Method for extracting blood RNA and application thereof

Abstract

The application relates to the technical field of nucleic acid extraction, in particular to a blood RNA extraction method and application thereof in fusion gene detection. The blood RNA extraction method can significantly improve RNA yield and purity, and has less DNA residue. Based on the fluorescence quantitative PCR technology of the extracted RNA, the detection sensitivity of leukemia related fusion genes can be improved, and a more reliable basis can be provided for clinical diagnosis. The scheme can realize automatic operation, and the extraction process can be completed within 1 hour without manual intervention. The traditional reagent method needs 24 hours, so the time of the test personnel is greatly saved.

Description

Technical Field

[0001] This application relates to the field of nucleic acid extraction technology, specifically to a blood RNA extraction method and its application in fusion gene detection. Background Technology

[0002] Leukemia is a type of hematologic malignancy characterized by the clonal malignant proliferation of hematopoietic stem / progenitor cells. Its diagnosis, classification, risk stratification, and minimal residual disease (MRD) monitoring all heavily rely on molecular biomarkers. Fusion genes formed by chromosomal translocations (such as BCR-ABL1, PML-RARA, RUNX1-RUNX1T1, TEL-AML1, etc.) are not only the core basis for WHO hematologic malignancy classification but also key targets for targeted therapy and efficacy evaluation. Therefore, rapid, sensitive, and stable detection of fusion gene transcripts (RNA) in the blood has significant clinical value in initial diagnosis, treatment, and relapse monitoring. Currently, the clinical laboratory procedure for detecting fusion genes in the blood typically includes "sample collection → RNA extraction → reverse transcription → real-time quantitative PCR (RT-qPCR) or next-generation sequencing (NGS)". However, fusion gene detection faces significant challenges in sample pretreatment: clinical samples are usually whole blood or bone marrow blood, with a low proportion of white blood cells (especially post-treatment samples), and RNA is easily degraded by RNases in plasma. Therefore, RNA extraction is the bottleneck that determines the sensitivity, accuracy, and reproducibility of subsequent detection.

[0003] Currently, the Trizol method is the most widely used blood RNA extraction method in clinical laboratories. This method is based on the phenol-chloroform extraction principle, releasing nucleic acids through cell lysis and utilizing the differences in the distribution of different molecules in the organic and aqueous phases to achieve RNA separation. According to the operating procedures, a single Trizol process requires sample homogenization, chloroform extraction, and isopropanol precipitation, with a standard operating time of approximately one hour. However, if auxiliary steps such as sample aliquoting, centrifugation, and reagent equilibration are included, the actual processing time often extends to 3-4 hours. Furthermore, Trizol cannot directly process whole blood; red blood cells must first be lysed and repeatedly centrifuged and washed, which adds nearly an hour or more to the processing time. This lengthy process not only reduces the turnaround efficiency of clinical testing but also leads to the loss of low-abundance fusion gene transcripts in the sample due to the susceptibility of RNA to RNase degradation in the in vitro environment. Furthermore, the Trizol method relies on manual operation, which is cumbersome and prone to subjective errors. For example, differences in centrifugation speed during chloroform separation (12,000×g vs. 2,600×g) can cause RNA yield fluctuations of over 20%, while organic phase residues may inhibit subsequent RT-qPCR reactions, reducing detection sensitivity by approximately 30%. These drawbacks are particularly prominent in the detection of minimal residual disease (MRD) in leukemia, often leading to false negative results and delaying treatment decisions.

[0004] To overcome the limitations of manual operation, magnetic bead extraction has been introduced into clinical practice as an emerging nucleic acid extraction technology. Its principle involves using magnetic nanoparticles with surface-modified functional groups to specifically bind to RNA in a specific buffer system, achieving purification through magnetic field separation. Compared to the Trizol method, magnetic bead extraction eliminates the need for centrifugation and can achieve high-throughput processing via automated workstations, meeting the needs of clinical laboratories for batch sample testing. However, existing magnetic bead methods for blood RNA extraction still face significant technical bottlenecks: First, the extraction yield and purity are greatly affected by the sample matrix. High concentrations of hemoglobin, bilirubin, and other substances in the blood can non-specifically adsorb onto the surface of magnetic beads, leading to a decrease in RNA adsorption efficiency. Furthermore, residual hemoglobin after elution can inhibit reverse transcriptase activity, reducing RT-qPCR signals by 40%-60%. Second, existing magnetic bead kits exhibit poor yield stability, with extraction efficiency differences between different manufacturers reaching more than three times, and batch-to-batch coefficients of variation often exceeding 15%, making it difficult to meet the standardized requirements of clinical testing. Third, to achieve ideal extraction results, existing methods typically only extract 200-300 µL of anticoagulated blood samples, which significantly reduces the sensitivity for detecting fusion genes.

[0005] While the centrifugation column method, which runs parallel to the magnetic bead method, can achieve high RNA concentrations, it relies on the adsorption properties of silica membranes, remains limited by the centrifuge, and is difficult to automate. More importantly, the centrifugation column method has limited ability to remove impurities such as lipids and proteins from blood, and residual inhibitors in the extracted RNA can significantly reduce the sensitivity of downstream detection.

[0006] From a clinical perspective, leukemia diagnosis and treatment place three core requirements on RNA extraction technology: high efficiency (the entire process from sample reception to RNA acquisition must be completed within one hour to meet emergency testing needs); high sensitivity (avoiding missed detections of minimal residual disease); and automation compatibility (integration into automated laboratory systems to reduce the risk of contamination from manual intervention). However, current technologies cannot simultaneously meet these requirements: the Trizol method is inefficient and difficult to automate, the centrifugation column method cannot overcome equipment limitations, and existing magnetic bead methods are limited by sample matrix interference and yield stability.

[0007] In summary, the sample preprocessing stage for leukemia fusion gene detection still faces many technical challenges, and existing extraction methods struggle to balance efficiency, sensitivity, automation, and cost control. Summary of the Invention

[0008] To address the problems of existing technologies, this application aims to provide an optimized magnetic bead-based blood RNA extraction technology, which optimizes the lysis-binding buffer system to achieve efficient capture and purification of RNA in blood, while being compatible with fully automated operation, laying a key foundation for improving the level of molecular diagnosis of leukemia.

[0009] In a first aspect, this application provides a method for extracting RNA, the method comprising: adding a lysis binding solution, proteinase K and magnetic beads to a blood sample for incubation, obtaining the adsorbed magnetic beads through magnetic separation, and further washing and eluting.

[0010] The blood sample is peripheral blood, bone marrow blood, or umbilical cord blood.

[0011] The blood sample was whole blood.

[0012] In some embodiments, the blood sample is a fresh, anticoagulated whole blood sample from a clinical setting.

[0013] In some embodiments, the blood sample is peripheral whole blood, bone marrow whole blood, or umbilical cord whole blood.

[0014] The pyrolysis binding solution comprises polyglycerol-3 acetate, lithium perchlorate, and polyethylene glycol. Preferably, the pyrolysis binding solution further comprises one or more of sodium acetate, disodium ethylenediaminetetraacetate, hexadecyltrimethylammonium bromide, or isopropanol.

[0015] In some embodiments, the cleavage binding solution includes polyglycerol-3 acetate, lithium perchlorate, polyethylene glycol, sodium acetate, disodium ethylenediaminetetraacetate, hexadecyltrimethylammonium bromide, and isopropanol.

[0016] The pH of the lysis binding solution is 5.0-8.0, for example 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0; preferably 5.0-7.5, more preferably 5.5-7.0.

[0017] Preferably, the lysis binding solution comprises 5-15% (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15%, preferably 6-12.5% ​​or 8-12%) of polyglycerol-3 acetate, 1-8M (e.g., 1, 2, 3, 4, 5, 6 or 8, preferably 2-7M or 3-6M) of lithium perchlorate and 5-25% (e.g., 5, 15, 20 or 25%, preferably 8-22% or 10-20%) of polyethylene glycol.

