Blot hybridization is the process of binding a nucleic acid fragment to be tested to a certain solid phase support and then hybridizing it with a labeled probe present in the liquid phase.Northern blot is a method of analyzing RNA, and is interestingly referred to as such because it corresponds to the Southern blot technique. The technique quantifies the intensity of transcription of a specific gene, and also determines the size of the gene transcription product based on where it migrates. The technique is commonly used in studies of gene expression regulation, gene structure and function, genetic variation, and pathology.
Principle
The basic principle of Northern blotting is that RNA samples are first separated by denaturing agarose gel electrophoresis, and then transferred to solid-phase carriers, such as nylon membranes, and then hybridized with single-stranded RNA with specific base sequences by labeling specific DNA or RNA probes with radioisotopes, and then analyzed by radiographic autofluorescence after removal of non-specific hybridization signals to identify the content and size of specific RNA molecules. The hybridization signal is analyzed to identify the content and size of the specific RNA molecule.
Appliance
Northern blotting technology can be used for gene expression regulation, gene structure and function, genetic variation and pathology research.
Operation method
Northern Blotting Technology
Principle
The basic principle of Northern blotting is that RNA samples are first separated by denaturing agarose gel electrophoresis, and then transferred to solid-phase carriers, such as nylon membranes, and then hybridized with single-stranded RNA with specific base sequences by labeling specific DNA or RNA probes with radioisotopes, and then analyzed by radiographic autofluorescence after removal of non-specific hybridization signals to identify the content and size of specific RNA molecules. The hybridization signal is analyzed to identify the content and size of the specific RNA molecule.
Materials and Instruments
Equipment: Move The basic process of Northern blotting can be divided into the following steps: (1) 0.1% DEPC (diethyl pyrocarbonate). (2) 5x formaldehyde electrophoresis buffer 700 ml DEPC-treated sterilized water dissolve 20.9 g MOPS [3-(N-morpholino)propanesulfonic acid], adjust the pH value of the solution to 7.0 with 2 mol/L NaOH. add 10 ml DEPC-treated 1 mol/L CH3COONa solution and 10 ml DEPC-treated 0.5 mol/L EDTA (pH 8.0). Add 10 ml of DEPC-treated 1 mol/L CH3COONa solution and 10 ml of DEPC-treated 0.5 mol/L EDTA (pH 8.0), and then make a volume of 0.1% DEPC-treated sterilized water to 1 L. Filter to remove the bacteria, and then store it at room temperature and keep it away from light. The final concentration of 0.1 mol / L MOPS (pH 7.0), 10 mmol / L CH3COONa, 5 mmol / L EDTA (pH 8.0). (3) EB solution 0.5 μg/ml EB, 0.1 mol/L ammonium acetate. (7) 6xSSC, 2xSSC and 0.1xSSC were diluted with 20xSSC. (8) 50xDenhardt's solution 1% Ficoll-400, 1% PVP (polyvinylpyrrolidone), 1% BSA (bovine serum albumin), filtered and sterilized, stored at -20 ℃. Caveat (1) RNA is easily degraded by RNAase, so it is necessary to eliminate the contamination of exogenous RNAase and try to inhibit the activity of endogenous RNAase. All the reagents used in the experiment should be treated with DEPC (the final concentration of DEPC is 1%), the glassware should be dry baked at 200 ℃ for 4 h before use, and the plastic equipments should be soaked with water containing 0.1% DEPC at 37 ℃ overnight, and then autoclaved, so as to ensure the degradation of the RNA enzyme and to remove traces of DEPC on the equipments, The experimental personnel must wear disposable gloves and masks, and the environment of the experimental protocol should be clean and clean, so as to create an environment free of RNA enzyme. The experimental protocol environment should be clean and clear to create an RNAase-free environment.(2) EB should not be added to the gel because it will affect the binding of RNA to the nitrocellulose membrane. In order to determine the size of the fragments, molecular mass standards can be added to the same gel and electrophoresed together, and then the bands where the molecular mass standards are located can be cut off, stained and photographed. The sample gel is then subjected to Northern transfer.EB is a mutagen and skin cancer may occur in prolonged contact with the skin. Therefore, polyethylene gloves should be worn for protection when handling EB.