Analysis of mRNA degradation experiments by cell-free extracts of higher eukaryotes
Analysis of mRNA degradation experiments by cell-free extracts of higher eukaryotes
Changes in the environment of the cell, the state of the replication cycle, and the state of differentiation can cause changes in the half-life of some mRNAs. This experiment is from "RNA Lab Guidebook", edited by Xiaofei Zheng.
Operation method
Analysis of mRNA degradation experiments by cell-free extracts of higher eukaryotes
Principle
Changes in the environment of the cell, the state of the replication cycle, and the state of differentiation cause changes in the half-life of some mRNAs.
Materials and Instruments
DEPC Cells Polyribosomes RNA Substrate Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Potassium acetate Magnesium acetate DTT Tris-acetic acid Tissue culture medium Sterile deionized water Creatine phosphate ATP GTP Potassium acetate Arginine RNase inhibitor
High-temperature oven Homogenizer Pestle and mortar Low-speed centrifuge Ultracentrifuge Turning head Centrifuge tube Single-sided blade Magnetic stirrer Stirrer
1. DEPC treated glassware
(1) DEPC.
(2) High temperature oven.
2. Preparation of polyribosomes and post-polyribosomal supernatants from exponentially growing tissue culture cells S130
(1) Exponentially growing tissue culture cells.
(2) Solution A : 1 mmol/L potassium acetate, 2 mmol/L magnesium acetate, 2 mmol/L DTT, 10 mmol/L Tris-acetic acid (pH 7.6), stored at room temperature.
(3) Solution B: Solution A with 30% (m/V) RNase-free sucrose, sterilized.
(4) Serum and antibiotic free tissue culture solution.
(5) Clean Potte-Elvelijem homogenizer with Teflon pestle and mortar with a mallet tube clearance (elearance) of approximately 0.1 mm.
(6) 1 ml or 2 ml Dounce glass homogenizer with tightly packed mortar and pestle with 0.06 mm hammer tube clearance.
(7) Low-speed centrifuge, ultracentrifuge, and appropriate rotor head and tube.
(8) Single-sided blades.
3. Preparation of RSW from polyribosomes
(1) Isolated polyribosomes, step 2 "Preparation of polyribosomes from exponentially growing tissue culture cells and polyribosomes after supernatant S130".
(2) Solutions A and B and ultracentrifuge.
(3) DEPC.
(4) Magnetic stirrer and stirrer.
(5) 4.0 mol/L potassium acetate.
4. Degradation of endogenous polyribosomal mRNAs
(1) Solution C : 200 μl sterile deionized water, 200 μl 1.0 mol/L phosphocreatine, 40 μl 1 mol/L DTT, 100 μl 0.2 mol/L ATP (Tris salt), 20 μl 0.2 mol/L GTP ( Tris salt), 1 ml 2.0 mol/L potassium acetate, 800 μl 2.5 mmol/L arginine, 200 μl 1.0 mol/L Tris-acetate (pH 7.5 ). 200 μl 1.0 mol/L Tris-acetic acid (pH 7.5 ). Dispense into 0.2~0.4 ml and store at -70℃.
(2) Solution A and a concentration of 106 cells per ml of source polyribosomes.
(3) Solution D: 1 μl deionized sterile water, 3.2 μl solution C, 0.25 μl 4 mg/ml creatine kinase, 0.25 μl 40 U/μl RNase inhibitor. 16 μl solution A, 0.2 μl 0.1 mol/L magnesium acetate. Configure on ice before use, 25 μl per reaction.
5. Analysis of exogenous mRNA degradation using polyribosomes as a source of mRNase activity
(1) Solution A and a concentration of 106 cell-derived polyribosomes per milliliter.
(2) Solution C.
(3) 32P-labeled ( 107 cpm/μg, 5000~20,000 cpm/μl aqueous solution) and unlabeled RNA substrate ( 0.1~2.0 μg/μl aqueous solution).
(4) Solution E : 3.2 μl of Solution C, 0.25 μl of 4 mg/ml Creatine Kinase, 025 μl of 40 U/μl RNase Inhibitor, 16 μl of Solution A, 0.2 μl of 0.1 mol/L Magnesium Acetate, and 1.1 μl of RNA Substrate, configured on ice prior to use, 25 μl per reaction.
6. Analyze the degradation of exogenous mRNA using RSW as a source of mRNase activity.
(1) Solution A is the same as step (2) in Procedure 3, "Preparation of Polyribosomes from Exponentially Growing Tissue Culture Cells after Supernatant S130".
(2) RNA substrate is the same as procedure 5, "Analysis of exogenous mRNA degradation using polyribosomes as a source of mRNase activity".