[0018] Preferably, the lysis binding solution comprises 5-15% (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15%, preferably 6-12.5% ​​or 8-12%) of polyglycerol-3. Acetate, 100-400mM (e.g., 100, 125, 150, 175, 200, 250, 300, 350, 400mM, preferably 100-300mM or 100-250mM) sodium acetate, 1-8M (e.g., 1, 2, 3, 4, 5, 6 or 8, preferably 2-7M or 3-6M) lithium perchlorate, 10-50mM (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, preferably 15-40mM or 20-40mM) disodium ethylenediaminetetraacetate, 0.5-10% (e.g.) For example, 0.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10%, preferably 1-8% or 1.5-6%) of hexadecyltrimethylammonium bromide, 5-25% (e.g., 5, 7, 8, 10, 15, 19, 20, 21, 22, 23, 24, or 25%, preferably 8-22% or 10-20%) of polyethylene glycol, and 15-50% (e.g., 15, 20, 25, 30, 35, 40, 45, or 50%, preferably 20-40% or 20-45%) of isopropanol. The concentrations of the polyglycerol-3 acetate, hexadecyltrimethylammonium bromide, polyethylene glycol, and isopropanol are expressed as mass-volume percentages.

[0019] In some embodiments, the cleavage binder comprises: 6-12.5% ​​polyglycerol-3 acetate, 100-300 mM sodium acetate, 2-7 M lithium perchlorate, 15-40 mM disodium ethylenediaminetetraacetate, 1-8% hexadecyltrimethylammonium bromide, 8-22% polyethylene glycol, and 20-45% isopropanol.

[0020] In some embodiments, the cleavage binder comprises: 8-12% polyglycerol-3 acetate, 100-250 mM sodium acetate, 3-6 M lithium perchlorate, 20-40 mM disodium ethylenediaminetetraacetate, 1.5-6% hexadecyltrimethylammonium bromide, 10-20% polyethylene glycol, and 20-40% isopropanol.

[0021] In some embodiments, the cleavage binder comprises: 10% polyglycerol-3 acetate, 125 mM sodium acetate, 4 M lithium perchlorate, 25 mM disodium ethylenediaminetetraacetate, 2.5% hexadecyltrimethylammonium bromide, 15% polyethylene glycol, and 30% isopropanol.

[0022] Preferably, the polyethylene glycol has a molecular weight of 100-20000, for example, 100, 200, 300, 400, 500, 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000 or 20000; more preferably 500-10000, more preferably 1000-8000, and most preferably 2000-6000.

[0023] The polyglycerol-3 acetate in the lysis binding solution consists of a triglyceride backbone (polyhydroxy hydrophilic head) and acetate esterification groups (hydrophobic tail), exhibiting a high hydrophilic-lipophilic balance (HLB value approximately 8-12). It is a moderately hydrophilic nonionic surfactant and, as a composite adjuvant, combines protein dissolution and RNA stabilization. Sodium acetate provides a pH-stable buffer environment, maintaining the charge state of nucleic acid phosphate groups and protecting RNA bases. Lithium perchlorate is a dissociative agent that disrupts the three-dimensional structure of proteins, rapidly disrupting cell membranes or viruses, releasing nucleic acids, and denaturing proteins, thus allowing nucleic acids to escape protein entanglement. Simultaneously, lithium perchlorate is a strong inhibitor of nucleases, reducing and inhibiting nuclease activity. Furthermore, it provides a large number of monovalent cations (lithium ions) to establish salt bridges between nucleic acid phosphate groups and the silanol surfaces of magnetic beads. Hexadecyltrimethylammonium bromide (CTAB) is a cationic surfactant that dissolves lipids and promotes hydrogen bonding and cationic bridge formation, stabilizing proteins, particularly membrane proteins, and preserving their native conformation. In some embodiments, this application attempts to replace CTAB with surfactants such as Triton X-100, Tween 20, or SDS, but the results are not as good as CTAB. Polyethylene glycol is present as a nucleic acid precipitant, causing RNA to precipitate onto the magnetic microsphere carrier.

[0024] The volume of the lysis binding solution added is 1-3 times the volume of the blood sample, for example, 1, 1.5, 2, 2.5 or 3 times.

[0025] Proteinase K activity >30 U / mg.

[0026] The amount of proteinase K added is 0.4 mg to 1.2 mg per milliliter of blood sample, for example, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, or 1.2 mg.

[0027] The magnetic beads are silanol magnetic microspheres, glass-modified magnetic microspheres, or carboxyl-modified magnetic microspheres.

[0028] The magnetic beads have a particle size of 0.1-10 μm, for example 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μm, preferably 0.2-5 μm.

[0029] The amount of magnetic beads added is 1-4 mg (e.g., 1, 2, 3, 4 mg) per milliliter of blood sample.

[0030] The incubation time is 5-30 minutes, for example, 5, 10, 15, 20, 25 or 30 minutes.

[0031] The incubation temperature is 20-30℃, for example 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30℃, preferably room temperature.

[0032] In some embodiments, the incubation is performed at 20-30°C for 5-30 minutes by centrifugation at a speed of 500-1500 rpm.

[0033] The magnetic separation operation is a routine operation, and those skilled in the art are fully capable of determining whether the magnetic separation is complete. Generally, the magnetic separation is considered complete when the magnetic beads are completely adsorbed or the liquid becomes clear.

[0034] The magnetic separation is performed using a magnetic rack or a nucleic acid extractor.

[0035] The cleaning is performed at least once, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. The cleaning solution used for each cleaning can be the same or different.

[0036] The cleaning solution used for the cleaning includes urea and sorbitol. Preferably, it also includes one or more of tris(hydroxymethyl)aminomethane, magnesium chloride, or calcium chloride.

[0037] In some embodiments, the cleaning solution includes urea, sorbitol, tris(hydroxymethyl)aminomethane, magnesium chloride, and calcium chloride.

[0038] The pH of the cleaning solution is 7.0-8.0, for example 7.0, 7.5 or 8.0.

[0039] In some embodiments, the cleaning solution comprises: 1-4M (e.g., 1, 2, 3, or 4M, preferably 1-2M) urea, 20-200mM (e.g., 20, 30, 40, 50, 60, 70, 80, 100, or 200mM, preferably 25-100mM) tris(hydroxymethyl)aminomethane, 1-20mM (e.g., 1, 3, 5, 10, 12, 15, or 20mM, preferably 3-10mM) magnesium chloride, 1-5mM (e.g., 1, 2, 3, 4, or 5mM, preferably 1-4mM) calcium chloride, and 2-25% (e.g., 2, 5, 10, 15, 20, or 25%, preferably 5-20%) sorbitol, wherein the concentration of the sorbitol is by weight-volume percentage, and the pH is 7.0-8.0.

[0040] In some embodiments, the cleaning solution comprises 1-2M urea, 25-100mM tris(hydroxymethyl)aminomethane, 3-10mM magnesium chloride, 1-4mM calcium chloride and 5-20% sorbitol, with a pH of 7.0-8.0.

[0041] In some embodiments, the cleaning solution comprises 1.5M urea, 50mM tris(hydroxymethyl)aminomethane, 5mM magnesium chloride, 2mM calcium chloride and 15% sorbitol, with a pH of 7.0-8.0.

[0042] In some embodiments, the cleaning is performed four times, wherein,

[0043] The cleaning solution 1 used for the first cleaning includes guanidine salt and isopropanol, and preferably has a pH of 7.0-8.0 (e.g., 4.0, 5.0, 6.0, 7.0 or 8.0, preferably 4.0-7.0).

[0044] The cleaning solution 2 used for the second cleaning includes lithium chloride, tris(hydroxymethyl)aminomethane and anhydrous ethanol, preferably with a pH of 5.0-8.0 (e.g. 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0).

[0045] The third cleaning uses cleaning solution 3 and deoxyribonuclease I (DNase I). Cleaning solution 3 includes urea, tris(hydroxymethyl)aminomethane, magnesium chloride, calcium chloride, and sorbitol, preferably with a pH of 7.0-8.0 (e.g., 7.0, 7.5, or 8.0).

[0046] The fourth cleaning uses a cleaning solution 4 comprising tris(hydroxymethyl)aminomethane and anhydrous ethanol, preferably with a pH of 5.0-8.0 (e.g., 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0).

[0047] In some embodiments, the cleaning is performed four times, wherein,

[0048] The cleaning solution 1 used for the first cleaning comprises: 1-5M (e.g., 1, 1.5, 2, 2.5, 3, 4, or 5M, preferably 2-4M) guanidine isothiocyanate and 30-80% (e.g., 30, 40, 50, 55, 60, 65, 70, or 80%, preferably 35-60%) isopropanol, with a pH of 4.0-8.0, wherein the concentration of the isopropanol is in mass-volume percentage. In some embodiments, it comprises 2-4M guanidine isothiocyanate and 35-60% isopropanol, with a pH of 4.0-8.0. In some embodiments, it comprises 2.5M guanidine isothiocyanate and 50% isopropanol, with a pH of 4.0-8.0.