(3) If the agarose concentration is higher than 1%, or the thickness of the gel is greater than 0.5 cm, or the RNA to be analyzed is greater than 2.5 kb, the gel should be soaked in 0.05 mol/L NaOH for 20 min to partially hydrolyze the RNA and improve the transfer efficiency. After soaking, rinse the gel with DEPC-treated water and soak the gel in 20xSSC for 45 min before transferring to nitrocellulose membrane.(4) Wear cotton gloves when handling the gel and the nitrocellulose membrane to prevent contamination by dirt and grease, which may lead to transfer failure.(5) Can not make the nitrocellulose membrane on the filter paper contact with the gel under the filter paper, otherwise the formation of short-circuit flow to make the transfer are. Available cling film to seal the gel edge around.(6) the nitrocellulose membrane spread on the gel, the membrane once in contact with the gel can not be moved. Because from the moment of contact, the transfer has begun.(7) When hybridization, do not leave too much area to waste the prehybridization solution and hybridization solution, and at the same time, do not leave air bubbles in the plastic bag, otherwise the prehybridization and hybridization are not uniform.(8) Before incubation, check whether the plastic bag is closed, do not let the liquid in the bag flow out, as well as the liquid in the water bath box.(9) The intensity of rinsing can be determined according to the size of the hybridized molecules, the size of the homologous region, and can be adjusted appropriately to the rinsing liquid strength (change the ionic strength), the rinsing temperature and rinsing time, and at the same time, can also be used to detect rinsing by the radioactivity detector.(10) Gloves should be worn for protection during hybridization and radiation autoradiography. Hybridized isotope film wrapped in plastic wrap, do not contaminate other utensils.(11) If the nitrocellulose membrane dries out during storage after hybridization, the probe will irreversibly bind to the membrane and cannot be eluted off. Therefore, it should be kept moist and sealed in a plastic bag during rinsing, radiation autoradiography and storage. For more product details, please visit Aladdin Scientific website.
① electrophoresis apparatus, electrophoresis tank, glue-making mold, comb plate, glass plate, tray
② ultraviolet spectrophotometer, ultraviolet detector
③ centrifuge, constant temperature water bath, vacuum oven
④ cellulose nitrate filter membrane, Whatman 3 MM filter paper, blotting paper, plastic wrap, hybridization bag
⑤ Micropipette (20 μl), tip (sterilized), 1.5 ml Eppendorf tube (sterilized)
⑥ Radiographic autoradiography cassette, X-ray film
Reagents:
① Materials: RNA, labeled probes
② 37% formaldehyde, 5x formaldehyde electrophoresis buffer, formaldehyde gel spiking buffer
① 37% formaldehyde, 5x formaldehyde electrophoresis buffer, formaldehyde gel spiking buffer
④ RNA molecular quality standard (Marker)
⑤ 20xSSC, 6xSSC, 2xSSC, and 0.1xSSC, 50xDenhardt's solution
⑤ 20xSSC, 6xSSC, 2xSSC and 0.1xSSC, 50xDenhardt's solution
(vii) Pre-hybridization solution
⑧ 0.1% SDS
0.1% SDS ⑨ Anhydrous ethanol, TE, sterilized water
(i) Reagent preparation
(4) Formaldehyde gel spiking buffer 0.25% bromophenol blue, 0.25% xylene cyanine, 50% glycerol, 1 mmol / LEDTA (pH 8.0), with 0.1% DEPC 37 ℃ treatment overnight, autoclaved, stored at room temperature.
(5) 37% formaldehyde 12.3 mol/L, pH greater than 4.0.
(6) 20xSSC 800 ml H2O Dissolve 175.3 g NaCl, 88.2 g sodium citrate, 14 mol/L HCI adjust the pH to 7.0, with H2O to 1 L, the final concentration of 3 mol / L NaCl, 0.3 mol / L sodium citrate.
(9) Pre-hybridization solution 6xSSC, 5xDenhardt's solution, 0.5% SDS, 100 mg/ml salmon sperm DNA, 50% formamide.
(10) Hybridization solution Add the denaturing probe to the pre-hybridization solution.
(11) 20% SDS 900 ml H2O Dissolve 200 g of SDS (heating to 68 ℃ and stirring with a magnetic stirrer will help dissolve), adjust the pH to 7.2 with concentrated HCl, and then make a volume of 1 L with H2O, and store at room temperature. Dilute proportionally when using.
(ii) Formaldehyde denaturing agarose gel electrophoresis
(1) Wash the electrophoresis tank with detergent, rinse with distilled water, rinse and dry with anhydrous ethanol, treat with 3% H2O2 for 10 min, and finally rinse with DEPC-treated triple-distilled water thoroughly to remove RNAase in the electrophoresis tank.
