1. Transfer 200 μl of blood to a 1.5 ml centrifuge tube. Add 20 μl of proteinase K and vortex to mix.

[0116] 2. Subsequent operating steps are the same as steps 2-11 in Example 1.

[0117] The obtained RNA was detected by RT-PCR using primers designed to amplify only ACTB RNA and primers designed to amplify only ACTB DNA. The detection results are as follows:

[0118] Table 9

[0119]

[0120] Based on the RT-PCR results of this embodiment, the proportion of DNA in the experimental group (treated with Dnase I) was much lower than that in the control group (not treated with Dnase I), meaning that the proportion of DNA decreased after Dnase I treatment.

[0121] Example 7: Test of the effect of magnetic bead method on removing DNA contamination during tissue RNA extraction

[0122] This embodiment sets up two groups: an experimental group (treated with Dnase I) and a control group (not treated with Dnase I). The formulations of all other components are the same and identical to those of Group 1 in Example 1. That is, the experimental group performs step 6 of the specific operation process described below, while the control group does not perform step 6. Apart from this, all other steps are the same.

[0123] 1. Take 10-20 mg of tissue, cut it into small pieces as much as possible, add 20 μl of proteinase K and 200 μl of proteinase K reaction solution, and grind the tissue thoroughly using an electric homogenizer (at low temperature). Shake to mix, and incubate at 56°C for 30 min, inverting and mixing 3 times during the process, 3-5 times each time.

[0124] 2. Subsequent operating steps are the same as steps 2-11 in Example 1.

[0125] The obtained RNA was detected by RT-PCR using primers designed to amplify only ACTB RNA and primers designed to amplify only ACTB DNA. The detection results are as follows:

[0126] Table 10

[0127]

[0128] Based on the RT-PCR results of this embodiment, the proportion of DNA in the experimental group (treated with Dnase I) was much lower than that in the control group (not treated with Dnase I), meaning that the proportion of DNA decreased after Dnase I treatment.

[0129] Example 8: Test of the effect of magnetic bead method on removing DNA contamination when extracting RNA from oral and nasopharyngeal swab samples. This example sets up two groups: an experimental group (treated with Dnase I) and a control group (not treated with Dnase I). The formulations of all other components are the same and identical to those of Group 1 in Example 1. That is, the experimental group performs step 6 of the specific operation process described below, while the control group does not perform step 6. Apart from this, all other steps are the same.

[0130] 1. Swab samples containing preservation solution (virus preservation solution or PBS): Take 200 μl of swab sample into a 1.5 ml centrifuge tube, add 20 μl of proteinase K, and vortex for 10 seconds to mix.

[0131] 2. Subsequent operating steps are the same as steps 2-11 in Example 1.

[0132] The obtained RNA was detected by RT-PCR using primers designed to amplify only ACTB RNA and primers designed to amplify only ACTB DNA. The detection results are as follows:

[0133] Table 11

[0134]

[0135] Based on the RT-PCR results of this embodiment, the proportion of DNA in the experimental group (treated with Dnase I) was much lower than that in the control group (not treated with Dnase I), meaning that the proportion of DNA decreased after Dnase I treatment.

[0136] Example 9: Test of the effect of magnetic bead method on removing DNA contamination when extracting RNA from urine samples

[0137] This embodiment sets up two groups: an experimental group (treated with Dnase I) and a control group (not treated with Dnase I). The formulations of all other components are the same and identical to those of Group 1 in Example 1. That is, the experimental group performs step 6 of the specific operation process described below, while the control group does not perform step 6. Apart from this, all other steps are the same.

[0138] 1. After centrifuging the collected urine sample at 1600g for 10 min at 4℃, remove the supernatant and keep about 1 ml of the lower layer of precipitate. Transfer it to a 1.5 ml centrifuge tube, centrifuge at 12000g for 10 min at 4℃, remove the supernatant and keep about 200 μl of the lower layer of precipitate suspension, add 20 μl of proteinase K, and vortex for 10 sec to mix.

[0139] 2. Subsequent operating steps are the same as steps 2-11 in Example 1.

[0140] The obtained RNA was detected by RT-PCR using primers designed to amplify only ACTB RNA and primers designed to amplify only ACTB DNA. The detection results are as follows:

[0141] Table 12

[0142]

[0143] Based on the RT-PCR results of this embodiment, the proportion of DNA in the experimental group (treated with Dnase I) was much lower than that in the control group (not treated with Dnase I), meaning that the proportion of DNA decreased after Dnase I treatment.

[0144] Example 10: DNA removal treatment during RNA extraction using magnetic beads applied to the detection of HPV virus E6 / E7 mRNA in cervical cells.

[0145] Following the procedure outlined in Example 4, RNA was extracted from 20 clinically collected cervical cells. The extracted RNA was detected using an HPV virus E6 / E7 mRNA detection reagent. The results were compared with those obtained from clinical colposcopy examinations, and the statistics are as follows:

[0146] Table 13

[0147]

[0148]

[0149] The results of this embodiment show that the technique of removing DNA during RNA extraction using magnetic beads can be effectively applied to the detection of HPV virus E6 / E7 mRNA in cervical cells.

Claims

1. A kit for extracting RNA, characterized in that, The product comprises the following components: proteinase K, lysis binding buffer, magnetic beads, DNase reaction solution, washing buffer, and elution buffer. The DNase reaction solution includes Dnase I and Dnase I buffer. The Dnase I buffer contains an aqueous phase, an oil phase, and an emulsifier. The oil phase is dimethyl silicone oil, accounting for 75% of the total volume of the two phases. The emulsifier is Tween 20, accounting for 1% of the total volume of the two phases. The aqueous phase consists of 10 mM pH 7.5 Tris-HCl buffer, 2.5 mM MgCl2, 0.5 mM CaCl2, and 5% PEG.