[0049] The second cleaning solution 2 used for the second cleaning comprises: 20-250 mM (e.g., 20, 30, 50, 80, 100, 150, 200, or 250 mM, preferably 20-100 mM) Tris-HCl, 20-200 mM (e.g., 20, 30, 40, 50, 60, 70, 80, 100, or 200 mM, preferably 40-100 mM) lithium chloride, and 40-80% (e.g., 40, 50, 75, or 80%) anhydrous ethanol, with a pH of 5.0-8.0, wherein the concentration of the anhydrous ethanol is by mass-volume percentage. In some embodiments, it comprises 20-100 mM Tris-HCl, 40-100 mM lithium chloride, and 40-80% anhydrous ethanol, with a pH of 5.0-8.0. In some embodiments, it contains 50 mM Tris-HCl, 50 mM lithium chloride and 75% anhydrous ethanol, with a pH of 5.0-8.0.

[0050] The third cleaning uses cleaning solution 3 and DNase I, wherein in some embodiments, the cleaning solution 3 comprises: 1-4M (e.g., 1, 2, 3, or 4M, preferably 1-2M) urea, 20-200mM (e.g., 20, 30, 40, 50, 60, 70, 80, 100, 200mM, preferably 25-100mM) tris(hydroxymethyl)aminomethane, 1-20mM (e.g., 1, 3, 5, 10, 12, 15, 20mM, preferably 3-10mM) magnesium chloride, 1-5mM (e.g., 1, 2, 3, 4, 5mM, preferably 1-4mM) calcium chloride and 2-25% (e.g., 2, 5, 10, 15, 20, or 25%, preferably 5-20%) sorbitol, wherein the concentration of the sorbitol is by weight-volume percentage and the pH is 7.0-8.0. In some embodiments, the cleaning solution 3 comprises 1-2M urea, 25-100mM tris(hydroxymethyl)aminomethane, 3-10mM magnesium chloride, 1-4mM calcium chloride, and 5-20% sorbitol, with a pH of 7.0-8.0. In some embodiments, the cleaning solution 3 comprises 1.5M urea, 50mM tris(hydroxymethyl)aminomethane, 5mM magnesium chloride, 2mM calcium chloride, and 15% sorbitol, with a pH of 7.0-8.0.

[0051] The fourth cleaning solution used comprises: 25-250 mM (e.g., 25, 30, 50, 80, 100, 200, or 250 mM, preferably 20-80 mM) Tris-HCl and 50-80% (e.g., 50, 55, 60, 65, 70, 75, or 80%) anhydrous ethanol, with a pH of 5.0-8.0, wherein the concentration of the anhydrous ethanol is by mass-volume percentage. In some embodiments, it comprises 20-80 mM Tris-HCl and 50-80% anhydrous ethanol, with a pH of 5.0-8.0. In some embodiments, it comprises 25 mM Tris-HCl and 75% anhydrous ethanol, with a pH of 5.0-8.0.

[0052] Preferably, the cleaning solution 1 used in the first cleaning is added at a rate of 320-400 μL per mg of magnetic beads, for example, 320, 350, 380, or 400 μL.

[0053] Preferably, the amount of cleaning solution 2 used in the second cleaning is 320-400 μL per mg of magnetic beads, for example, 320, 350, 380, or 400 μL.

[0054] Preferably, the amount of cleaning solution 3 used in the third cleaning is 40-160 μL per mg of magnetic beads, for example, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 or 160 μL.

[0055] Preferably, the amount of DNase I added is 1-10 U (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 U) per milliliter of blood.

[0056] Preferably, the DNase I can be 1-10 U / μL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 U / μL).

[0057] In some embodiments, the amount of DNase I added is 1-10 U (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 U) per milliliter of blood, with a concentration of 1-10 U / μL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 U / μL).

[0058] Preferably, the amount of cleaning solution 4 used in the fourth cleaning is 320-400 μL per mg of magnetic beads, for example, 320, 350, 380, or 400 μL.

[0059] The cleaning process also includes an alcohol removal step, followed by elution.

[0060] The elution solution used is nuclease-free water or DEPC water.

[0061] Preferably, the amount of eluent added is 20-80 μL per mg of magnetic beads, for example, 20, 30, 40, 50, 60, 70 or 80 μL.

[0062] The elution temperature is 25-60°C (e.g., 25, 30, 35, 40, 45, 50, 55 or 60°C).

[0063] The elution time is 3-10 min (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 min, preferably 3-8 min).

[0064] In some embodiments, the elution includes adding an eluent, mixing, and then incubating with shaking at 25-60°C (e.g., 25, 30, 35, 40, 45, 50, 55, or 60°C) for 5-10 minutes (e.g., 5, 6, 7, 8, 9, or 10 minutes).

[0065] In some embodiments, the extraction method includes:

[0066] (1) Sample preparation: Add the blood sample to a centrifuge tube;

[0067] (2) Sample lysis: Add lysis binding solution, proteinase K solution and magnetic bead suspension to the centrifuge tube of step (1), mix and incubate at 20-30℃ (preferably room temperature) for 5-30 min (e.g. 5, 10, 15, 20, 25 or 30 min); the preferred centrifugation speed is 500-1500 rpm;

[0068] (3) Nucleic acid adsorption: Magnetic separation is performed using a magnetic rack or nucleic acid extractor. The liquid obtained after magnetic separation is discarded to obtain magnetic beads after adsorption.

[0069] (4) First cleaning: Add the cleaning solution 1 to the adsorbed magnetic beads, mix well, and then use a magnetic rack or nucleic acid extractor to perform magnetic separation. Then discard the liquid obtained after magnetic separation to obtain the first cleaned magnetic beads.

[0070] (5) Second cleaning: Add the cleaning solution 2 to the first cleaning magnetic beads and mix well. Then use a magnetic rack or nucleic acid extractor to perform magnetic separation. Then discard the liquid obtained after magnetic separation to obtain the second cleaning magnetic beads.

[0071] (6) Third cleaning: Add the cleaning solution 3 and DNase I to the second cleaning magnetic beads and mix well. Then use a magnetic rack or nucleic acid extractor to perform magnetic separation. Then discard the liquid obtained after magnetic separation to obtain the third cleaning magnetic beads.

[0072] (7) Fourth cleaning: Add the cleaning solution 4 to the third cleaning magnetic beads, then use a magnetic rack or nucleic acid extractor for magnetic separation, and then discard the liquid obtained after magnetic separation to obtain the fourth cleaning magnetic beads;

[0073] (8) Sample elution: Add elution buffer to the fourth washing magnetic bead, mix well, and incubate with shaking at 25-60℃ (e.g., 25, 30, 35, 40, 45, 50, 55 or 60℃) for 5-10 min (e.g., 5, 6, 7, 8, 9 or 10 min). Collect the RNA solution obtained after elution.

[0074] Preferably, the magnetic bead suspension comprises water and magnetic beads. The water is not particularly limited, as long as it does not adversely affect the extraction of nucleic acids from biological samples. However, the water is preferably nuclease-free water.

[0075] Preferably, the magnetic bead suspension contains magnetic beads with a mass-volume percentage of 1-10% (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%).

[0076] In some embodiments, the extraction method includes:

[0077] (1) Sample preparation: Add the blood sample to a centrifuge tube;

[0078] (2) Sample lysis: Add lysis binding solution, proteinase K and magnetic beads to the centrifuge tube of step (1), mix and incubate at 20-30℃ (preferably room temperature) for 5-30 min (e.g. 5, 10, 15, 20, 25 or 30 min); the preferred centrifugation speed is 500-1500 rpm, wherein the volume of the added lysis binding solution is 1-3 times the volume of the blood sample, the amount of added magnetic beads is 1-4 mg per milliliter of blood sample, and the amount of added proteinase K is 0.4 mg-1.2 mg per milliliter of blood sample;

[0079] (3) Nucleic acid adsorption: Magnetic separation is performed using a magnetic rack or nucleic acid extractor. The liquid obtained after magnetic separation is discarded to obtain magnetic beads after adsorption.

