Efficient extraction method and application of pancreatic tissue RNA
By employing techniques such as liquid nitrogen precooling, the trypsin inhibitor ulinastatin, and low-temperature high-speed centrifugation, the problems of easy degradation and contamination of pancreatic RNA were solved, achieving efficient and stable RNA extraction and improving RNA quality and integrity.
Patent Information
- Application Number
- CN202511096485.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-06
- Publication Date
- 2025-10-28
AI Technical Summary
Pancreatic tissue RNA is easily degraded, and RNase contamination is severe during extraction, resulting in poor RNA quality and integrity. Strict experimental conditions are required, and existing methods have not been able to completely solve this problem.
The extraction process was carried out under low temperature conditions using liquid nitrogen pre-cooling, the trypsin inhibitor ulinastatin, increased lysis buffer volume, low-temperature high-speed centrifugation, and purification steps, combined with chloroform and ethanol treatment, and a highly efficient RNA purification kit.
It significantly improved the extraction quality and integrity of pancreatic RNA, reduced the risk of RNA degradation during the extraction process, and improved extraction efficiency and success rate.
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Figure CN120843499A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, and in particular relates to a highly efficient method for extracting RNA from pancreatic tissue and its application. Background Technology
[0002] RNA extraction from glandular tissue is a crucial step in biological, medical research, and clinical diagnosis. As a vital endocrine and exocrine organ, the pancreas contains abundant genetic information in its RNA, which is essential for understanding pancreatic function, disease mechanisms, and developing related drugs. However, pancreatic RNA is highly susceptible to degradation due to its instability, RNase contamination, and the high activity of pancreatic enzymes in pancreatic tissue, thus increasing the difficulty of pancreatic RNA extraction. Despite significant advancements in RNA-related biological extraction technologies, the inherent instability of RNA, the presence of endogenous and exogenous RNases, RNase contamination during extraction, and the abundance and activation of enzymes in the tissue all present challenges for tissue RNA extraction. Furthermore, different tissue types and experimental conditions require different RNA extraction methods. In particular, the high activity of pancreatic enzymes in pancreatic tissue leads to highly susceptible RNA degradation; therefore, researchers need to explore suitable extraction methods and select appropriate reagent kits based on the specific characteristics of the pancreas.
[0003] However, current extraction methods have the following problems: 1. RNA is easily degraded: Pancreatic cancer is the most dangerous type of cancer, characterized by short survival and poor prognosis. However, its pathogenesis remains unclear. RNA extraction from pancreatic tissue is a necessary tool for in-depth research on pancreatic cancer. However, due to the inherent instability of RNA, the presence of endogenous and exogenous RNases in pancreatic tissue, RNase contamination during extraction, and the high concentration and activation of pancreatic enzymes in the tissue, RNA is easily degraded during extraction, affecting its quality and integrity, making subsequent experiments impossible. This limits in-depth research on diseases of related pancreatic organs.
[0004] 2. Strict requirements for experimental conditions: Pancreatic tissue RNA extraction has high requirements for experimental conditions, such as techniques, experimental progress speed, temperature, sterility, and RNase-free environment. Slight negligence can lead to substandard quality of pancreatic RNA extraction, resulting in experimental failure.
[0005] To address the above issues, existing solutions mainly focus on: 1. Improve the extraction performance of the reagent kit, but there are technical limitations and barriers.
[0006] 2. Mechanical grinding of pancreatic tissue was used, but the heat generated during the grinding process led to RNA degradation.
[0007] 3. Reduce exposure to environmental RNases: This includes pipettes, workbenches, glassware, and gel casting equipment. It is essential to ensure that every piece of experimental equipment that comes into contact with purified RNA is free of RNase contamination.
[0008] However, the above methods have not completely solved the problem of low quality of RNA extraction from pancreatic tissue, so further research and optimization are still needed. Summary of the Invention
[0009] The purpose of this invention is to provide an optimized method for efficient extraction of RNA from pancreatic tissue, aiming to solve the problems of high RNA degradation risk and poor quality and integrity of extracted RNA in the prior art, and to provide a more efficient and stable method for extracting RNA from pancreatic tissue.
