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WO2022121621A1 - Kit d'extraction d'arn et son procédé - Google Patents

Kit d'extraction d'arn et son procédé Download PDF

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Publication number
WO2022121621A1
WO2022121621A1 PCT/CN2021/130327 CN2021130327W WO2022121621A1 WO 2022121621 A1 WO2022121621 A1 WO 2022121621A1 CN 2021130327 W CN2021130327 W CN 2021130327W WO 2022121621 A1 WO2022121621 A1 WO 2022121621A1
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Prior art keywords
kit
rna
final concentration
tris
washing liquid
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Chinese (zh)
Inventor
王源舒
王岩
林明芳
吴文辉
王寅
吴�琳
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Berryoncology Precision Medical Device Co Ltd
Berry Oncology Co Ltd
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Berryoncology Precision Medical Device Co Ltd
Berry Oncology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

  • the present disclosure relates to the technical field of nucleic acid extraction, and in particular, to a kit for extracting RNA and a method thereof.
  • Circulating nucleic acid (Cell Free Nucleic Acid, cfNA) exists in both normal and patient plasma. Circulating nucleic acid includes cfDNA and cfRNA, which are closely related to tumors, pregnancy-related diseases and autoimmune diseases. With the development of molecular biology, cfNA detection as an effective non-invasive diagnostic technology, its clinical application scope is expanding, including early diagnosis, staging, treatment monitoring, prognosis judgment and prenatal genetic diagnosis and many other aspects.
  • cf-mRNA is a fragmented, less abundant mRNA that can be packaged in e.g. exosomes, microparticles, microvesicles, or multivesicular bodies despite increased levels of RNA hydrolase (RNase) in the blood. In the structure, it can effectively prevent the degradation by RNases.
  • Placenta-derived mRNA can be detected in maternal plasma during pregnancy. Some studies believe that the detection of placental cfRNA can reveal the expression level of fetal genes, which can be used for fetal pathological diagnosis, including chromosomal aneuploidy and preeclampsia. Preeclampsia is a potentially lethal pregnancy disorder, and direct placental tissue monitoring is not feasible due to safety concerns with placental tissue sampling.
  • CCND1 mRNA cyclin D1-encoding RNA
  • Miura's team measured TERT mRNA and EGFR mRNA levels in the serum of lung cancer patients and found that TERT concentrations were associated with tumor size, metastasis, disease recurrence, and smoking.
  • EGFR mRNA concentrations were associated with advanced clinical stage, and postoperative EGFR and TERT levels were significantly decreased.
  • Non-small cell lung cancer patients with EML4-ALK gene rearrangement are sensitive to crizotinib.
  • TEPs Tumor-educated blood platelets
  • RNA expression profiles By sequencing the mRNA of 283 platelet samples, Best et al identified 228 localized and metastatic tumors from 55 healthy individuals with an accuracy rate of 96 percent. %. Across six different tumor types, the location of the primary tumor was correctly identified with 71 percent accuracy. Through mRNA sequencing, researchers can tell the origin of the tumor, and suggest tumor type and molecular subclass.
  • nucleic acid extraction methods mainly include phenol/chloroform extraction method represented by TRIzol, silica gel membrane adsorption column method represented by Qiagen, and silica magnetic bead nucleic acid extraction method represented by MagMax.
  • phenol/chloroform extraction method is cheap, but the reagents are highly toxic, and the operation repeatability between the experimenters is poor, so it is not suitable for the extraction of trace nucleic acids;
  • the silica membrane adsorption column method has high extraction efficiency, but requires manual operation. , the steps are cumbersome, and it is only suitable for the processing of a small number of samples.
  • the commercial magnetic bead technology is mature, and the magnetic bead microspheres are monodispersed and uniform in size and shape. Based on its morphological and structural characteristics, it has a large specific surface area, can bind nucleic acids more efficiently, has higher yield and reproducibility in nucleic acid extraction, and is very suitable for automated applications. Compared with the adsorption column method, it has no risk of being blocked by particles in the sample, and is suitable for nucleic acid extraction of cell-free body fluid samples such as plasma with high protein content and large volume. Magnetic bead nucleic acid extraction method is widely used in the field of biotechnology. However, its application in the field of cfRNA extraction is quite low.
  • the present disclosure provides a kit for extracting RNA, which includes an extraction solution; the extraction solution is composed of an RNase inhibitor, a surfactant, a reducing agent and a buffer;
  • the final concentration of the RNase inhibitor is 0.01-10.00M; the mass percentage concentration of the surfactant is 0.05%-40%; the final concentration of the reducing agent is 0.1-1000mM ; The final concentration of the buffer is 1 to 1000 mM.
  • the present disclosure provides a composition for extracting RNA, the composition comprising an extraction solution;
  • the extraction solution is composed of an RNase inhibitor, a surfactant, a reducing agent and a buffer;
  • the final concentration of the RNase inhibitor is 0.01-10.00M; the mass percentage concentration of the surfactant is 0.05%-40%; the final concentration of the reducing agent is 0.1-1000mM ; The final concentration of the buffer is 1 to 1000 mM.
  • the final concentration of the RNase inhibitor is 1.00-8.00M; the mass percentage concentration of the surfactant is 0.5%-20%; the final concentration of the reducing agent is 0.1-100mM; The final concentration of the buffer is 1 to 500 mM;
  • the final concentration of the RNase inhibitor is 1.00-5.00M; the mass percentage concentration of the surfactant is 0.5%-5%; the final concentration of the reducing agent is 1-5. 50mM; the final concentration of the buffer is 50-260mM.