[0080] (4) First cleaning: Add the cleaning solution 1 to the adsorbed magnetic beads, mix well, and then use a magnetic rack or nucleic acid extractor for magnetic separation. Then discard the liquid obtained after magnetic separation to obtain the first cleaned magnetic beads. The amount of cleaning solution 1 added is 320-400 μL per mg of magnetic beads.

[0081] (5) Second cleaning: Add the cleaning solution 2 to the first cleaning magnetic beads and mix well. Then use a magnetic rack or nucleic acid extractor to perform magnetic separation. Then discard the liquid obtained after magnetic separation to obtain the second cleaning magnetic beads. The amount of cleaning solution 2 added is 320-400 μL per mg of magnetic beads.

[0082] (6) Third washing: Add the washing solution 3 and DNase I to the second washing magnetic beads and mix well. Then use a magnetic rack or nucleic acid extractor for magnetic separation. Then discard the liquid obtained after magnetic separation to obtain the third washing magnetic beads. The amount of washing solution 3 added is 40-160 μL per mg of magnetic beads, and the amount of DNase I added is 1-10 U of DNase I per milliliter of blood.

[0083] (7) Fourth cleaning: Add the cleaning solution 4 to the third cleaning magnetic beads, and then use a magnetic rack or nucleic acid extractor for magnetic separation. Then discard the liquid obtained after magnetic separation to obtain the fourth cleaning magnetic beads. The amount of cleaning solution 4 added is 320-400 μL per mg of magnetic beads.

[0084] (8) Sample elution: Add elution buffer to the fourth washing magnetic beads, mix well, and incubate with shaking at 25-60℃ (e.g., 25, 30, 35, 40, 45, 50, 55 or 60℃) for 5-10 min (e.g., 5, 6, 7, 8, 9 or 10 min). Collect the RNA solution obtained after elution. The amount of elution buffer added is 20-80 μL per mg of magnetic beads, for example, 20, 30, 40, 50, 60, 70 or 80 μL.

[0085] In some embodiments, in step (1), the volume of the blood sample can be 0.5-3 mL. In this case, the volume of the blood sample added can be 1-3 times (e.g., 1, 1.5, 2, 2.5, or 3 times) of the lysis binding solution; in step (2), 20-80 μL (e.g., 20, 25, 30, 40, 50, 60, 70, or 80 μL) of the magnetic bead suspension and 20-60 μL (e.g., 20, 25, 30, 40, 50, or 60 μL) of the proteinase K solution can be added; in step (4), 800-1000 μL of the washing solution 1 can be added; in step (5), 8 00-1000 μL of the cleaning solution 2; in step (6), 100-400 μL of the cleaning solution 3 may be added; in step (7), 800-1000 μL of the cleaning solution 4 may be added; in step (8), 50-200 μL (e.g., 50, 100 or 150 μL) of the elution solution may be added, and the mixture is incubated and eluted at 25-60°C (e.g., 25, 30, 35, 40, 45, 50, 55 or 60°C) for 3-10 min (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 min, preferably 3-8 min).

[0086] In some implementations, in step (1), the volume of the blood sample is 1000 μL. In step (2), add 1-3 times the volume of the lysis binding buffer (1000-3000 μL) of the blood sample; in step (2), add 20-80 μL of the magnetic bead suspension and 20-60 μL of proteinase K solution; in step (4), add 800 μL of the washing solution 1; in step (5), add 800 μL of the washing solution 2; in step (6), add 200 μL of the washing solution 3 and 1-10 U of DNase I (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 U, concentration can be 1-10 U / μL); in step (7), add 800 μL of the washing solution 4; in step (8), add 50-200 μL of the elution buffer and incubate at 25-60℃ for 3-8 min.

[0087] In some embodiments, in step (1), the volume of the blood sample is 1000 μL. In step (2), 1.5 times the volume of the blood sample is added to the lysis binding buffer (1500 μL); in step (2), 50 μL of the magnetic bead suspension and 50 μL of proteinase K solution are added; in step (4), 800 μL of the washing solution 1 is added; in step (5), 800 μL of the washing solution 2 is added; in step (6), 200 μL of the washing solution 3 and 5 U of DNase I (2 U / μL) are added; in step (7), 800 μL of the washing solution 4 is added; in step (8), 100 μL of the elution buffer is added, and the mixture is eluted and incubated at 50°C for 5 min.

[0088] A second aspect of this application provides an RNA extraction kit comprising a lysis binding buffer, wherein the lysis binding buffer comprises polyglycerol-3 acetate, lithium perchlorate, and polyethylene glycol. Preferably, the lysis binding buffer further comprises one or more of sodium acetate, disodium ethylenediaminetetraacetate, hexadecyltrimethylammonium bromide, or isopropanol.

[0089] In some embodiments, the cleavage binding solution includes polyglycerol-3 acetate, lithium perchlorate, polyethylene glycol, sodium acetate, disodium ethylenediaminetetraacetate, hexadecyltrimethylammonium bromide, and isopropanol.

[0090] The pH of the lysis binding solution is 5.0-8.0, for example 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 or 8.0; preferably 5.0-7.5, more preferably 5.5-7.0.

[0091] Preferably, the lysis binding solution comprises 5-15% (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15%, preferably 6-12.5% ​​or 8-12%) of polyglycerol-3 acetate, 1-8M (e.g., 1, 2, 3, 4, 5, 6 or 8, preferably 2-7M or 3-6M) of lithium perchlorate and 5-25% (e.g., 5, 15, 20 or 25%, preferably 8-22% or 10-20%) of polyethylene glycol.

[0092] Preferably, the lysis binding solution comprises 5-15% (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15%, preferably 6-12.5% ​​or 8-12%) of polyglycerol-3. Acetate, 100-400mM (e.g., 100, 125, 150, 175, 200, 250, 300, 350, 400mM, preferably 100-300mM or 100-250mM) sodium acetate, 1-8M (e.g., 1, 2, 3, 4, 5, 6 or 8, preferably 2-7M or 3-6M) lithium perchlorate, 10-50mM (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, preferably 15-40mM or 20-40mM) disodium ethylenediaminetetraacetate, 0.5-10% (e.g.) For example, 0.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, or 10%, preferably 1-8% or 1.5-6%) of hexadecyltrimethylammonium bromide, 5-25% (e.g., 5, 7, 8, 10, 15, 19, 20, 21, 22, 23, 24, or 25%, preferably 8-22% or 10-20%) of polyethylene glycol, and 15-50% (e.g., 15, 20, 25, 30, 35, 40, 45, or 50%, preferably 20-40% or 20-45%) of isopropanol. The concentrations of the polyglycerol-3 acetate, hexadecyltrimethylammonium bromide, polyethylene glycol, and isopropanol are expressed as mass-volume percentages.

[0093] In some embodiments, the cleavage binder comprises: 6-12.5% ​​polyglycerol-3 acetate, 100-300 mM sodium acetate, 2-7 M lithium perchlorate, 15-40 mM disodium ethylenediaminetetraacetate, 1-8% hexadecyltrimethylammonium bromide, 8-22% polyethylene glycol, and 20-45% isopropanol.

[0094] In some embodiments, the cleavage binder comprises: 8-12% polyglycerol-3 acetate, 100-250 mM sodium acetate, 3-6 M lithium perchlorate, 20-40 mM disodium ethylenediaminetetraacetate, 1.5-6% hexadecyltrimethylammonium bromide, 10-20% polyethylene glycol, and 20-40% isopropanol.

[0095] In some embodiments, the cleavage binder comprises: 10% polyglycerol-3 acetate, 125 mM sodium acetate, 4 M lithium perchlorate, 25 mM disodium ethylenediaminetetraacetate, 2.5% hexadecyltrimethylammonium bromide, 15% polyethylene glycol, and 30% isopropanol.

[0096] Preferably, the polyethylene glycol has a molecular weight of 100-20000, for example, 100, 200, 300, 400, 500, 800, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000 or 20000; more preferably 500-10000, more preferably 1000-8000, and most preferably 2000-6000.

[0097] The RNA extraction kit further includes a washing solution comprising urea and sorbitol. Preferably, it also includes one or more of tris(hydroxymethyl)aminomethane, magnesium chloride, or calcium chloride.