[0010] The present invention adopts the following technical solution: A highly efficient method for extracting RNA from pancreatic tissue for non-diagnostic and therapeutic purposes includes the following steps: S1. Pre-cooling treatment of grinding tools; S2. Tissue grinding: Cut an appropriate amount of pancreatic tissue sample, quickly put the trypsin inhibitor ulinastatin into a pre-cooled grinding instrument for cutting and grinding, and replenish liquid nitrogen as needed during the grinding process until the tissue is powdered. S3, lysis: Add lysis buffer to the ground tissue for lysis, transfer to EP centrifuge tubes, cool on dry ice, and separate RNA by vortexing and low-temperature high-speed centrifugation. S4. RNA purification: Under uniform dry ice working environment, RNA is further purified by adding chloroform and ethanol, combined with low temperature high speed centrifugation and filtration. S5. RNA elution and preservation: The purified RNA was eluted into enzyme-free water, and its concentration and purity were determined. Finally, the RNA was stored in a -80°C freezer for later use.
[0011] In the above technical solution, in step S1, an appropriate amount of liquid nitrogen is poured into the mortar to pre-cool the mortar and pestle.
[0012] In the above technical solution, in step S2, 10-30 mg of pancreatic tissue sample is cut using sterile, enzyme-free tissue scissors. The tissue sample is then quickly transferred to a pre-cooled mortar containing pure liquid nitrogen. 0.6 ml of 30,000 units of the trypsin inhibitor ulinastatin is added. The pancreatic tissue is then cut into small pieces using a pre-cooled pestle. The pieces are then scraped together by the pestle. Liquid nitrogen is added continuously during the grinding process until the tissue is powdered.
[0013] In the above technical solution, in step S3, 3 ml of Trizol lysis buffer is added to the ground tissue for lysis, transferred to an EP centrifuge tube, placed on dry ice for 5 minutes, and RNA is separated by vortexing and low-temperature high-speed centrifugation at 4℃ and 12000 rpm.
[0014] In the above technical solution, in step S3, after adding Trizol lysis solution, Trizol lysis solution is quickly frozen into blocks, and then an appropriate amount of liquid nitrogen is poured in. The blocks of Trizol are crushed in step S3 with a grinding rod and ground into powder. The grinding continues for about 5 minutes until it is completely melted into a liquid state.
[0015] In the above technical solution, in step S4, RNA is further purified by adding chloroform and ethanol under a uniform dry ice working environment, combined with low-temperature high-speed centrifugation at 4℃ and 12000rpm and filtration.
[0016] In the above technical solution, step S4, the low-temperature high-speed centrifugation and filtration steps are as follows: S401. Transfer the mixture to an RNA column, centrifuge at 12000 rpm for 1 min at 4°C, and discard the filtrate; S402. Add 600 μL of RW1 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate. S403. Add 650 μL of RW2 to the RNA solution, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate. S404. Add 650 μL of RW2 to the RNA column, discard the filtrate, and replace with a new collection tube. Centrifuge in an empty tube at 4℃ and 12000 rpm for 2 minutes using S405.
[0017] In the above technical solution, in step S5, 50uL of enzyme-free water is added to the center of the RNA column membrane, and the membrane is centrifuged at 12000rpm for 1min at 4℃. The resulting eluent is the RNA sample.
[0018] The second objective of this invention is to provide an application of the above-described efficient method for extracting pancreatic tissue RNA for non-diagnostic and non-therapeutic purposes in the extraction / detection of pancreatic tissue genomic RNA.
[0019] Combining all the above technical solutions, the advantages and positive effects of this invention are as follows: This invention extracts RNA from pancreatic tissue by mincing the pancreatic tissue, applying the trypsin inhibitor ulinastatin, pre-cooling the grinding equipment with liquid nitrogen, increasing the volume of lysis buffer, controlling the temperature throughout the extraction process, and accelerating the experimental process to improve the quality of RNA extraction. This method can significantly improve the quality of RNA extraction, significantly reduce the risk of RNA degradation during the extraction process, and significantly improve the integrity and purity of RNA.
[0020] In addition, the inventive step evidence for this invention is also reflected in the following important aspects: 1. After the technical solution of this invention is transformed, it can be prepared into an improved pancreatic RNA extraction kit, targeting researchers and clinical medical laboratory personnel. It is expected that the technical solution of this invention will generate substantial benefits after transformation.