(3) RSW (ribosomal salt wash) as in procedure 3 "Preparation of RSW from polyribosomes".
(4) Solution F : 1.2 ml sterile deionized water, 200 μl 1.0 mol/L phosphocreatine, 40 μl 1.0 mol/L DTT, 100 μl 0.2 mol/L ATP ( Tris salt), 20 μl 0.2 mol/L GTP ( Tris salt), 800 μl 2.5 mmol/L arginin, 200 μl 1.0 mol/L Tris-acetic acid (pH 7.5 ), 0.2~0.4 ml dispensed and stored at -70°C.
(5) Solution G, 25 μl of each reaction was prepared on ice before use: 3.2 μl of solution F, 0.25 μl of 4 mg/ml creatine kinase, 0.25 μl of KNase inhibitor, 15 μl of solution A, 0.2 μl of 0.1 mol/L magnesium acetate and 1.1 μl of RNA substrate.
7. The degradation of exogenous mRNA was analyzed using the mRNA-dependent rabbit reticulocyte translation system as a source of mRNase activity.
(1) Commercialized rabbit reticulocytes pretreated with micrococcal nuclease.
(2) Cell-derived or in vitro transcription-derived mRNA was lysed in RNase-free sterile water at a concentration of 0.1 to 5.0 μg/μl.
(3) Placenta or recombinant RNase inhibitor (40 U/μl).
(4) 1 mmol/L amino acid mixture.
(5) Radiolabeled amino acids (optional).
II. Operating Procedures
1. DEPC-treated glassware
(1) Add a small amount of DEPC solution to each glassware.
(2) Turn the glassware so that all inner surfaces are exposed to DEPC.
(3) Pour off the DEPC and rinse thoroughly with sterile deionized water until the distinct DEPC odor is completely eliminated, at least 10 times.
(4) Cover the glassware with aluminum foil and bake in an oven for at least 1 h, preferably overnight.
2. Preparation of polyribosomes and post-polyribosomal supernatants from exponentially growing tissue culture cells S130
(1) Cell counting.
(2) Centrifuge at 4°C, 500 r/min for 4 min with a centrifugal rotor with a horizontal radius of 8 cm.
(3) Aspirate the supernatant.
(4) Gently resuspend the cells in serum- and antibiotic-free tissue culture medium.
(5) Centrifuge as in step (2).
(6) Resuspend cells in Solution A.
(7) Transfer cells to a pre-cooled Potter-Elvehjcm homogenizer.
(5) Place the homogenizer in an ice-water bath and move the mortar and pestle rapidly up and down while twisting it from side to side to crush the cells.
(9) Centrifuge the homogenate at 8300 g for 10 min (equivalent to 9000 r/min) with a centrifugal rotor radius of 9.1 cm.
(10) Carefully aspirate the supernatant (S8), avoiding any precipitates (nuclei and membranes), leaving a small amount of supernatant in the centrifuge tube to avoid contamination of S8 with precipitated components.
(11) Add approximately 25% of Solution B to the pre-cooled ultracentrifuge tube and carefully spread S8 over Solution B. If the amount of S8 supernatant is insufficient, add the supernatant to the tube. Add Solution A if the amount of S8 supernatant is insufficient.
(12) Centrifuge at 2°C, 130,000 g for 2.5 h. If the S130 supernatant is to be collected, request an automatic deceleration at the end of the centrifugation without applying the brake.
(13) To recover the S130 supernatant from the top of the Sucrose Solution, care should be taken to prevent collection of white material from the top of the Sucrose Solution, and if necessary, end up with a small amount of supernatant left in the centrifuge tube without aspirating. Do not worry about whether some of the white, sticky liquid at the top of the centrifuge tube will be recovered along with the S130. Store the recovered S130 in a pre-cooled centrifuge tube.
(14) Aspirate the remaining liquid in the centrifuge tube, including the sucrose solution. A pale blue-white precipitate of polyribosomes is visible in the bottom center of the tube.
(15) Discard the top by making an incision with a razor blade in the cold chamber nearly 2/3 of the way from the top of the heart tube.
(16) Gently add about 1 ml of Solution A to the bottom of the remaining centrifuge tube, gently move the tube back and forth to wash away any residual liquid, including sucrose, and aspirate the liquid.
(17) Repeat the washing procedure, which does not need to be done too quickly as the polysaccharides will not dissolve in the wash solution, but the liquid should be added carefully and slowly to avoid dislodging the polysaccharide precipitate from the bottom of the centrifuge tube.