[0098] In some embodiments, the cleaning solution includes urea, sorbitol, tris(hydroxymethyl)aminomethane, magnesium chloride, and calcium chloride.

[0099] The pH of the cleaning solution is 7.0-8.0, for example 7.0, 7.5 or 8.0.

[0100] In some embodiments, the cleaning solution comprises: 1-4M (e.g., 1, 2, 3, or 4M, preferably 1-2M) urea, 20-200mM (e.g., 20, 30, 40, 50, 60, 70, 80, 100, or 200mM, preferably 25-100mM) tris(hydroxymethyl)aminomethane, 1-20mM (e.g., 1, 3, 5, 10, 12, 15, or 20mM, preferably 3-10mM) magnesium chloride, 1-5mM (e.g., 1, 2, 3, 4, or 5mM, preferably 1-4mM) calcium chloride, and 2-25% (e.g., 2, 5, 10, 15, 20, or 25%, preferably 5-20%) sorbitol, wherein the concentration of the sorbitol is by weight-volume percentage, and the pH is 7.0-8.0.

[0101] In some embodiments, the cleaning solution comprises 1-2M urea, 25-100mM tris(hydroxymethyl)aminomethane, 3-10mM magnesium chloride, 1-4mM calcium chloride and 5-20% sorbitol, with a pH of 7.0-8.0.

[0102] In some embodiments, the cleaning solution comprises 1.5M urea, 50mM tris(hydroxymethyl)aminomethane, 5mM magnesium chloride, 2mM calcium chloride and 15% sorbitol, with a pH of 7.0-8.0.

[0103] In some embodiments, the RNA extraction kit includes washing solution 1, washing solution 2, washing solution 3, and washing solution 4.

[0104] The cleaning solution 1 comprises guanidine salt and isopropanol, preferably with a pH of 4.0-8.0 (e.g., 4.0, 5.0, 6.0, 7.0, or 8.0, preferably 4.0-7.0). More preferably, the cleaning solution 1 comprises guanidine isothiocyanate and isopropanol, with a pH of 4.0-8.0. More preferably, the cleaning solution 1 comprises: 1-5M (e.g., 1, 1.5, 2, 2.5, 3, 4, or 5M, preferably 2-4M) guanidine isothiocyanate and 30-80% (e.g., 30, 40, 50, 55, 60, 65, 70, or 80%, preferably 35-60%) isopropanol, with a pH of 4.0-8.0, wherein the concentration of the isopropanol is by weight-volume percentage. In some embodiments, it comprises 2-4M guanidine isothiocyanate and 35-60% isopropanol, with a pH of 4.0-8.0. In some embodiments, it contains 2.5M guanidine isothiocyanate and 50% isopropanol, with a pH of 4.0-8.0.

[0105] The cleaning solution 2 comprises lithium chloride, tris(hydroxymethyl)aminomethane, and anhydrous ethanol, preferably with a pH of 5.0-8.0 (e.g., 5, 5.5, 6.0, 6.5, 7.0, 7.5, or 8). More preferably, the cleaning solution 2 comprises: 20-250 mM (e.g., 20, 30, 50, 80, 100, 150, 200, or 250 mM, preferably 20-100 mM) Tris-HCl, 20-200 mM (e.g., 20, 30, 40, 50, 60, 70, 80, 100, or 200 mM, preferably 40-100 mM) lithium chloride, and 40-80% (e.g., 40, 50, 75, or 80%) anhydrous ethanol, with a pH of 5.0-8.0, wherein the concentration of the anhydrous ethanol is by mass-volume percentage. In some embodiments, the mixture comprises 20-100 mM Tris-HCl, 40-100 mM lithium chloride, and 40-80% anhydrous ethanol, with a pH of 5.0-8.0. In some embodiments, the mixture comprises 50 mM Tris-HCl, 50 mM lithium chloride, and 75% anhydrous ethanol, with a pH of 5.0-8.0.

[0106] The cleaning solution 3 includes urea, tris(hydroxymethyl)aminomethane, magnesium chloride, calcium chloride, and sorbitol, and preferably has a pH of 7.0-8.0 (e.g., 7.0, 7.5, or 8.0). More preferably, the cleaning solution 3 comprises: 1-4M (e.g., 1, 2, 3, or 4M, preferably 1-2M) urea, 20-200mM (e.g., 20, 30, 40, 50, 60, 70, 80, 100, 200mM, preferably 25-100mM) tris(hydroxymethyl)aminomethane, 1-20mM (e.g., 1, 3, 5, 10, 12, 15, 20mM, preferably 3-10mM) magnesium chloride, 1-5mM (e.g., 1, 2, 3, 4, 5mM, preferably 1-4mM) calcium chloride, and 2-25% (e.g., 2, 5, 10, 15, 20, or 25%, preferably 5-20%) sorbitol, wherein the concentration of the sorbitol is by weight-volume percentage, and the pH is 7.0-8.0. In some embodiments, the cleaning solution 3 comprises 1-2M urea, 25-100mM tris(hydroxymethyl)aminomethane, 3-10mM magnesium chloride, 1-4mM calcium chloride, and 5-20% sorbitol, with a pH of 7.0-8.0. In some embodiments, the cleaning solution 3 comprises 1.5M urea, 50mM tris(hydroxymethyl)aminomethane, 5mM magnesium chloride, 2mM calcium chloride, and 15% sorbitol, with a pH of 7.0-8.0.

[0107] The cleaning solution 4 comprises tris(hydroxymethyl)aminomethane and anhydrous ethanol, preferably with a pH of 5.0-8.0 (e.g., 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0). More preferably, the cleaning solution 4 comprises: 25-250 mM (e.g., 25, 30, 50, 80, 100, 200, or 250 mM, preferably 20-80 mM) Tris-HCl and 50-80% (e.g., 50, 55, 60, 65, 70, 75, or 80%) anhydrous ethanol, with a pH of 5.0-8.0, wherein the concentration of the anhydrous ethanol is by weight-volume percentage. In some embodiments, it comprises 20-80 mM Tris-HCl and 50-80% anhydrous ethanol, with a pH of 5.0-8.0. In some embodiments, it contains 25 mM Tris-HCl and 75% anhydrous ethanol, with a pH of 5.0-8.0.

[0108] Preferably, the RNA extraction kit further includes one or more of magnetic beads, proteinase K, DNase I, or elution buffer.

[0109] The magnetic beads are silanol magnetic microspheres, glass-modified magnetic microspheres, or carboxyl-modified magnetic microspheres.

[0110] The magnetic beads have a particle size of 0.1-10 μm, for example 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μm, preferably 0.2-5 μm.

[0111] Proteinase K activity >30 U / mg.

[0112] The DNase I can be 1-10 U / μL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 U / μL).

[0113] The elution buffer is nuclease-free water or DEPC water.

[0114] In some embodiments, the RNA extraction kit includes a lysis binding buffer, magnetic beads, proteinase K solution, washing buffer 1, washing buffer 2, washing buffer 3, DNase I, washing buffer 4, and elution buffer.

[0115] Preferably, the concentration of proteinase K in the proteinase K solution is 10-30 mg / mL, for example, 10, 15, 20, 25 or 30 mg / mL.

[0116] The RNA extraction kit mentioned is a blood RNA extraction kit.

[0117] The blood in question is peripheral blood, bone marrow blood, or umbilical cord blood.

[0118] The blood in question is whole blood.

[0119] The blood in question is fresh, anticoagulated whole blood obtained clinically.

[0120] The blood in question is peripheral whole blood, bone marrow whole blood, or umbilical cord whole blood.

[0121] A third aspect of this application provides an application of the aforementioned RNA extraction kit, the application comprising:

[0122] A) Applications in RNA extraction;

[0123] B) Applications in gene testing; and / or,

[0124] C) Application in the preparation of products for diagnosing diseases.

[0125] Preferably, the disease is leukemia.

[0126] Preferably, the diagnosis of the disease includes the detection of disease biomarkers.

[0127] Preferably, the marker is one or a combination of two or more of WT1, fusion gene 210, fusion gene 190, DEK-CAN, NPM1-ALK, NUP98-HOXA9, MLL-AF6-3, MLL-ELL-3, MLL-AF9, AML1-ETO, or BCR-ABL1.

[0128] A fourth aspect of this application provides a method for gene detection, the method comprising obtaining RNA using the extraction method described in the first aspect, and then amplifying and detecting the target gene.