[0021] 2. Extracting pancreatic RNA is a highly demanding technique that requires extensive experience and skill, especially for beginners who often need to experiment multiple times. This solution ensures a high success rate for pancreatic RNA extraction, stabilizes extraction efficiency, and simplifies and makes the notoriously difficult technique of pancreatic RNA extraction simple and successful. Attached Figure Description
[0022] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this disclosure and, together with the description, serve to explain the principles of this disclosure; Figure 1 This is a flowchart of the method for efficient RNA extraction from pancreatic tissue in the embodiments; Figure 2 This is a flowchart illustrating the efficient RNA extraction method from pancreatic tissue in this embodiment. Figure 3 This is a comparison graph of gel electrophoresis results obtained by extracting RNA using four different methods in the comparative analysis. Detailed Implementation
[0023] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0024] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Example
[0025] An optimized method for high-efficiency RNA extraction from pancreatic tissue is proposed. The key to this method is that all equipment in contact with the pancreatic tissue is sterilized, enzyme-free, and cryogenically treated with liquid nitrogen. The entire extraction process is conducted at low temperatures, and pre-chilled ulinastatin is added to inhibit trypsin activation in the pancreatic tissue. Specifically, the pancreatic tissue is first minced in liquid nitrogen using cryogenically treated scissors. Then, liquid nitrogen is added to encapsulate the pancreatic tissue and pre-chilled ulinastatin, and the mixture is rapidly ground by hand using a pre-chilled mortar and pestle (this step must be completed quickly). A large volume of lysis buffer (approximately 2-3 ml, exceeding the usual dosage) is added, and centrifugation is performed at low temperature and high speed to ensure RNA integrity and high purity. This method includes the following steps: 1. Preparation stage: Ensure that all equipment and reagents used are sterile and enzyme-free (choose imported consumables if possible), and prepare the necessary instruments and consumables, such as ice makers, low-temperature high-speed centrifuges, mortars and pestles pre-cooled with liquid nitrogen, tissue scissors, etc.
[0026] 2. Tissue grinding: After cutting approximately 10-30 mg of fresh pancreatic tissue, quickly place 0.6 ml of 30,000 units of the trypsin inhibitor ulinastatin into a pre-cooled mortar containing liquid nitrogen for cutting and grinding. Liquid nitrogen should be added continuously during the process until the tissue is powdered. This step must be completed quickly to reduce the various risks of RNA degradation.
[0027] 3. Trizol lysis: Add 3 ml of Trizol to the ground tissue for lysis, transfer to an EP centrifuge tube, place on dry ice for 5 minutes, and then separate the RNA using vortexing and low-temperature high-speed centrifugation at 4℃ and 12000 rpm. This step requires strict control of time and temperature to ensure RNA integrity and high purity. Adding 3 ml of Trizol lysis buffer solves the problem of insufficient lysis buffer due to evaporation during grinding in liquid nitrogen. Placing the lysed tissue on dry ice for 5 minutes after lysis ensures RNA integrity and high purity.
[0028] 4. RNA purification: Under uniform dry ice conditions, RNA was further purified by adding reagents such as chloroform and ethanol, combined with high-speed centrifugation at 12,000 rpm at 4°C and filtration. This step aims to remove impurities and improve RNA purity.
[0029] 5. RNA elution and storage: The purified RNA was eluted into enzyme-free water, and its concentration and purity were determined. Finally, the RNA was stored at -80°C for later use.
[0030] The above-described extraction method was used to extract RNA from pancreatic tissue. The quality of RNA extraction was improved by mincing the pancreatic tissue, using the trypsin inhibitor ulinastatin, pre-cooling the grinding equipment with liquid nitrogen, increasing the volume of lysis buffer, controlling the temperature throughout the extraction process, and accelerating the experimental procedure. This method significantly improves the pancreatic RNA extraction capacity, significantly reduces the risk of degradation during extraction, and significantly improves the integrity and purity of the pancreatic RNA.
[0031] Application example: An optimized method for efficient RNA extraction from pancreatic tissue includes the following steps: 1. Pour an appropriate amount of liquid nitrogen into the mortar and pestle to pre-cool the mortar and pestle.