(18) Resuspend the polysome precipitate in a small volume of Solution A. The volume of Solution A depends on the amount of polysomes and the subsequent experimental requirements, and is typically resuspended in 1 μl of solution for every 105 cell-derived polysomes.
(19) Determine the optical density of polysomes and S130 at 260 nm and 280 nm using a spectrophotometer, dispense 100~200 μl and store at -70℃.
(20) A small portion of the preparation can be taken to recover RNA, and the integrity of the prepared polyribosomes can be analyzed by agarose gel electrophoresis. rRNA can be observed by EB staining, and if the 28S rRNA and 18S rRNA bands are clear and the intensity ratio is close to 2:1 (28S:18S), it indicates that the rRNA is intact.
3. Preparation of RSW from polyribosomes
(1) If possible, RSW preparation should be done in a cold room. It is not necessary to use freshly prepared polysomes, but it is possible to use frozen polysomes for experiments. Resuspend polysomes in Solution A at a concentration greater than 105 cells/μl. A suitable starting concentration is 2X104~5X107 cells/ml .
(2) Transfer the polysaccharide into a tube or beaker containing a stirrer. Soak the stirrer in DEPC for 3~5 min before use, and then rinse it thoroughly with water.
(3) Add enough 4.0 mol/L potassium acetate dropwise, and stir gently until the final concentration is 0.5 mol/L. The stirrer should be used as a buffer.
(4) Stir gently for 15 min in a cold room to avoid air bubbles.
(5) Add Solution B to the homogeneous heterogeneous polymerization (polyallomer) ultracentrifuge tube to about 25% of its volume, and carefully spread the salt-washed polysaccharide on Solution B, with the final liquid level a few millimeters from the top of the tube, and centrifuge at 130,000 g ultracentrifugation speed for 2.5 h at 2℃.
(6) Recover the RSW on the sucrose solution layer and retain the salt-washed polysaccharide precipitate in the centrifuge tube.
(7) Mix the RSW gently and thoroughly and determine its protein concentration.
(8) Dispense RSW into 100~200 μl and store at -70℃.
(9) If desired, salt-washed polyribosomes at the bottom of the centrifuge tube can be recovered using Operating Procedure 2, "Preparation of Polyribosomes from Exponentially Growing Tissue Culture Cells and Polyribosomes Post Supernatant S130".
4 Degradation of endogenous polyribosomal mRNAs
(1) Add 21 μl of Solution D and 4 μl of polyribosomes to a centrifuge tube placed in an ice bath.
(2) Gently mix the liquid in the tube and keep it warm at 37°C, taking care not to shake the tube.
(3) After incubation for a certain period of time, store the reaction tube at -70℃ or extract the RNA directly for analysis. If you want to analyze the degradation of the target mRNA at different time points, you should take the reaction tube out of the water bath and place it directly on dry ice, and then you can use any method of RNA extraction, but you should avoid non-specific RNase contamination.
(4) Analyze and quantify endogenous destination mRNA.
(5) Analyze exogenous mRNA degradation using polyribosomes as a source of mRNase activity
(1) Add 21 μl of Solution E and 4 μl of Polysaccharide to a centrifuge tube in an ice bath.
(2) Incubate the tube at the appropriate temperature for a certain period of time.
(3) After incubation for a certain period of time, store the reaction tube at -70°C or extract the RNA directly for analysis. To analyze the degradation of the target mRNA at different time points, remove the reaction tube from the water bath and place it directly on dry ice, then any method of RNA extraction can be used, but non-specific RNase contamination should be avoided.
(4) Analyze and quantify endogenous target mRNA.
6. Analyze the degradation of exogenous mRNA using RSW as a source of mRNase activity.
(1) Take 20 μl of Solution G and 5 μl of RSW and add to a centrifuge tube in an ice bath.
(2) Incubate the reaction tube at the appropriate temperature for a certain period of time.
(3) After a certain period of incubation, store the reaction tube at -70°C or extract the RNA directly for analysis. To analyze degradation of the target mRNA at different time points, remove the tubes from the water bath and place them directly on dry ice, then any method of RNA extraction can be used, but non-specific RNase contamination should be avoided.
(4) Analyze and quantify endogenous target mRNA.
7. Analyze exogenous mRNA degradation using the mRNA-dependent rabbit reticulocyte translation system as a source of mRNase activity.
(1) Add 35 μl of nuclease-treated lysate, 1 μl of RNase inhibitor, 1 μl of amino acid mixture, radiolabeled amino acid, and aqueous mRNA to a total volume of 50 μl of water in a centrifuge tube placed in an ice bath.
(2) Mix gently and react at 30°C for a certain period of time.
(3) Store the reaction product at -70°C or extract the RNA immediately.