  • the RNase inhibitor is selected from one or more of guanidine hydrochloride, urea, guanidine isothiocyanate, vanadyl ribonucleoside complex and 8-hydroxyquinoline;
  • the surface active The agent is selected from one or more of PEG200, Triton X-100, Tween 20, SDS, LDS, SLS and NP-40;
  • the reducing agent is selected from dithiothreitol, cysteine, glutathione
  • the buffer solution includes one or more of Tris-HCl, NaCl, PBS and NaOH.
  • the RNase inhibitor is guanidine isothiocyanate and the surfactant is SLS.
  • the kit for extracting RNA according to any one of the above, further comprising magnetic beads.
  • the particle size of the magnetic beads is 0.05-5 ⁇ m
  • the magnetic beads comprise Oligo dT magnetic beads.
  • the kit further comprises: at least one of proteinase K, diluent, washing solution I, washing solution II, washing solution III, washing solution IV and eluent.
  • the diluent includes one or more of Tris-HCl, NaCl, LiCl, KCl, EDTA, PBS and NaOH;
  • the washing liquid I includes one or more of Tris-HCl, LiCl, EDTA, NaCl, NaOH, LDS, SDS, dithiothreitol and 2-mercaptoethanol;
  • the washing liquid II includes one or more of Tris-HCl, LiCl, EDTA, NaCl, NaOH, LDS and SDS;
  • the washing liquid III includes one or more of Tris-HCl, LiCl, EDTA, NaCl and NaOH;
  • the washing liquid IV includes: one or more of Tris-HCl, NaCl, MgCl 2 , KCl, dithiothreitol and 2-mercaptoethanol.
  • the diluent comprises: 10-100 mM Tris-HCl pH 7.0-8.0, 0.1-10.0 M LiCl and 1-40 mM EDTA.
  • the diluent comprises: 20-40 mM Tris-HCl pH 7.0-8.0, 0.5-5.0 M LiCl and 10-20 mM EDTA.
  • the washing liquid I comprises: 80-120 mM Tris-HCl pH 7.0-8.0, 480-520 mM LiCl, 5-20 mM EDTA, the mass percentage concentration is 0.05-1.5% LDS and 1- 10mM DTT.
  • the mass percentage concentration of LDS is 0.05-0.25%.
  • the washing liquid II comprises: 5-15 mM Tris-HCl pH 7.0-8.0, 100-200 mM LiCl, 0.05-2.0 mM EDTA and a mass percentage concentration of 0.05-1% LDS.
  • the washing liquid III comprises: 5-15mM Tris-HCl pH 7.0-8.0, 100-200mM LiCl and 0.05-2.0mM EDTA.
  • the wash solution IV comprises: 30-70 mM Tris-HCl pH 8.0-8.5, 1-10 mM MgCl 2 , 50-90 mM KCl and 1-20 mM DTT.
  • the present disclosure provides a method for extracting nucleic acid, which comprises using the RNA extraction kit according to any one of claims 1 to 5 to extract RNA in a sample.
  • the method includes: mixing the extract in the kit with the sample, so that the concentration of the RNase inhibitor in the mixed solution is 0.5-1.5 mol/L.
  • the concentration of the RNase inhibitor is 0.8-1.3 mol/L.
  • the method further comprises mixing and digesting proteinase K with the extract and the sample to obtain the mixed solution.
  • the final concentration of proteinase K is 0.1-20 mg/ml.
  • the conditions of the digestion are: 55-75° C., 10-30 min.
  • the method when the kit includes a diluent, the method includes mixing the mixed solution with the diluent, so that the concentration of the RNase inhibitor in the diluted mixed solution is 0.35-0.71 mol/L.
  • the method further comprises mixing and incubating the mixed solution or the diluted mixed solution with magnetic beads.
  • the mixed incubation time is 10-180 min.
  • the incubation time is 30-90 min.
  • the sample is selected from any of plasma samples, serum samples, tissue samples, cell samples, urine, tissue culture supernatants, and FFPE samples.
  • the method according to any one of the above further comprising: performing DNA extraction on the sample after RNA extraction.
  • the present disclosure provides use of the kit for extracting RNA described in any one of the above or the composition described above for extracting RNA from a sample.
  • the present disclosure provides use of the RNA extraction kit described in any one of the above or the composition described above for detecting RNA in a sample.
  • the use is the detection of circulating RNA (cfRNA) in a sample.
  • cfRNA circulating RNA
  • the sample is any one of plasma samples, serum samples, tissue samples, cell samples, urine, tissue culture supernatants, and FFPE samples.
  • the sample is a plasma sample or a serum sample.
  • Fig. 1 is the qPCR detection result of verification example 1;
  • Fig. 2 is the qPCR detection result of verification example 2
  • Fig. 3 is the qPCR detection result of verification example 3.
  • Fig. 4 is the qPCR detection result of different concentrations of proteinase K of verification example 4, wherein, group 1 is the proteinase K concentration of 0.6 mg/mL, and group 2 is the proteinase K concentration of 1.2 mg/mL;
  • Fig. 5 is the qPCR detection result of proteinase K of verification example 4 at different action time
  • Fig. 6 is the qPCR detection result of verification example 5.
  • Fig. 7 is the detection result of directly extracting cfDNA and extracting cfDNA after extracting cfRNA (co-extraction) in verification example 7;
  • Fig. 8 is the analysis result after second-generation sequencing of the extracted product in verification example 7;
  • Fig. 9 is the qPCR detection result in verification example 8.