[0032] 2. After euthanizing the mice, disinfect the surface with 75% alcohol. Obtain the pancreatic tissue by dissection. Use sterile, enzyme-free tissue scissors to precisely cut a pancreatic tissue sample of about 10-30 mg (about the size of a mung bean, ensuring no impurities or hair contamination). Quickly transfer the tissue sample to a pre-cooled mortar containing pure liquid nitrogen. Add 0.6 ml of 30,000 units of the trypsin inhibitor ulinastatin. Crush the pancreatic tissue and quickly crush it into small pieces using a pre-cooled pestle. Then, scrape the pieces together with the pestle.
[0033] 3. Once the liquid nitrogen is almost completely evaporated, immediately add 3 mL of Trizol (the lysis buffer from the kit). The Trizol will quickly freeze into a block. Then pour in an appropriate amount of liquid nitrogen, use a mortar and pestle to break up the blocky Trizol and grind it into powder. Continue grinding for about five minutes until it completely melts into a liquid state (ensure ventilation, as Trizol is irritating and toxic). (See...) Figure 2 Operations 1 to 4).
[0034] 4. Measure 800 μL of the melted liquid homogenate from the mortar and transfer it to a 1.5 mL enzyme-free EP tube. Place the EP tube on dry ice in a foam box and let it stand for 5 minutes, then perform vortex lysis for 30 seconds to 1 minute (see [link to EP tube]). Figure 2 Operation 5).
[0035] 5. Centrifuge at 4℃ and 12000rpm for 5 minutes, then transfer the supernatant to a new enzyme-free EP tube.
[0036] 6. Add 1 / 5 of the total volume of lysis buffer to the supernatant above, mix thoroughly, and let stand on dry ice for 2-5 minutes (the time can be adjusted according to the actual situation). Centrifuge at 12,000 rpm for 5 minutes at 4°C. (Chloroform is toxic, this step must be performed in a biosafety cabinet).
[0037] 7. Add an equal volume of 70% ethanol (diluted with sterile ddH2O or enzyme-free water) to the supernatant above, mix well, and use a pipette to mix the flocculent or precipitated RNA.
[0038] 8. Transfer the above mixture to an RNA column, centrifuge at 4°C and 12,000 rpm for 1 min, and discard the filtrate.
[0039] 9. Add 600 μL of RW1 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate.
[0040] 10. Add 650 μL of RW2 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate.
[0041] 11. Add 650 μL of RW2 to the RNA column, discard the filtrate, and replace with a new collection tube.
[0042] Centrifuge in an empty tube at 12.4℃ and 12000rpm for 2 minutes.
[0043] 13. Add 50 μL of enzyme-free water to the center of the RNA column membrane, centrifuge at 12,000 rpm for 1 min at 4 °C, and the resulting eluent is the RNA sample. Take a small amount of RNA for subsequent concentration determination and experiments, and store the remaining RNA in a -80 °C freezer for later use.
[0044] Comparative Example: RNA extracted using four different methods Extraction Method 1: RNA extraction without additives (i.e., conventional RNA extraction kits available on the market) 1) Place the mortar and pestle on ice at 0 degrees Celsius to pre-cool.
[0045] 2) After euthanizing the mice, disinfect the surface with 75% alcohol, obtain the pancreatic tissue by dissection, and accurately cut a pancreatic tissue sample of about 10-30mg (about the size of a mung bean) with sterile, enzyme-free tissue scissors, ensuring that there are no impurities or hair contamination. Quickly transfer the tissue sample to a pre-cooled mortar, cut the pancreatic tissue into small pieces, and quickly crush the pancreatic tissue into small pieces with a pre-cooled pestle. Then, scrape the pieces together with the pestle.
[0046] 3) Add 1.5 mL of Trizol (the lysis buffer from the kit) and continue grinding for about five minutes until it becomes a slurry (keep the ventilation open, as Trizol is irritating and toxic).
[0047] 4) Measure 800 μL of the thawed liquid homogenate from the mortar and transfer it to a 1.5 mL enzyme-free EP tube. Place the EP tube on ice at 0 degrees Celsius in a foam box and let it stand for 5 minutes. Then perform vortex lysis for 30 seconds to 1 minute.