2. The RNA extraction kit according to claim 1, characterized in that, The concentration of Dnase I in the DNase reaction solution is 40 U / mL-120 U / mL.

3. The RNA extraction kit according to claim 1, characterized in that, The lysis binding buffer comprises the following components: a dissociation salt, a nucleic acid flocculant, a nuclease inhibitor, a reducing agent, a surfactant, isopropanol, and a pH buffer. The dissociation salt comprises one or more of the following components: guanidine hydrochloride, guanidine isothiocyanate, urea, and sodium perchlorate, at a concentration of 3-6 M. The nucleic acid flocculant comprises one or more of the following components: 5%-20% PEG, 2-4 M LiCl, 0.1-0.5 mg / mL Glycogen, and 2-10 μg / μL Carrier RNA. The nuclease inhibitor comprises one or more of the following components: 5 mM-50 mM EDTA, 5 mM-50 mM MEGTA, 0.5-10 mM ATA, 50 mM-200 mM glyceraldehyde, and 1 mg / ml-5 mg / ml NaF. The reducing agent comprises one or more of the following components: 20 mM-200 mM tris(2-carboxyethyl)phosphine hydrochloride, 2%-10% β-mercaptoethanol, and 50-400 mM... DTT; the surfactant includes one or more of the following components: Tween 20, Triton X-100, Nonidet P40, Brij35, at a content of 1%-10%; isopropanol content is 30-60%; the pH buffer includes one or more of the following components: 50mM-250mM pH 6.6 NaAc-HAc buffer, 50mM-250mM pH 6.6 Tris-HCl buffer; the solvent is RNase-free water; The magnetic beads include at least one of hydroxyl magnetic beads and carboxyl magnetic beads; The washing solution is divided into washing solution 1 and washing solution 2; Washing solution 1 comprises the following components: a dissociation salt, ethanol, a pH buffer, and a surfactant; the dissociation salt comprises one or more of the following components: guanidine hydrochloride, guanidine isothiocyanate, urea, and sodium perchlorate, with a concentration of 1-4M; the ethanol content is 30-60%; the pH buffer comprises one or more of the following components: 50mM-250mM pH 6.6 NaAc-HAc buffer and 50mM-250mM pH 6.6 Tris-HCl buffer; the surfactant comprises one or more of the following components: Tween 20, Triton X-100, Nonidet P40, and Brij 35, with a content of 1%-10%; Washing solution 2 comprises the following components: 70%-80% ethanol, pH buffer, and 400-800 mM NaCl; the pH buffer comprises one or more of the following components: 10 mM-50 mM pH 7.5 NaAc-HAc buffer and 10 mM-50 mM pH 7.5 Tris-HCl buffer. The eluent consists of the following components: 0.1% DEPC-treated Water.

4. The use of the kit according to any one of claims 1-3 in RNA extraction.

5. The application according to claim 4, characterized in that, Includes the following steps: 1) Place the collected samples into centrifuge tubes with a sample buffer volume of 150-250 µl, then add 18-22 µl of proteinase K and vortex to mix. 2) After centrifuging at a low speed of 500-2000 rpm for at least 5 seconds, add 250-350 µl of lysis binding solution and 18-22 µl of magnetic beads, shake to mix, and lyse the binding at 56℃ for 8-12 minutes, inverting the container to mix during the process. 3) After centrifuging at a low speed of 500-2000 rpm for at least 5 seconds, place the centrifuge tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear. Then carefully aspirate the liquid. 4) Add 550-650 μl of washing buffer 1 and shake to mix for at least 1 min; 5) After centrifuging at a low speed of 500-2000 rpm for at least 5 seconds, place the centrifuge tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear. Then carefully aspirate the liquid. 6) Gently premix DNase I and DNase I Buffer in the specified ratio. Add 250-700 μl of the premix and gently mix. React at room temperature for 10-20 min. Centrifuge at 500-2000 rpm for at least 5 s. Let the centrifuge tube stand until the magnetic beads are completely adsorbed. Carefully remove the liquid. Add 550-650 μl of Washing Buffer 1 and vortex to mix for at least 1 min. Centrifuge at 500-2000 rpm for at least 5 s. Place the centrifuge tube on a magnetic rack and let it stand for 2-5 min until the solution is completely clear. Carefully remove the liquid. 7) Add 550-650 μl of washing solution 1, shake to mix for at least 1 min, centrifuge at a low speed of 500-2000 rpm for at least 5 s, place the centrifuge tube on a magnetic rack and let it stand for 2-5 min until the solution is completely clear, then carefully aspirate the liquid. 8) Add 550-650 μl of washing solution 2, shake to mix; centrifuge at low speed of 500-2000 rpm for at least 5 seconds, then place the centrifuge tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear, then carefully aspirate the liquid. 9) After centrifuging at a low speed of 500-2000 rpm for at least 5 seconds, place the centrifuge tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear. Then carefully remove all the liquid. 10) Allow the centrifuge tubes to air dry at room temperature for 3-5 minutes; 11) Add 50-100 μl of elution buffer to the centrifuge tube, vortex to mix, and incubate at room temperature for at least 5 min, inverting the tube occasionally to mix. 12) Place the centrifuge tube on a magnetic rack and let it stand for 2-5 minutes until the solution is completely clear. Then carefully transfer the supernatant to a new centrifuge tube for storage.

6. The application according to claim 4, characterized in that, RNA can be extracted from cells, tissues, and urine samples.

7. The application according to claim 4, characterized in that, RNA can be extracted from samples including blood, cervical swabs, oral swabs, and nasopharyngeal swabs.

Images