  • Fig. 10 is the result of the completeness of biological information analysis in verification example 9;
  • FIG. 11 is the analysis results of the efficiency and stability of plasma cfRNA extracted by the kit of Example 1 in Verification Example 9;
  • Figure 12 is the analysis results of the efficiency and stability of plasma cfRNA extracted by the exosome kit in Validation Example 9;
  • Figure 13 is the analysis result of the efficiency and stability of plasma cfRNA extracted by the adsorption column kit in Validation Example 9;
  • Figure 14 is the analysis result of the efficiency and stability of plasma cfRNA extracted by the silicon adsorption magnetic bead kit in Verification Example 9;
  • Figure 15 is the qPCR detection result in verification example 10.
  • Fig. 16 is the RNA total amount detection result of embodiment 6 in verification example 11 and Qiagen AllPrep column extraction;
  • Figure 17 shows the qPCR detection results of extracting 4 gradient tissues (0.5mg, 1.0mg, 5.0mg and 10mg) by two methods in Verification Example 11;
  • FIG. 18 shows the results of qPCR detection in Verification Example 12.
  • An embodiment of the present disclosure provides a kit for extracting RNA, which includes an extraction solution; the extraction solution is composed of an RNase inhibitor, a surfactant, a reducing agent and a buffer;
  • the final concentration of the RNase inhibitor is 0.01-10.00M; the mass percentage concentration of the surfactant is 0.05%-40%; the final concentration of the reducing agent is 0.1-1000mM ; The final concentration of the buffer is 1 to 1000 mM.
  • compositions for extracting RNA characterized in that the composition includes an extraction solution; the extraction solution is composed of an RNase inhibitor, a surfactant, a reducing agent and a buffer;
  • the final concentration of the RNase inhibitor is 0.01-10.00M; the mass percentage concentration of the surfactant is 0.05%-40%; the final concentration of the reducing agent is 0.1-1000mM ; The final concentration of the buffer is 1 to 1000 mM.
  • RNA contained in biological samples including tissue samples, cell samples, urine, tissue culture supernatant and FFPE, etc., especially for some samples containing only trace amounts of RNA (such as plasma samples and serum samples) can also effectively extract RNA. If the sample still contains DNA, DNA extraction can be continued after RNA extraction.
  • the RNase inhibitor is selected from one or more of guanidine hydrochloride, urea, guanidine isothiocyanate, vanadyl ribonucleoside complex and 8-hydroxyquinoline;
  • the surfactant is selected from PEG200 one or more of , Triton X-100, Tween 20, SDS, LDS, SLS and NP-40;
  • the reducing agent is selected from dithiothreitol, cysteine, glutathione, 2- One or more of mercaptoethanol and tris(2-formylethyl) phosphine hydrochloride;
  • the buffer solution includes one or more of Tris-HCl, NaCl, PBS and NaOH.
  • the final concentration of the RNase inhibitor can be 0.01-10.00M, 1.00-8.00M, 1.00-6.00M or 1.00-5.00M, such as 0.01M, 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M or 10M.
  • the mass percentage concentration of the surfactant can be 0.05%-40%, 0.5%-20%, 0.5%-15%, 0.5%-10% or 0.5%-5%, such as 0.05%, 0.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40%.
  • the final concentration of the reducing agent may be 0.1-1000 mM, 0.1-200 mM, 0.1-100 mM or 1-50 mM, such as 0.1 mM, 1 mM, 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM or 1000mM;
  • the final concentration of the buffer can be 1-1000mM, 1-500mM, 50-500mM or 50-260mM, such as 1mM, 50mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM or 1000mM.
  • the final concentration of the RNase inhibitor is 1.00-8.00M; the mass percentage concentration of the surfactant is 0.5%-20%; the final concentration of the reducing agent is 0.1-20% 100mM; the final concentration of the buffer is 1-500mM.
  • the final concentration of the RNase inhibitor is 1.00-5.00M; the mass percentage concentration of the surfactant is 0.5%-5%; the final concentration of the reducing agent is 1-5. 50mM; the final concentration of the buffer is 50-260mM.
  • the stability of the extraction system is higher, and it can better cooperate with magnetic beads, especially Oligo dT magnetic beads, to extract RNA more efficiently.
  • the RNase inhibitor is selected from one or more of guanidine hydrochloride, urea, guanidine isothiocyanate, vanadyl ribonucleoside complex and 8-hydroxyquinoline. It is believed, without being bound by theory, that guanidine salts are RNase inhibitors and decoupling agents, a class of powerful protein denaturants that unravel the secondary structure of proteins, degrade cellular structures, and allow Nucleoproteins are rapidly separated from nucleic acids. However, in the case of high protein content in the sample, guanidine salts may lead to episodes of protein, affecting the subsequent hybridization and adsorption of RNA and magnetic beads.
  • the surfactant is selected from PEG200, Triton X-100, Tween 20, Sodium Lauryl Sulfate (SDS), Lithium Lauryl Sulfate (LDS), Sodium N-Lauroyl Sarcosinate (SLS) and NP - One or more of 40.
  • the surfactant disrupts the membrane structure, while it is also an effective protein solubilizer, reducing protein aggregation in the presence of guanidine salts.
  • SDS is a commonly used ionic detergent that disintegrates cell membranes, binding to the hydrophobic portion of membrane proteins and separating them from the membrane.
  • the effect of LDS is similar to that of SDS, and the solubility of LDS is higher than that of SDS at low temperature.
  • the reducing agent is selected from one or more of dithiothreitol (DTT), cysteine, glutathione, 2-mercaptoethanol and tris(2-formylethyl)phosphine hydrochloride .
  • reducing agents can open protein disulfide bonds, making disulfide bond-containing proteins such as RNases more susceptible to changes in higher order structure, more inhibited function and more susceptible to protease degradation. Its reducing power is affected by pH value, and it can only play a reducing effect when the pH value is greater than 7.