[0048] 5) Centrifuge at 4℃ and 12000rpm for 5 minutes, then transfer the supernatant to a new enzyme-free EP tube.
[0049] 6) Add 1 / 5 of the total volume of lysis buffer to the supernatant above, mix thoroughly, and let stand on ice at 0 degrees for 2-5 minutes (the time can be adjusted according to the actual situation). Centrifuge at 12,000 rpm for 5 minutes at 4 degrees. (Chloroform is toxic, this step must be performed in a biosafety cabinet).
[0050] 7) Add an equal volume of 70% ethanol (diluted with sterile ddH2O or enzyme-free water) to the supernatant above, mix well, and use a pipette to mix the flocculent or precipitated RNA.
[0051] 8) Transfer the above mixture to an RNA column, centrifuge at 4°C and 12,000 rpm for 1 min, and discard the filtrate.
[0052] 9) Add 600 μL of RW1 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4 °C, and discard the filtrate.
[0053] 10) Add 650 μL of RW2 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4 °C, and discard the filtrate.
[0054] 11) Add 650 μL of RW2 to the RNA column, discard the filtrate, and replace with a new collection tube.
[0055] 12) Centrifuge in an empty tube at 12000 rpm for 2 minutes at 4℃.
[0056] 13) Add 50 μL of enzyme-free water to the center of the RNA column membrane, centrifuge at 12,000 rpm for 1 min at 4 °C, and the resulting eluent is the RNA sample. Take a small amount of RNA for subsequent concentration determination and experiments, and store the remaining RNA in a -80 °C freezer for later use.
[0057] Extraction Method 2: Extraction of pancreatic RNA by adding the pancreatic enzyme inhibitor ulinastatin 1) Use 0-degree ice to pre-cool the mortar and pestle.
[0058] 2) After euthanizing the mice, the surface was disinfected with 75% alcohol. The pancreatic tissue was obtained by dissection. Using sterile, enzyme-free tissue scissors, a pancreatic tissue sample of about 10-30 mg (about the size of a mung bean, ensuring no impurities or hair contamination) was precisely cut. The tissue sample was quickly transferred to a pre-cooled mortar, and 0.6 ml of 30,000 units of the trypsin inhibitor ulinastatin was added. The pancreatic tissue was cut into small pieces and quickly crushed into small fragments using a pre-cooled pestle. The fragments were then aggregated by scraping with the pestle.
[0059] 3) Add 1.5 mL of Trizol lysis buffer and continue grinding for about five minutes until it is completely dissolved into a slurry (note that ventilation should be maintained, as Trizol is irritating and toxic).
[0060] 4) Measure 800 μL of the thawed liquid homogenate from the mortar and transfer it to a 1.5 mL enzyme-free EP tube. Place the EP tube on ice at 0 degrees Celsius in a foam box and let it stand for 5 minutes. Then perform vortex lysis for 30 seconds to 1 minute (see [link to EP tube]). Figure 2 Operation 5).
[0061] 5) Centrifuge at 4℃ and 12000rpm for 5 minutes, then transfer the supernatant to a new enzyme-free EP tube.
[0062] 6) Add 1 / 5 of the total volume of lysis buffer to the supernatant above, mix thoroughly, and let stand on ice at 0 degrees for 2-5 minutes (the time can be adjusted according to the actual situation). Centrifuge at 12,000 rpm for 5 minutes at 4 degrees. (Chloroform is toxic, this step must be performed in a biosafety cabinet).
[0063] 7) Add an equal volume of 70% ethanol (diluted with sterile ddH2O or enzyme-free water) to the supernatant above, mix well, and use a pipette to mix the flocculent or precipitated RNA.
[0064] 8) Transfer the above mixture to an RNA column, centrifuge at 4°C and 12,000 rpm for 1 min, and discard the filtrate.
[0065] 9) Add 600 μL of RW1 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate.
[0066] 10) Add 650 μL of RW2 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate.
[0067] 11) Add 650 μL of RW2 to the RNA column, discard the filtrate, and replace with a new collection tube.
[0068] 12). Centrifuge in an empty tube at 12000 rpm for 2 minutes at 4℃.