[0129] The amplification includes polymerase chain reaction (PCR), real-time fluorescence PCR, ligation-mediated PCR (LM-PCR), isothermal amplification (RPA), strand displacement amplification (SDA), or primer extension pre-amplification (PEP).

[0130] The detection can be sequencing or detection of fluorescence signals.

[0131] A fifth aspect of this application provides a method for diagnosing a disease, the method comprising obtaining RNA using the extraction method described in the first aspect, and then amplifying and detecting disease biomarkers.

[0132] The preferred disease is leukemia.

[0133] The disease biomarker is preferably one or a combination of two or more of the following: WT1, fusion gene 210, fusion gene 190, DEK-CAN, NPM1-ALK, NUP98-HOXA9, MLL-AF6-3, MLL-ELL-3, MLL-AF9, AML1-ETO, or BCR-ABL1.

[0134] Compared with the prior art, this application has the following beneficial technical effects:

[0135] (1) Provide a magnetic bead method blood RNA extraction kit that can be used with an automated nucleic acid extractor to achieve rapid and high-throughput nucleic acid extraction or purification.

[0136] (2) The extraction method provided in this application can fully and automatically extract nucleic acids without pausing the process and remove genomic DNA interference during the process.

[0137] (3) The extraction method provided in this application combines pyrolysis in one step, which can be directly operated on the machine and has a high degree of automation. Attached Figure Description

[0138] The embodiments of this application will now be described in detail with reference to the accompanying drawings, wherein:

[0139] Figure 1The images show gel electrophoresis diagrams of the bone marrow RNA extraction products obtained by the method of this application and the Trizol method. A1-8 are electrophoresis diagrams of the bone marrow RNA products extracted from 4 patients in this application, and B1-8 are electrophoresis diagrams of the bone marrow RNA products extracted from the same 4 patients using the Trizol method. Detailed Implementation

[0140] The present application will be further described below with reference to the embodiments.

[0141] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, in the following description, different "an embodiment" or "an embodiment" do not necessarily refer to the same embodiment. Different embodiments can be substituted or combined, and for those skilled in the art, other implementation methods can be obtained based on these embodiments without creative effort.

[0142] To further understand this application, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of this application and are not intended to limit the scope of the claims. Unless otherwise specified, the experimental methods described in the following examples are conventional methods; and the materials described, unless otherwise specified, are commercially available.

[0143] Before describing this teaching in detail, it should be understood that this disclosure is not limited to specific compositions or process steps, as these can vary. It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise.

[0144] When this document provides a range of values, unless otherwise expressly stated, the range is intended to include a starting value and an ending value, as well as the values ​​or ranges of values ​​in between. For example, "from 0.2 to 0.5" can include 0.2, 0.3, 0.4, 0.5, etc.; the ranges in between are such as 0.2-0.3, 0.3-0.4, 0.2-0.4, 0.2-0.5, 0.4-0.5, or 0.3-0.5; the increments in between are such as 0.25, 0.35, 0.220, 0.325, 0.49; the increment ranges in between are such as 0.26-0.39, and so on.

[0145] It should be understood that the terms "about" are implied before the temperature, mass, weight, volume ratio, concentration, time, etc., discussed in this application, so that minor and non-substantial deviations are within the scope of the teachings herein. Generally, the term "about" indicates a non-substantial change in the amount of a component in the composition that has no significant effect on the effect or stability of the composition. Furthermore, the use of "comprising," "containing," and "including" is not intended to be restrictive. It should be understood that the foregoing general and detailed descriptions are exemplary and explanatory only and are not intended to limit the teachings herein. In the event of any inconsistency between any material incorporated by reference and the statements herein, the statements shall prevail.

[0146] Unless otherwise specified, embodiments described in the specification as “comprising” various components are also considered to include “composed of said components” or “substantially composed of said components”; embodiments described in the specification as “composed of various components” are also considered to “comprising” or “substantially composed of said components”.

[0147] "Nucleic acid" refers to a polymeric compound containing two or more covalently bonded nucleosides or nucleoside analogs having nitrogen-containing heterocyclic bases or base analogs, wherein the nucleosides are linked together by phosphodiester bonds or other bonds to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides and their analogs. The nucleic acid "backbone" can be composed of a variety of linkages, including one or more sugar-phosphodiester bonds and peptide-nucleic acid bonds. Nucleic acids may include modified bases to alter their function or behavior, such as adding a 3'-terminal dideoxynucleotide to prevent the addition of additional nucleotides to the nucleic acid. Synthetic methods for the in vitro preparation of nucleic acids are well known in the art, although nucleic acids can be purified from natural sources using conventional techniques.

[0148] In this application, "extraction," "separation," or "purification" refers to the removal or separation of one or more components of a sample from other sample components. Sample components contain target nucleic acids typically found in an aqueous solution phase, and may also contain cellular fragments, proteins, carbohydrates, lipids, salt ions, metal ions, and other nucleic acids. "Extraction," "separation," or "purification" does not imply any degree of purification. Typically, at least 70%, 80%, or 90% of the target nucleic acids are separated or purified from other sample components.

[0149] In this example, the RNA concentration was detected using NanoDrop. TM The 8000 spectrophotometer was purchased from ThermoFisher Scientific, model number ND8000LAPTOP.

[0150] In this example, Qubit RNA detection uses Qubit.TM 4. Fluorometer, purchased from Thermo Fisher Scientific, item number Q33238.

[0151] In this example, the kit used for Qubit RNA detection is Qubit. TM RNA high sensitivity (HS), wide range (BR), and extended range (XR) quantification kits were purchased from Thermo Fisher Scientific, catalog number Q32852.

[0152] WT1 stands for Wilms Tumor Gene 1.

[0153] The fusion gene 210 is of type b3a2, which is the fusion of exon 3 of the BCR gene and exon a2 of the ABL gene.

[0154] The fusion gene 190 is of type b2a2, which is the fusion of exon 2 of the BCR gene and exon a2 of the ABL gene.

[0155] Example 1: Magnetic Bead Blood RNA Extraction Kit

[0156] The kit includes: lysis binding buffer, magnetic bead suspension, proteinase K solution, washing buffer 1, washing buffer 2, washing buffer 3, DNase I, washing buffer 4, and elution buffer.

[0157] The lysis binder consists of: 10% polyglycerol-3 acetate, 125 mM sodium acetate, 4 M lithium perchlorate, 25 mM disodium ethylenediaminetetraacetate, 2.5% hexadecyltrimethylammonium bromide, 15% polyethylene glycol 5000, and 30% isopropanol. The pH of the lysis binder is 6.5. The concentrations of polyglycerol-3 acetate, hexadecyltrimethylammonium bromide, polyethylene glycol 5000, and isopropanol are expressed as a percentage by weight (volume percentage), for example, 10% polyglycerol-3 acetate is 100 mg / mL polyglycerol-3 acetate.

[0158] The magnetic bead suspension contains water and 5% by mass (i.e., 50 mg / mL) silanol magnetic microspheres with a particle size of 1 μm, derived from MagH1N Silica (Cat#BMD00751) from Suzhou Cretaceous Biotechnology Co., Ltd.

[0159] The proteinase K solution was obtained from Suzhou Cretaceous Biotechnology Co., Ltd. (Cat#CNA02120), with a concentration of 20 mg / mL and an activity >30 U / mg.

[0160] DNase I was obtained from Suzhou Cretaceous Biotechnology Co., Ltd. (Cat#CNA02128S) at a concentration of 2 U / μL.

[0161] Cleaning solution 1 consists of 2.5M guanidine isothiocyanate and 50% isopropanol, with a pH of 5.5.

[0162] Cleaning solution 2 consists of 50mM Tris-HCl, 50mM lithium chloride and 75% anhydrous ethanol, with a pH of 7.

[0163] The cleaning solution consists of 1.5M urea, 50mM tris(hydroxymethyl)aminomethane, 5mM magnesium chloride, 2mM calcium chloride and 15% sorbitol, with a pH of 7.5.

[0164] Cleaning solution 4 contains 25 mM Tris-HCl and 75% anhydrous ethanol, with a pH of 7.5.

[0165] The eluent was DEPC water.

[0166] Comparative Example 1:

[0167] The lysis binder does not contain polyglycerol-3 acetate, and the remaining components are consistent with those in Example 1.