  • the buffer includes one or more of Tris-HCl, NaCl, PBS and NaOH.
  • the RNase inhibitor is guanidine isothiocyanate and the surfactant is SLS.
  • the extraction system composed of the combination is more stable and can effectively reduce or inhibit the occurrence of side reactions.
  • the extract contains: 1.0-5.0 M guanidine isothiocyanate, 100-300 mM Tris-HCl pH 7.5-8.0, 1.0-4.50% SLS, and 5.0-25.0 mM DTT.
  • the particle size of the magnetic beads ranges from 50 nm to 5 ⁇ m.
  • the particle size of the magnetic beads can be 100nm ⁇ 5 ⁇ m, 200nm ⁇ 4 ⁇ m, 300nm ⁇ 3 ⁇ m or 500nm ⁇ 2.5 ⁇ m, for example, the particle size of the magnetic beads can be 50nm, 100nm, 200nm, 400nm, 600nm, 800nm, 1 ⁇ m, 1.2 ⁇ m, 1.4 ⁇ m, 1.6 ⁇ m, 1.8 ⁇ m, 2.0 ⁇ m, 2.2 ⁇ m, 2.4 ⁇ m, 2.6 ⁇ m, 3 ⁇ m, 4 ⁇ m and 5 ⁇ m.
  • the magnetic beads can be uniformly sized and monodispersed superparamagnetic particles, which are formed by encapsulating magnetic materials with any one of SiO 2 , polystyrene or agarose polymer layers.
  • the magnetic beads comprise Oligo dT magnetic beads.
  • Oligo dT magnetic beads combined with the reagents in the above kits, can effectively extract RNA from samples containing a small amount of RNA, especially mRNA.
  • Oligo dT magnetic beads can be used commercially available Oligo dT 25 magnetic beads, which can optionally include but are not limited to the following: Oligo d(T) 25 Magnetic Beads (NEB, S1419S), Dynabeads Oligo d(T) 25 (Thermo, 61005), Enriching Oligo dT Magnetic Beads (MPS100/Oligo dT), BioMag-Oligo(dT) (BMDT1000) and Oligo dT Magnetic Beads (MS04dT).
  • the kit further comprises: at least one of proteinase K, diluent, washing solution I, washing solution II, washing solution III, washing solution IV and eluent.
  • proteinase K is a serine protease with broad cleavage activity that has the ability to strongly degrade and digest proteins, and in nucleic acid extraction, proteinase K is able to digest proteins bound to RNA, releasing RNA and dissolved in solution.
  • proteinase K can exist stably in various protein denaturing agents, such as SDS, urea, EDTA chelating agent and reducing agent (sulfhydryl reagent), and has the ability to dissolve protein, which can solve the problem of protein precipitation.
  • proteinase K can also degrade RNA hydrolase and irreversibly inactivate RNA hydrolase, thereby protecting RNA from degradation.
  • the extract is not directly pre-mixed with proteinase K in advance, but is added to the extract and mixed with the sample only during operation.
  • the diluent includes one or more of Tris-HCl, NaCl, LiCl, KCl, EDTA, PBS and NaOH. It is believed, without being bound by theory, that EDTA acts as a metal chelator, chelating metal ions such as Mg 2+ and Ca 2+ , inhibiting the degradation of RNA by RNA hydrolase (RNA hydrolase requires a certain amount of metal ions to act as an auxiliary base). A certain concentration of lithium chloride (LiCl) can specifically reduce the solubility of RNA without affecting the solubility of proteins or DNA, and facilitate the binding of RNA to magnetic beads.
  • LiCl lithium chloride
  • the diluent comprises: 10-100 mM Tris-HCl pH 7.0-8.0, 0.1-10.0 M LiCl and 1-40 mM EDTA. In some typical embodiments, the diluent comprises: 20-40 mM Tris-HCl pH 7.0-8.0, 0.5-5.0 M LiCl and 10-20 mM EDTA.
  • the washing liquid I includes one or more of Tris-HCl, LiCl, EDTA, NaCl, NaOH, LDS, SDS, dithiothreitol and 2-mercaptoethanol.
  • the mass percentage concentration of LDS is 0.05-0.25%, for example, 0.05-0.20%, 0.1-0.20% or 0.15-0.20%.
  • the washing liquid I comprises: 80-120 mM Tris-HCl pH 7.0-8.0, 480-520 mM LiCl, 5-20 mM EDTA, the mass percentage concentration is 0.05-1.5% LDS and 1- 10mM DTT.
  • the washing liquid II includes one or more of Tris-HCl, LiCl, EDTA, NaCl, NaOH, LDS and SDS.
  • the washing liquid II comprises: 5-15 mM Tris-HCl pH 7.0-8.0, 100-200 mM LiCl, 0.05-2.0 mM EDTA and a mass percentage concentration of 0.05-1% LDS.
  • the washing liquid III includes one or more of Tris-HCl, LiCl, EDTA, NaCl and NaOH.
  • the washing liquid III comprises: 5-15mM Tris-HCl pH 7.0-8.0, 100-200mM LiCl and 0.05-2.0mM EDTA.
  • the washing liquid IV includes: one or more of Tris-HCl, NaCl, MgCl 2 , KCl, dithiothreitol and 2-mercaptoethanol.
  • the wash solution IV comprises: 30-70 mM Tris-HCl pH 8.0-8.5, 1-10 mM MgCl 2 , 50-90 mM KCl and 1-20 mM DTT.
  • An embodiment of the present disclosure also provides a method for extracting nucleic acid, which includes using the RNA extraction kit according to any of the foregoing embodiments to extract RNA from a sample.