[0069] 13) Add 50 μL of enzyme-free water to the center of the RNA column membrane, centrifuge at 12,000 rpm for 1 min at 4 °C, and the resulting eluent is the RNA sample. Take a small amount of RNA for subsequent concentration determination and experiments, and store the remaining RNA in a -80 °C freezer for later use.
[0070] Extraction Method 3: Extraction of Pancreatic RNA using Liquid Nitrogen Cryogenic Operation 1) Pour an appropriate amount of liquid nitrogen into the mortar and pestle to pre-cool the mortar and pestle.
[0071] 2) After euthanizing the mice, the surface was disinfected with 75% alcohol. The pancreatic tissue was obtained by dissection. A pancreatic tissue sample of about 10-30 mg (about the size of a mung bean, ensuring no impurities or hair contamination) was precisely cut using sterile, enzyme-free tissue scissors. The tissue sample was quickly transferred to a pre-cooled mortar containing pure liquid nitrogen. The pancreatic tissue was shredded and quickly crushed into small pieces using a pre-cooled pestle. The pieces were then scraped together by the pestle.
[0072] 3) Once the liquid nitrogen is about to evaporate, immediately add 1.5 mL of Trizol lysis buffer. At this point, the Trizol will quickly freeze into blocks. Then, pour in an appropriate amount of liquid nitrogen, break up the blocks of Trizol with a mortar and pestle, and grind it into powder. Continue grinding for about five minutes until it is completely melted into a liquid state (be careful to keep the ventilation open, as Trizol is irritating and toxic).
[0073] 4) Measure 800 μL of the thawed liquid homogenate from the mortar and transfer it to a 1.5 mL enzyme-free EP tube. Place the EP tube on ice at 0 degrees Celsius in a foam box and let it stand for 5 minutes. Then perform vortex lysis for 30 seconds to 1 minute (see [link to EP tube]). Figure 2 Operation 5).
[0074] 5) Centrifuge at 4℃ and 12000rpm for 5 minutes, then transfer the supernatant to a new enzyme-free EP tube.
[0075] 6) Add 1 / 5 of the total volume of lysis buffer to the supernatant above, mix thoroughly, and let stand on ice at 0 degrees for 2-5 minutes (the time can be adjusted according to the actual situation). Centrifuge at 12,000 rpm for 5 minutes at 4 degrees. (Chloroform is toxic, this step must be performed in a biosafety cabinet).
[0076] 7) Add an equal volume of 70% ethanol (diluted with sterile ddH2O or enzyme-free water) to the supernatant above, mix well, and use a pipette to mix the flocculent or precipitated RNA.
[0077] 8) Transfer the above mixture to an RNA column, centrifuge at 4°C and 12,000 rpm for 1 min, and discard the filtrate.
[0078] 9) Add 600 μL of RW1 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate.
[0079] 10) Add 650 μL of RW2 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate.
[0080] 11) Add 650 μL of RW2 to the RNA column, discard the filtrate, and replace with a new collection tube.
[0081] 12). Centrifuge in an empty tube at 12000 rpm for 2 minutes at 4℃.
[0082] 13) Add 50 μL of enzyme-free water to the center of the RNA column membrane, centrifuge at 12,000 rpm for 1 min at 4 °C, and the resulting eluent is the RNA sample. Take a small amount of RNA for subsequent concentration determination and experiments, and store the remaining RNA in a -80 °C freezer for later use.
[0083] Extraction Method 4: Method for extracting pancreatic RNA by adding liquid nitrogen and the trypsin inhibitor ulinastatin 1) Pour an appropriate amount of liquid nitrogen into the mortar and pestle to pre-cool the mortar and pestle.
[0084] 2) After euthanizing the mice, the surface was disinfected with 75% alcohol. The pancreatic tissue was obtained by dissection. A pancreatic tissue sample of about 10-30 mg (about the size of a mung bean, ensuring no impurities or hair contamination) was precisely cut using sterile, enzyme-free tissue scissors. The tissue sample was quickly transferred to a pre-cooled mortar containing pure liquid nitrogen. 0.6 ml of 30,000 units of the trypsin inhibitor ulinastatin was added. The pancreatic tissue was cut into small pieces and quickly crushed into small fragments using a pre-cooled pestle. The fragments were then scraped together by the pestle.