[0168] Comparative Example 2:

[0169] The pyrolysis binder does not contain lithium perchlorate, and the remaining components are consistent with those in Example 1.

[0170] Comparative Example 3:

[0171] The pyrolysis binder does not contain polyethylene glycol 5000, and the remaining components are consistent with those in Example 1.

[0172] Comparative Example 4:

[0173] The cleaning solution 3 does not contain urea, and the remaining components are the same as those in Example 1.

[0174] Comparative Example 5:

[0175] Cleaning solution 3 does not contain sorbitol, and the remaining components are the same as those in Example 1.

[0176] Example 2: Blood RNA Extraction Method Using Magnetic Beads

[0177] This embodiment uses the kits prepared in Example 1 and Comparative Examples 1-5 to extract RNA from different blood samples. The specific steps are as follows:

[0178] (1) Sample preparation: Add 1000 μL of whole blood sample to a centrifuge tube;

[0179] (2) Sample lysis: Add 1500 μL of the lysis binding solution, 50 μL of magnetic bead suspension and 50 μL of proteinase K solution to the centrifuge tube of step (1), mix and incubate at 1000 rpm at room temperature for 15 min;

[0180] (3) Nucleic acid adsorption: Magnetic separation is performed using a magnetic rack or nucleic acid extractor. The liquid obtained after magnetic separation is discarded to obtain magnetic beads after adsorption.

[0181] (4) First cleaning: Add 800 μL of the cleaning solution 1 to the magnetic beads after adsorption, mix for 2 min, and then use a magnetic rack or nucleic acid extractor for magnetic separation. Then discard the liquid obtained after magnetic separation to obtain the first cleaning magnetic beads.

[0182] (5) Second cleaning: Add 800 μL of the cleaning solution 2 to the first cleaning magnetic beads and mix for 2 min. Then use a magnetic rack or nucleic acid extractor to perform magnetic separation. Then discard the liquid obtained after magnetic separation to obtain the second cleaning magnetic beads.

[0183] (6) Third cleaning: Add 200 μL of the cleaning solution 3 and 5 U of DNase I to the second cleaning magnetic beads and mix for 15 min. Then use a magnetic rack or nucleic acid extractor for magnetic separation. Then discard the liquid obtained after magnetic separation to obtain the third cleaning magnetic beads.

[0184] (7) Fourth cleaning: Add 800 μL of the cleaning solution 4 to the third cleaning magnetic beads and mix for 2 min. Then use a magnetic rack or nucleic acid extractor for magnetic separation. Then discard the liquid obtained after magnetic separation to obtain the fourth cleaning magnetic beads. Volatilize to remove alcohol for 5 min.

[0185] (8) Sample elution: Add 100 μL of elution buffer to the fourth washing magnetic beads, mix well, and incubate at 50 °C with shaking for 5 min. Collect the RNA solution obtained after elution.

[0186] Example 3: Comparison of RNA extraction from fresh bone marrow blood

[0187] Using the magnetic bead method whole blood RNA extraction kit formulated in Example 1 and Comparative Examples 1-5, RNA was extracted from freshly collected bone marrow blood from 6 patients using the extraction method described in Example 2. 10 mL of anticoagulated bone marrow whole blood was collected from each patient, and 1 mL of each sample was used for RNA extraction. The elution volume was 100 μL. The concentration and purity of the extracted RNA were detected by Nanodrop, and the RNA from the extracted products was detected by Qubit RNA assay.

[0188] Table 1 shows the Nanodrop concentration values. The RNA concentration extracted using the kit in Example 1 was significantly higher than that in Comparative Examples 1-5. The results from Comparative Examples 2 and 3 show that lithium perchlorate and polyethylene glycol play a crucial role in binding RNA in the blood. Data from Comparative Examples 4 and 5 show that when urea and sorbitol were absent in washing solution 3, RNA was lost during the DNA removal process in the third washing step. Sorbitol's role was more significant than urea's. The combined effect of urea and sorbitol in the third washing step kept the RNA on the magnetic beads bound, while the DNA on the beads was degraded by DNase I. This has a crucial impact on this application, enabling fully automated RNA extraction. Table 2 shows the Nanodrop purity results, indicating that the A260 / 280 ratio of the RNA extracted using the kit in Example 1 was around 2.0 or greater, representing good RNA purity. Table 3 shows the Qubit RNA results, which are consistent with the concentration data in Table 1. In addition, Qubit DNA residue detection showed that the residual DNA was relatively low when RNA was extracted using the kit in Example 1, which provides a good RNA template for downstream DNA-sensitive experiments.

[0189] Table 1: Nanodrop concentration (ng / μL) of RNA extracted from bone marrow blood of 6 patients

[0190]

[0191] Table 2: Nanodrop purity of RNA extracted from bone marrow blood of 6 patients (A260 / 280)

[0192]

[0193] Table 3: Qubit RNA concentration (ng / μL) extracted from bone marrow blood of 6 patients

[0194]

[0195] Example 4: Comparison of RNA extraction from bone marrow blood with the Trizol method

[0196] Using the magnetic bead-based blood RNA extraction kit formulated in Example 1, RNA extraction was performed on bone marrow blood samples collected from four patients on the same day using the extraction method described in Example 2. Five mL of EDTA-anticoagulated whole blood was collected from each patient, and 1 mL of each sample was used for RNA extraction. Each sample was prepared as a duplicate, with an elution volume of 100 μL, labeled as Group A. The concentration and purity of the extracted RNA were determined using Nanodrop, and gel electrophoresis was also performed. Additionally, RNA extraction from 1 mL of whole bone marrow blood was performed using the Trizol method. First, red blood cells were lysed from 1 mL of whole blood. Then, the white blood cell pellet was lysed using Trizol, followed by chloroform separation, isopropanol precipitation, alcohol washing, evaporation to remove alcohol, and elution. The elution volume was also 100 μL, labeled as Group B. The entire process was performed manually, taking 4.5 hours to extract 8 samples.

[0197] like Figure 1 As shown, A1 and A2 are samples extracted using the method of this application (sample 1), A3 and A4 are samples extracted using the method of this application (sample 2), A5 and A6 are samples extracted using the method of this application (sample 3), and A7 and A8 are samples extracted using the method of this application (sample 4). Similarly, B1 and B2 are samples extracted using the Trizol method (sample 1), B3 and B4 are samples extracted using the Trizol method (sample 2), B5 and B6 are samples extracted using the Trizol method (sample 3), and B7 and B8 are samples extracted using the Trizol method (sample 4). The gel electrophoresis results show that the 28S and 18S RNA extracted from bone marrow using the method of this application is clearly defined, while the bone marrow RNA extracted using the Trizol method shows faint bands and obvious dragging and degradation. Table 4 shows that the Nanodrop values ​​indicate a concentration at least three times higher than that obtained using the Trizol method. Combining Nanodrop and gel electrophoresis, it can be concluded that the method provided in this application has the advantages of good RNA integrity and high concentration in bone marrow RNA extraction.

[0198] Table 4: Nanodrop concentration (ng / μL) of RNA extracted from bone marrow blood of 4 patients

[0199]

[0200] Example 5: Detection of ABL and WT1 gene RNA in bone marrow serum of leukemia patients

[0201] RNA was extracted from bone marrow blood of 32 patients using the magnetic bead-based blood RNA extraction kit formulated in Example 1 and the extraction protocol in Example 2. 5 mL of EDTA-anticoagulated whole blood was collected from each patient, and 1 mL of each sample was used for RNA extraction, with an elution volume of 100 μL. Additionally, RNA was extracted from 1 mL of whole blood using the Trizol method. The RNA products extracted by both methods were then used for cDNA synthesis using the Script IV 1st Strand cDNA Synthesis Kit (CPC02590). The synthesized cDNA products were then detected by quantitative real-time PCR using a commercially available WT1 detection kit. The Ct values ​​of the internal control RNA and the WT1 RNA gene were also measured.