  • the method includes: mixing the extract in the kit with the sample, so that the concentration of the RNase inhibitor in the mixed solution is 0.5-1.5 mol/L. Within this concentration range, it is beneficial to fully inhibit the activity of RNA hydrolase in the stage of RNA release, and at the same time, in the stage of hybridization and adsorption, it does not affect the adsorption of RNA by magnetic beads.
  • the concentration of RNase inhibitor in the mixed solution, is 0.5mol/L, 0.7mol/L, 0.9mol/L, 1.1mol/L, 1.3mol/L and 1.5mol/L any of the . In a typical embodiment, in the mixed solution of the extract and the sample, the concentration of the RNase inhibitor is 0.8-1.3 mol/L.
  • the method further comprises mixing and digesting proteinase K with the extract and the sample to obtain the mixed solution.
  • the final concentration of proteinase K is 0.1-20 mg/ml.
  • the final concentration of proteinase K can be selected from: 0.5-20 mg/ml, 0.5-15 mg/ml or 1.0-20 mg/ml, such as 0.1 mg/ml, 0.3 mg/ml, 0.6 mg/ml, 0.9 mg/ml, 1 mg/ml, 3 mg/ml, 6 mg/ml, 9 mg/ml, 11 mg/ml, 13 mg/ml, 16 mg/ml or 20 mg/ml.
  • the concentration of proteinase K in the mixed solution is 0.6 mg/ml.
  • the conditions of the digestion are: 55-75° C., 10-30 min.
  • the method when the kit includes a diluent, the method includes mixing the mixed solution with the diluent, so that the concentration of the RNase inhibitor in the diluted mixed solution is 0.35 ⁇ 0.71mol/L.
  • RNA release stage a certain concentration of RNase inhibitors is required to depolymerize proteins, release RNA, and inhibit RNase activity, but high concentrations of RNase inhibitors (such as GTC) will also affect the base ratio, and added
  • RNase inhibitors such as GTC
  • the diluent reduces the concentration of RNase inhibitor in the solution, so that the system can not only release RNA, but also effectively carry out magnetic bead hybridization and adsorption of RNA. In this concentration range, the extraction effect is better.
  • the method further comprises mixing and incubating the mixed solution or the diluted mixed solution with magnetic beads.
  • the mixed incubation time is 10-180 min. In some typical embodiments, the mixed incubation time is 30-90 min.
  • the sample may be selected from any of plasma samples, serum samples, tissue samples, cell samples, urine, tissue culture supernatants, and FFPE samples.
  • the method further comprises: performing DNA extraction on the sample after RNA extraction.
  • the method of DNA extraction can be performed by using any existing reagents and methods for DNA extraction, which will not be repeated here.
  • One embodiment of the present disclosure provides the use of a kit or composition for extracting RNA for extracting RNA from a sample.
  • One embodiment of the present disclosure provides a kit for extracting RNA or use of the composition for detecting RNA in a sample.
  • the use is to detect circulating RNA (cfRNA) in a sample.
  • cfRNA circulating RNA
  • the sample is any one of plasma samples, serum samples, tissue samples, cell samples, urine, tissue culture supernatants, and FFPE samples.
  • the sample is a plasma sample or a serum sample.
  • the present disclosure provides a kit for extracting RNA and a method thereof.
  • the kit for extracting RNA is composed of an RNase inhibitor, a surfactant, a reducing agent and a buffer.
  • the RNase inhibitor The final concentration of the reducing agent is 0.01-10.00M; the mass percentage concentration of the surfactant is 0.05%-40%; the final concentration of the reducing agent is 0.1-1000mM; the final concentration of the buffer is 1-1000mM ,
  • the kit can efficiently extract RNA contained in biological samples, including tissue samples, cell samples, urine, tissue culture supernatant and FFPE, etc., especially for samples that are difficult to extract RNA (such as plasma samples and serum samples).
  • RNA has the advantages of simple operation, short operation time, high operation stability, strong repeatability and high extraction efficiency.
  • the use of the kit will not affect the DNA in the sample, and the DNA extraction can be continued after RNA extraction, which effectively improves the utilization rate of biological samples.
  • This embodiment provides a kit, which includes the following reagents: extracting liquid, diluent, washing liquid I, washing liquid II, washing liquid III, washing liquid IV, eluent, proteinase K and magnetic beads.
  • the extract contained: 4.14M guanidine isothiocyanate, 255mM Tris-HCl pH 8.0, 4.14% SLS and 20.7mM DTT.
  • Dilutions contained: 35 mM Tris-HCl pH 8.0, 1.28 M LiCl and 12.8 mM EDTA.
  • Wash Solution I contains: 100 mM Tris-HCl pH 7.5, 500 mM LiCl, 10 mM EDTA, 0.1% LDS and 5 mM DTT.
  • Wash Solution II contains: 10 mM Tris-HCl pH 7.5, 150 mM LiCl, 1 mM EDTA and 0.1% LDS.
  • Wash Solution III contains: 10 mM Tris-HCl pH 7.5, 150 mM LiCl and 1 mM EDTA
  • Wash Solution IV contained: 50 mM Tris-HCl pH 8.3, 3 mM MgCl2 , 75 mM KCl and 10 mM DTT.
  • the eluent contained: 10 mM Tris-HCl pH 7.5.
  • the concentration of proteinase K was 20 mg/mL
  • the magnetic beads are Oligo dT magnetic beads with a particle size of 1 ⁇ m or 2.8 ⁇ m.