[0085] 3) Once the liquid nitrogen is almost completely evaporated, immediately add 3 mL of Trizol lysis buffer. At this point, the Trizol will quickly freeze into blocks. Then, pour in an appropriate amount of liquid nitrogen, use a mortar and pestle to break up the Trizol blocks and grind them into powder. Continue grinding for about five minutes until it completely melts into a liquid state (be sure to maintain ventilation, as Trizol is irritating and toxic) (see...). Figure 2 Operations 1 to 4).
[0086] 4) Measure 800 μL of the melted liquid homogenate from the mortar and transfer it to a 1.5 mL enzyme-free EP tube. Place the EP tube on dry ice in a foam box and let it stand for 5 minutes, then perform vortex lysis for 30 seconds to 1 minute (see [link to EP tube]). Figure 2 Operation 5).
[0087] 5) Centrifuge at 4℃ and 12000rpm for 5 minutes, then transfer the supernatant to a new enzyme-free EP tube.
[0088] 6) Add 1 / 5 of the total volume of lysis buffer to the supernatant above, mix thoroughly, and let stand on dry ice for 2-5 minutes (the time can be adjusted according to the actual situation). Centrifuge at 12,000 rpm for 5 minutes at 4°C. (Chloroform is toxic, this step must be performed in a biosafety cabinet).
[0089] 7) Add an equal volume of 70% ethanol (diluted with sterile ddH2O or enzyme-free water) to the supernatant above, mix well, and use a pipette to mix the flocculent or precipitated RNA.
[0090] 8) Transfer the above mixture to an RNA column, centrifuge at 4°C and 12,000 rpm for 1 min, and discard the filtrate.
[0091] 9) Add 600 μL of RW1 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate.
[0092] 10) Add 650 μL of RW2 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate.
[0093] 11) Add 650 μL of RW2 to the RNA column, discard the filtrate, and replace with a new collection tube.
[0094] 12). Centrifuge in an empty tube at 12000 rpm for 2 minutes at 4℃.
[0095] 13) Add 50 μL of enzyme-free water to the center of the RNA column membrane, centrifuge at 12,000 rpm for 1 min at 4 °C, and the resulting eluent is the RNA sample. Take a small amount of RNA for subsequent concentration determination and experiments, and store the remaining RNA in a -80 °C freezer for later use.
[0096] RNA was extracted from pancreatic tissue using the method described in the comparative example above, and the extracted RNA was subjected to gel electrophoresis.
[0097] Result: As Figure 3 As shown, Figure 3 This is a comparison of gel electrophoresis results obtained using the four different methods described above. Figure 3 The order of sample loading for medium gel electrophoresis was as follows: 1. Marker; 2. RNA extracted without any additives; 3. RNA extracted with the trypsin inhibitor ulinastatin; 4. RNA extracted under liquid nitrogen cryogenic conditions; 5. RNA extracted with the addition of liquid nitrogen and the trypsin inhibitor ulinastatin.
[0098] Conclusion: Comparison shows that the RNA bands extracted using extraction method 4, which involves sequentially adding liquid nitrogen and the trypsin inhibitor ulinastatin (e.g., ...), are superior. Figure 3 The middle band (shown in image 5) is the clearest and brightest, indicating that the pancreatic RNA has minimal degradation, minimal contamination, the highest concentration, and the best quality. This demonstrates that the addition of liquid nitrogen and the pancreatic enzyme inhibitor ulinastatin effectively inhibited RNA instability, prevented RNase contamination, and controlled the activation of pancreatic enzymes in pancreatic tissue, thereby ensuring the integrity of the pancreatic RNA and improving the efficiency and success rate of pancreatic RNA extraction.
[0099] Note: The marker is a DNA marker, ranging in size from 500bp to 15000bp; it is only used to detect whether the genome is degraded or contaminated, and does not represent the size of the RNA band.
[0100] The above description is merely a preferred embodiment of the present invention. It should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments, but can be used in various other combinations, modifications, and environments. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.