[0202] The WT1 gene (Wilms Tumor 1) is one of the most commonly used universal molecular markers for clinical monitoring of minimal residual disease (MRD) in leukemia. It is typically significantly overexpressed in leukemia, therefore, changes in the Ct value of WT1 are closely related to the severity of leukemia, relapse risk, and treatment efficacy. Newly diagnosed AML (acute myeloid leukemia) patients often show high WT1 expression, with Ct values ​​significantly lower than normal. In newly diagnosed positive patients, the Ct value of WT1 is usually as low as 18-25, indicating a higher proportion of tumor cells. After drug remission, an increase in Ct signifies tumor reduction; most patients achieving molecular remission have a Ct ≥ 32-35. Therefore, the Ct value of WT1 is extremely important for patient diagnosis and medication. Table 5 shows that this application provides more stable Ct values ​​for ABL, indicating stable extraction of RNA from bone marrow blood, and lower WT1 Ct values, leading to more accurate clinical diagnosis. Using the Trizol method, the WT1 gene cannot be stably detected in many samples (No Ct), which carries the risk of misdiagnosis in clinical practice.

[0203] In addition, for patient number 6848, the Ct values ​​of WT1 detected by the two methods differed greatly. If this were diagnostic data after medication, the Trizol method would easily lead clinicians to believe that the medication was effective, when in fact the treatment effect was poor. The Ct value of WT1 detected by the method of this application was 23.87, which indicates that the medication or treatment effect on this patient was not significant, consistent with reality, and the treatment plan should be changed. This shows that this application provides a reliable pre-processing solution for precision medicine.

[0204] Table 5: Bone marrow serum RNA ABL and RNA WT1 gene Ct values ​​of 32 actual patients

[0205]

[0206] Example 6: Detection of ABL gene and fusion gene in bone marrow RNA of leukemia patients

[0207] RNA was extracted from bone marrow blood of 10 patients using the magnetic bead-based blood RNA extraction kit formulated in Example 1 and the extraction method described in Example 2. 5 mL of EDTA-anticoagulated whole blood was collected from each patient, and 1 mL of each sample was used for RNA extraction, with an elution volume of 100 μL. Additionally, RNA was extracted from 1 mL of whole blood using the Trizol method. cDNA synthesis was performed on the RNA products extracted by both methods using the Script IV 1st Strand cDNA Synthesis Kit (CPC02590). The synthesized cDNA products were then detected by quantitative real-time PCR using a commercially available fusion gene detection kit. The Ct values ​​of the internal control RNA and different fusion gene RNAs were also measured.

[0208] The detection data are shown in Table 6 below. The RNA extracted by the method of this application has a lower ABL Ct value, and the protocol is more stable. The Ct values ​​of different fusion genes are lower than those of the Trizol method. This indicates that the method of this application provides a more accurate and reliable way to diagnose leukemia. At the same time, its fully automated operation process saves clinicians time and effort. The method of this application saves time and costs for clinical blood RNA extraction and has great potential for clinical molecular detection applications.

[0209] Table 6: Bone marrow serum RNA ABL and RNA WT1 gene Ct values ​​of 10 actual patients

[0210]

[0211] The above description of the embodiments is merely for the purpose of helping to understand the method and core ideas of this application. It should be noted that those skilled in the art can make various improvements and modifications to this application without departing from the principles of this application, and these improvements and modifications also fall within the protection scope of the claims of this application. The above description of the disclosed embodiments enables those skilled in the art to implement or use this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0212] It should be noted that the above embodiments can be freely combined as needed. The above description is only a preferred embodiment of this application and is not intended to limit this application. For those skilled in the art, this application can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for extracting RNA, characterized in that, The extraction method includes: adding a lysis binding solution, proteinase K, and magnetic beads to a blood sample for incubation; obtaining the adsorbed magnetic beads through magnetic separation; and further washing and eluting. The lysis binding solution includes 5-15% polyglycerol-3 acetate, 100-400mM sodium acetate, 1-8M lithium perchlorate, 10-50mM disodium ethylenediaminetetraacetate, 0.5-10% cetyltrimethylammonium bromide, 5-25% polyethylene glycol, and 15-50% isopropanol, with a pH of 5.0-8.

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2. The extraction method according to claim 1, characterized in that, The cleaning solution used for cleaning includes 1-4M urea, 20-200mM tris(hydroxymethyl)aminomethane, 1-20mM magnesium chloride, 1-5mM calcium chloride and 2-25% sorbitol, with a pH of 7.0-8.

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3. The extraction method according to claim 1, characterized in that, The cleaning process consists of four cycles, in which... The first cleaning solution used contains: 1-5M guanidine isothiocyanate and 30-80% isopropanol, with a pH of 4.0-8.0; The second cleaning solution used contains: 20-250 mM Tris-HCl, 20-200 mM lithium chloride and 40-80% anhydrous ethanol, with a pH of 5.0-8.0; The third cleaning uses cleaning solution 3 and DNase I, wherein cleaning solution 3 contains: 1-4M urea, 20-200mM tris(hydroxymethyl)aminomethane, 1-20mM magnesium chloride, 1-5mM calcium chloride and 2-25% sorbitol, with a pH of 7.0-8.0; The fourth cleaning solution used contains 25-250 mM Tris-HCl and 50-80% anhydrous ethanol, with a pH of 5.0-8.

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4. The extraction method according to claim 3, characterized in that, The volume of the lysis binding solution added is 1-3 times the volume of the blood sample. The amount of magnetic beads added is 1-4 mg per milliliter of blood sample, and the amount of proteinase K added is 0.4 mg-1.2 mg per milliliter of blood sample. The amount of washing solution 1 used in the first wash is 320-400 μL per mg of magnetic beads. The amount of washing solution 2 used in the second wash is 320-400 μL per mg of magnetic beads. The amount of washing solution 3 used in the third wash is 40-160 μL per mg of magnetic beads. The amount of DNase I added is 1-10 U of DNase I per milliliter of blood. The amount of washing solution 4 used in the fourth wash is 320-400 μL per mg of magnetic beads.

5. The extraction method according to claim 1, characterized in that, The incubation is carried out at 20-30℃ for 5-30 minutes by centrifugation at a speed of 500-1500 rpm.

6. The extraction method according to claim 1, characterized in that, The blood sample is peripheral blood, bone marrow blood, or umbilical cord blood.

7. An RNA extraction kit, characterized in that, The RNA extraction kit includes a lysis binding buffer comprising 5-15% polyglycerol-3 acetate, 100-400 mM sodium acetate, 1-8 M lithium perchlorate, 10-50 mM disodium ethylenediaminetetraacetate, 0.5-10% cetyltrimethylammonium bromide, 5-25% polyethylene glycol, and 15-50% isopropanol, with a pH of 5.0-8.

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8. The RNA extraction kit according to claim 7, characterized in that, The RNA extraction kit also includes a washing solution comprising 1-4M urea, 20-200mM tris(hydroxymethyl)aminomethane, 1-20mM magnesium chloride, 1-5mM calcium chloride and 2-25% sorbitol, with a pH of 7.0-8.

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9. The RNA extraction kit according to claim 7, characterized in that, The RNA extraction kit includes a lysis binding buffer, magnetic beads, proteinase K, washing buffer 1, washing buffer 2, washing buffer 3, DNase I, washing buffer 4, and elution buffer, wherein: The pyrolysis binding solution comprises 5-15% polyglycerol-3 acetate, 100-400mM sodium acetate, 1-8M lithium perchlorate, 10-50mM disodium ethylenediaminetetraacetate, 0.5-10% cetyltrimethylammonium bromide, 5-25% polyethylene glycol, and 15-50% isopropanol, with a pH of 5.0-8.

0. The magnetic beads are silanol magnetic microspheres, glass-modified magnetic microspheres, or carboxyl-modified magnetic microspheres. Cleaning solution 1 contains: 1-5M guanidine isothiocyanate and 30-80% isopropanol, with a pH of 4.0-8.0; Cleaning solution 2 contains: 20-250 mM Tris-HCl, 20-200 mM lithium chloride and 40-80% anhydrous ethanol, with a pH of 5.0-8.0; Cleaning solution 3 contains: 1-4M urea, 20-200mM tris(hydroxymethyl)aminomethane, 1-20mM magnesium chloride, 1-5mM calcium chloride and 2-25% sorbitol, with a pH of 7.0-8.

0. Cleaning solution 4 contains 25-250 mM Tris-HCl and 50-80% anhydrous ethanol, with a pH of 5.0-8.

0.

10. The application of an RNA extraction kit according to any one of claims 7-9, characterized in that, The applications include: A) Applications in RNA extraction; B) Application in the preparation of gene detection products; and / or, C) Application in the preparation of products for diagnosing diseases.

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