  • This example provides a kit, which is roughly the same as Example 1, except that the extraction solution is different: the extraction solution contains: 1.625M guanidine isothiocyanate, 100mM Tris-HCl pH 8.0, 1.625% SLS and 8.125mM DTT.
  • This example provides a kit, which is roughly the same as Example 1, except that the extraction solution is different: the extraction solution contains: 3.25M guanidine isothiocyanate, 100mM Tris-HCl pH 7.5, 1.625% SLS and 8.125mM DTT.
  • This example provides a kit, which is roughly the same as Example 1, except that it does not contain washing liquid IV, and the eluent is different: the eluent contains: RNase-free water.
  • This example provides a kit, which is roughly the same as Example 1, except that the extraction solution is different: the extraction solution contains: 1M guanidine isothiocyanate, 100mM Tris-HCl pH 7.0, 1.625% SLS and 8.125mM DTT.
  • This embodiment provides a method for extracting RNA.
  • the kit provided in the above embodiment can be used to extract RNA from a plasma sample, which specifically includes the following steps.
  • the system can refer to Table 2, after fully mixing, invert at room temperature Incubate for 30min.
  • washing liquid I 1 mL of washing liquid I, fully resuspend, transfer to a new clean 1.5 mL low-adsorption centrifuge tube, place it on a magnetic stand for adsorption for 5 min or until it is clear, discard the supernatant;
  • washing liquid III 0.5 mL of washing liquid III, fully resuspend, and place it on a magnetic stand for adsorption for 10 min or until it becomes clear, and discard the supernatant.
  • Washing Solution III 0.2 mL of Washing Solution III, and after fully mixing by blowing and suction, transfer the solution to a new clean 0.2 mL PCR tube, put it on the shelf for 5 min or until it is clear, and discard the supernatant.
  • the product obtained after washing with washing liquid IV can be directly used for RT-PCR or next-generation sequencing library construction, or refer to the eluent volume shown in Table 3, add the eluent, heat at 75 °C for 2 min, and quickly put Adsorb on a magnetic rack for 10s, and immediately transfer the supernatant to a clean new PCR tube to obtain an RNA elution product.
  • the eluted products were subjected to quality inspection or library construction, or stored at -80°C.
  • This example provides a method for extracting RNA, which is used for RNA extraction from tissue samples.
  • the difference from Example 6 is that in the hybridization adsorption stage, 200 ⁇ L of Ligo dT magnetic beads are used for extraction, and 80 ⁇ L of eluent is used for elution. , and other steps are the same as in Example 6.
  • This embodiment provides a method for extracting nucleic acid, which specifically includes the following steps.
  • step (3) Directly extract cfDNA from plasma according to the instructions of plasma cell-free DNA extraction kit (magnetic bead method) (R0011) and QIAamp Circulating Nucleic Acid Kit (55114). It is also possible to add proteinase K to the supernatant obtained in step (2), and then add SLS (if LDS or SDS is added, cfDNA cannot be carried out) according to the ratio described in the instructions of MagMax Cell-Free DNA (cfDNA) Isolation (A29319). extraction) for digestion. DNA extraction was then performed according to MagMax (A29319) automated extraction instructions.
  • MagMax MagMax
  • This example provides a method for extracting RNA, which is roughly the same as the method in Example 6, except that the RNA release stage is different.
  • the specific steps are as follows: according to the volume of the plasma sample, add the extract and proteinase K into the low adsorption centrifuge tube in sequence. , the specific system can refer to Table 8. After fully mixing, incubate at 65°C for 10-30min, and mix once every 5min to obtain a sample mixed solution.
  • the extract of the above-mentioned test group was used and the proportions of other components except the variables in the kit of Example 1 were used, and the method in Example 6 was used to detect the ACTB gene in the extracted product by qPCR.
  • the quantitative results of qPCR please refer to Figure 1, from the quantitative results of qPCR, it is found that the system stability of v5 is better.
  • the above concentration is the final concentration of each component in the RNA release stage in Example 6.
  • the mass percentage concentration of SLS is 1%
  • the DTT concentration is 5 mM, which correspond to the parameters of the extract in Example 1.
  • the extract of the above-mentioned test group was used, and the proportions of other components except the variables in the kit of Example 1 were used, and the ACTB gene in the extracted product was detected by qPCR using the method in Example 6.
  • the detection results are shown in Figure 2 shown.
  • a in Figure 2 is the qPCR quantitative results of G1-G7
  • B in Figure 2 is the qPCR quantitative results of G8-G10.
  • the results showed that the extraction efficiency was related to the GTC concentration in the hybridization adsorption stage. Higher concentrations of GTC would affect the RNA extraction efficiency. When the GTC concentration in the hybridization adsorption stage was 0.35-0.71M, the extraction efficiency was better.
  • Example 6 Three groups of reaction systems for nucleic acid extraction were set up, and the LiCl concentration in the diluent of Example 1 was used as a variable to prepare different diluents, so that in the hybrid adsorption stage system, the LiCl concentrations were 0.4M, 0.5M and 0.6M, respectively.
  • the ACTB gene in the extracted product was detected by qPCR. Please refer to A in Figure 3 for the test results.
  • Example 1 Six groups of reaction systems for nucleic acid extraction were set up, and the kit of Example 1 was used. The difference was that the concentrations of Tris-HCl and EDTA in the diluent were used as variables to prepare different diluents, so that in the hybridization adsorption stage system, the Tris-HCl and EDTA concentrations were used as variables. The concentrations were 50 mM and 100 mM; the concentrations of EDTA were 1 mM, 5 mM and 10 mM, respectively, and the method in Example 6 was used to detect the ACTB gene in the extracted products by qPCR. Please refer to B in Figure 3 for the test results.