Claims
1. A highly efficient method for extracting RNA from pancreatic tissue for non-diagnostic and non-therapeutic purposes, characterized in that: Includes the following steps: S1. Pre-cooling treatment of grinding tools; S2. Tissue grinding: Cut an appropriate amount of pancreatic tissue sample, quickly put the trypsin inhibitor ulinastatin into a pre-cooled grinding instrument for cutting and grinding, and replenish liquid nitrogen as needed during the grinding process until the tissue is powdered. S3, lysis: Add lysis buffer to the ground tissue for lysis, transfer to EP centrifuge tubes, cool on dry ice, and separate RNA by vortexing and low-temperature high-speed centrifugation. S4. RNA purification: Under uniform dry ice working environment, RNA is further purified by adding chloroform and ethanol, combined with low temperature high speed centrifugation and filtration. S5. RNA elution and preservation: The purified RNA was eluted into enzyme-free water, and its concentration and purity were determined. Finally, the RNA was stored in a -80°C freezer for later use.
2. The method for efficient extraction of RNA from pancreatic tissue for non-diagnostic and non-therapeutic purposes according to claim 1, characterized in that: In step S1, an appropriate amount of liquid nitrogen is poured into the mortar and pestle to pre-cool the mortar and pestle.
3. The method for efficient extraction of RNA from pancreatic tissue for non-diagnostic and non-therapeutic purposes according to claim 1, characterized in that: In step S2, 10-30 mg of pancreatic tissue sample is cut using sterile, enzyme-free tissue scissors. The tissue sample is then quickly transferred to a pre-cooled mortar containing pure liquid nitrogen. 0.6 ml of 30,000 units of the trypsin inhibitor ulinastatin is added. The pancreatic tissue is then shredded and quickly crushed into small pieces using a pre-cooled pestle. The pieces are then scraped together by the pestle. Liquid nitrogen is added continuously during the grinding process until the tissue is powdered.
4. The method for efficient extraction of RNA from pancreatic tissue for non-diagnostic and non-therapeutic purposes according to claim 1, characterized in that: In step S3, 3 ml of Trizol lysis buffer was added to the ground tissue for lysis, transferred to an EP centrifuge tube, placed on dry ice for 5 minutes, and RNA was separated by vortexing and low-temperature high-speed centrifugation at 4℃ and 12000 rpm.
5. The method for efficient extraction of RNA from pancreatic tissue for non-diagnostic and non-therapeutic purposes according to claim 4, characterized in that: In step S3, after adding the Trizol lysis solution, the Trizol lysis solution quickly freezes into blocks. Then, an appropriate amount of liquid nitrogen is poured in, and the blocks of Trizol in step S3 are crushed with a mortar and pestle and ground into powder. The grinding continues for about 5 minutes until it is completely melted into a liquid state.
6. The method for efficient extraction of RNA from pancreatic tissue for non-diagnostic and non-therapeutic purposes according to claim 1, characterized in that: In step S4, RNA is further purified by adding chloroform and ethanol under a uniform dry ice working environment, combined with low-temperature high-speed centrifugation at 4℃ and 12000rpm and filtration.
7. The method for efficient extraction of RNA from pancreatic tissue for non-diagnostic and non-therapeutic purposes according to claim 6, characterized in that: In step S4, the low-temperature high-speed centrifugation and filtration steps are as follows: S401. Transfer the mixture to an RNA column, centrifuge at 12000 rpm for 1 min at 4°C, and discard the filtrate; S402. Add 600 μL of RW1 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate. S403. Add 650 μL of RW2 to the RNA column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the filtrate. S404. Add 650 μL of RW2 to the RNA column, discard the filtrate, and replace with a new collection tube. Centrifuge in an empty tube at 4℃ and 12000 rpm for 2 minutes using S405.
8. The method for efficient extraction of RNA from pancreatic tissue for non-diagnostic and non-therapeutic purposes according to claim 1, characterized in that: In step S5, add 50 μL of enzyme-free water to the center of the RNA column membrane, centrifuge at 12,000 rpm for 1 min at 4°C, and the resulting eluent is the RNA sample.
9. The application of the efficient method for extracting pancreatic tissue RNA for non-diagnostic and non-therapeutic purposes as described in any one of claims 1-8 in the extraction / detection of genomic RNA from pancreatic tissue.