  • the detection results showed that the extraction efficiency was better when the concentration of Tris-HCl in the hybrid adsorption system was 50 mM, the concentration of EDTA was 5 mM and the concentration of LiCl was 0.5 M. That is, corresponding to the parameters of the diluent in Example 1, when the concentration of Tris-HCl is 34.88 mM, the concentration of EDTA is 12.79 mM, and the concentration of LiCl is 1279 mM, the extraction effect is better.
  • PK proteinase K
  • Example 1 the concentration of proteinase K was 20 mg/mL
  • group 2 plasma samples were not digested with proteinase K.
  • Plasma contains a large amount of albumin, globulin and fibrinogen. Under the action of the extract, the plasma salt concentration changes, the plasma protein denatures, and the solubility decreases, resulting in the precipitation of plasma proteins.
  • Example 2 Referring to the kit of Example 1, and using the method in Example 6 to perform nucleic acid extraction. The difference is that the concentration of proteinase K was used as a variable, and two experimental groups were set up to extract RNA from the samples, so that in the RNA release system, the proteinase K concentration reached 0.6 mg/mL (group 1) and 1.2 mg/mL (group 2), respectively. ).
  • RNA content of the extracted products was determined by qPCR, and the results are shown in Figure 4. It can be seen from the results that when the concentration of proteinase K in the system reaches 0.6 mg/mL, plasma proteins can be fully hydrolyzed to release RNA.
  • Example 5 Referring to the kit of Example 1, and using the method in Example 6 to perform nucleic acid extraction. The difference is that the incubation time was used as a variable, and three experimental groups were set up. After adding protease, they were incubated for 10 min, 20 min and 30 min respectively, and the RNA content of the extracted products was determined by qPCR. The results are shown in Figure 5.
  • Example 6 Referring to the kit of Example 1, and using the method in Example 6 to perform nucleic acid extraction. The difference is that the pH in the extract and the diluent is used as a variable, and 4 groups of test groups are set up. RNA content. The results are shown in Figure 6.
  • Example 6 Referring to the kit of Example 1, and using the method in Example 6 to perform nucleic acid extraction. The difference is that, taking the LDS concentration of the washing liquid I as a variable, three groups of test groups were set up, and the LDS concentrations of the washing liquid I were 1%, 0.5% and 0.1% respectively.
  • RNA content of the two internal reference genes (ACTB and GAPDH) in the extracted product was detected by qPCR method, and the detection results of the internal reference genes in the washing liquid I_v3 are shown in Table 11.
  • Example 1 the plasma samples were subjected to extraction of cfDNA and cfRNA according to the method in Example 8. After extracting cfRNA, keep the supernatant and continue to extract cfDNA. At the same time, MagMax (A29319) kit was used to directly extract the cfDNA of the same amount of plasma, and the steps were referred to the instructions of the kit. Three parallel controls were set up for each method.
  • Example 1 Silicon adsorption magnetic bead kit (MagMax TM cfTNA Isolation kit, Thermo, A36716), adsorption column kit (Quick-cfRNA TM Serum&Plasma Kit, ZYMO RESEARCH, R1059), exosome kit (VEX Exosome Isolation Reagent, Vazyme) were used respectively.
  • R603 combined with FastPure TM Cell Tissue Total RNA Isolation Mini Kit, Vazyme, R101
  • the operation steps of Example 1 refer to the method of Example 6, and other reagents
  • the operating steps of the kit refer to the operating steps of the instructions of each commercial kit.
  • RNA The expression levels of genes such as ACTB, GAPDH, CD74 and SDC4 were detected by qPCR.
  • a pair of primers were designed at the 5' and 3' ends of the ACTB gene, and the expression levels of different regions of the ACTB gene were detected by qPCR. level, indicating the integrity of the RNA.
  • each parallel reaction was measured three times, and the results were averaged.
  • Figure 9 for the quantitative results of qPCR. It can be seen from the results that the extraction efficiency and stability of the kit provided in Example 1 are better than other commercial kits (the Ct value is the lowest), and can ensure the integrity of the RNA (ACTB gene). There is no significant difference in the content of the 3' and 5' ends).
  • Example 1 Using silicon adsorption magnetic bead kit (Thermo, A36716), adsorption column kit (ZYMO RESEARCH, R1059), exosome kit (Vazyme, R603 combined with R101) and the kit described in Example 1 of the present disclosure, extract the same
  • the operation steps of Example 1 refer to the method of Example 6, and the operation steps of other kits refer to the operation steps of the instructions of each commercial kit.
  • the cfRNA in 2 mL of plasma was extracted, the product was constructed into a transcriptome library, and the next-generation sequencing was performed.
  • the experimental results are shown in Table 12.
  • FIG. 10 The integrity of plasma cfRNA extracted by the four kits of Example 1, exosomes, adsorption columns and silicon magnetic beads is shown in Figure 10, respectively.
  • a in Figure 10 is the result of Example 1
  • B in Figure 10 is the result of the exosome kit
  • C in Figure 10 is the silicon magnetic bead kit
  • D in Figure 10 is the adsorption column kit.
  • Three parallel control transcriptome libraries were constructed for each kit. By analyzing the number of genes and the number of shared genes between the parallel controls, the efficiency and stability of the plasma cfRNA captured by the reaction kits were analyzed. The results are shown in Figures 11 to 14.
  • the kit of Example 1 can specifically extract RNA in plasma without affecting DNA in plasma.
  • the kit can be used to extract biological samples in the volume range of 0.5 to 4 mL, which can meet the requirements of different biological samples. Due to the high upper limit of RNA extraction, it can be applied not only to the extraction of trace amounts of RNA from plasma samples, but also to the extraction of samples with high RNA content such as cells, tissues, and platelet-rich plasma.
  • the kit is a magnetic bead nucleic acid extraction method. Compared with the extraction column method, it is suitable for automatic extraction.
  • Example 1 The same part of PRP was extracted by using silicon adsorption magnetic bead kit (MagMax TM A36716), adsorption column kit (Quick-cfRNA TM R1059), exosome kit (Vazyme R603&R101) and the kit described in Example 1 of the present disclosure, respectively.
  • silicon adsorption magnetic bead kit MagneticMax TM A36716)
  • adsorption column kit Quick-cfRNA TM R1059
  • exosome kit Vazyme R603&R101
  • RNA content in PRP plasma is much higher than that in ordinary plasma, and the extraction efficiency is affected by the limit of the kit. It can be seen from the results that the upper limit of RNA extraction by the kit of Example 1 of the present disclosure is higher than that of other kits.
  • Example 7 Take 10 mg, 5 mg, 1 mg and 0.5 mg of liver tissue samples, and use the method of Example 7 and Qiagen AllPrep DNA/RNA Mini Kit (Cat. No.: 80204) to extract RNA in the tissue samples, and determine by qPCR method.
  • RNA detection results of Example 7 and Qiagen AllPrep column extraction are shown with reference to Figure 16, and it can be seen from the results that both methods have a good linear relationship and are feasible.
  • Example 6 Using the kit of Example 1, based on the method provided in Example 6, 2 mL of plasma was automatically extracted, and the products of different extraction wells (A1, A6, B3, D1 and D6) were randomly extracted for qPCR detection. The results are shown in Figure 18 .
  • a transcriptome library was constructed, and the next-generation sequencing results are shown in Table 14.
  • kits N Use the commercial OligodT magnetic bead kit N and the kit described in Example 1 to extract the cfRNA of the same plasma, and use the extraction steps of the kit of Example 1 to refer to the method of Example 6.
  • the operation steps of the kit N are: Take two times, four times, and eight times the plasma volume of Kit N's LysisBuffer (components: 100mM Tris-HCl, 500mM LiCl, 10mM EDTA, 5mM DTT, and 1% LDS), add it to the plasma, and mix it. Use according to the LysisBuffer volume, according to the instructions for RNA extraction.
  • RNA content of two internal reference genes (ACTB and GAPDH) in the extracted products was detected by qPCR method, and the detection results are shown in Table 15.
  • the present disclosure provides a kit for extracting RNA and a method thereof, which can efficiently extract RNA contained in biological samples, including tissue samples, cell samples, urine, tissue culture supernatant and FFPE, etc.
  • RNA extraction samples (such as plasma samples and serum samples) can also be effectively extracted for RNA, which has the advantages of simple operation, short operation time, high operation stability, strong repeatability and high extraction efficiency.
  • the use of the kit will not affect the DNA in the sample, and the DNA extraction can be continued after RNA extraction, which effectively improves the utilization rate of biological samples and has a wide range of application value.

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Abstract

L'invention concerne un kit d'extraction d'ARN et son procédé. Le kit comprend une solution d'extraction composée d'un inhibiteur d'enzyme d'ARN, d'un tensioactif, d'un agent réducteur et une solution tampon, la concentration finale de l'inhibiteur d'enzyme d'ARN étant de 0,01 à 10,00 M, la concentration en pourcentage en masse du tensioactif étant de 0,05 % à 40 %, la concentration finale de l'agent réducteur étant de 0,1 à 1000 mM, et la concentration finale de la solution tampon étant de 1 à 1000 mM. Le kit peut être utilisé pour extraire l'ARN contenu dans un échantillon, et en particulier peut être utilisé pour extraire efficacement l'ARN d'échantillons tels que le plasma, les cellules et les tissus. De plus, l'utilisation du kit n'influence pas l'ADN dans l'échantillon, et l'ADN peut être extrait de façon continue après l'extraction d'ARN, de telle sorte que le taux d'utilisation de l'échantillon biologique est efficacement amélioré.
PCT/CN2021/130327 2020-12-11 2021-11-12 Kit d'extraction d'arn et son procédé Ceased WO2022121621A1 (fr)

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WO2024023335A1 (fr) * 2022-07-29 2024-02-01 Sanofi Procédés de purification d'arnm sans éthanol
CN118389492A (zh) * 2024-07-01 2024-07-26 广州凯普医药科技有限公司 一种磁珠法提取干血斑基因组dna的试剂盒及其应用
CN120992823A (zh) * 2025-10-22 2025-11-21 珠海市祥臻生物科技有限公司 一种毛发裂解液及其应用

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CN113549674B (zh) * 2021-04-22 2023-11-03 福建和瑞基因科技有限公司 一种检测样本中目标序列整合和突变的方法及其引物的设计方法和试剂盒
CN113444771B (zh) * 2021-05-20 2025-12-02 康码芯(上海)智能科技有限公司 适合dna或rna病毒直接扩增的裂解剂、试剂盒及其在病毒pcr检测中的应用
CN113930418B (zh) * 2021-10-14 2024-02-23 杭州迪安生物技术有限公司 核酸释放剂及其核酸释放方法
CN114107446A (zh) * 2021-12-16 2022-03-01 福建和瑞基因科技有限公司 一种核酸的检测试剂盒及其检测方法
CN115305246B (zh) * 2022-08-25 2025-09-30 武汉纺织大学 磁珠法提取完整型细菌总rna的试剂盒及其提取方法
CN116790577B (zh) * 2023-08-17 2023-11-17 中国海关科学技术研究中心 一种rna保存液及其用途

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