(2) Dissolve 1.5 g of agarose in 62 ml of DEPC-treated sterilized water, cool it down to 60 ℃, and add 20 ml of 5x formaldehyde electrophoresis buffer and 18 ml of formaldehyde to the final concentration of 1x and 2.2 mol/L, respectively. In a chemical fume hood, pour the melted gel into a gel-making mold, with the comb plate placed at one end, and the bottom of the comb plate and the mold left with an interspace of 0.5~1 mm, and the thickness of the gel was 3~5 mm. The thickness of the gel should be 3-5 mm.
(3) After the gel is solidified, put the gel-making mold into the electrophoresis tank and add 1x formaldehyde electrophoresis buffer into it, with the liquid level 1-2 mm above the surface of the gel, and pull out the comb plate carefully.
(4) In a 1.5 ml Eppendorf tube, mix the following reagents: 2 μl (20 μg) of sample RNA, 4 μl of 5x formaldehyde electrophoresis buffer, 3.5 μl of 37% formaldehyde, 10 μl of formamide, the total volume of 20 μl.
(5) Incubate the above samples at 65°C for 15 min, cool rapidly in an ice bath, centrifuge for 5 s, so that all the liquid in the tube is concentrated at the bottom. centrifuge for 5 s, so that all the liquid in the tube is concentrated at the bottom of the tube. Add 2 μl of formaldehyde gel spiking buffer and mix for use.
(6) Before adding samples, pre-electrophoresis the prepared gel for 5 min, then add the samples into the sample wells of the gel, and add RNA molecular quality standard into the outermost sample well of the gel.
(7) Turn on the power cord, with the sample wells at the cathode end of the electrophoresis tank, and turn on the power switch. Adjust the voltage to 3~4 V/cm, and mix the liquid in the electrophoresis tank with positive and negative poles every 1~2 h. (8) After electrophoresis, add RNA molecular quality standards into the outermost well of the gel.
(8) After electrophoresis, cut off the gel strip where the quality standard of RNA molecules is located, and immerse it in EB solution for 30-45 min. Under the ultraviolet light, observe the migration distance of RNA electrophoresis bands and take photos to record.
(C) Transfer
(1) Remove the useless part of the gel, and cut off a corner on one side of the spiking hole to mark the orientation of the gel.
(2) Rinse the gel several times with DEPC-treated water to remove formaldehyde.
(3) cut a sheet of nitrocellulose membrane, 2 to 4 sheets of 3 MM filter paper and some blotting paper (available toilet paper), are the same size as the gel (nitrocellulose membrane and blotting paper can not be larger than the gel, otherwise it is easy to form a bypass). Cut off a corner of the nitrocellulose membrane to make the corresponding orientation marks. Then wet it completely with sterile water and soak it in 20xSSC. Wear rubber gloves when touching both the adhesive and the nitrocellulose membrane.
(4) Place a plate larger than the gel in the transfer tray and place a 3 mm filter paper on top, soaking both sides of the paper in 20xSSC buffer. Remove any air bubbles between the filter paper and the plate.
(5) Place the gel on the filter paper so that the side that is up during electrophoresis is facing down, removing any air bubbles that appear between the two layers. The wetted nitrocellulose membrane was then laid accurately on the gel one at a time, aligned. Lay the membrane down gradually from one side to prevent air bubbles. If there are air bubbles, use a pipette to drive them out, and do not allow the membrane to come into direct contact with the filter paper under the adhesive. Cover the nitrocellulose membrane with 2 sheets of 3 MM filter paper soaked in 2xSSC buffer, again to drive out air bubbles.
(6) Place a stack of dry blotting paper on top of the 3 mm filter paper, place a glass plate on top of the blotting paper, and pressurize with a weight of approximately 500 g. (7) Transfer
(7) Transfer The above steps are completed and RNA transfer begins. By adsorption of the filter paper, the buffer in the pan is moved up through the adhesive, and the RNA is blotted onto the membrane. This process lasts for 6 to 18 h. If the blotting paper is too wet during this period, it should be replaced with a new dry blotting paper.
(8) After fixation transfer, remove the nitrocellulose membrane and soak it in 6xSSC solution for 5 min to remove agarose gel debris. The membrane was air dried and then baked in a vacuum oven at 80°C for 2 h. The baked membrane could be dried and stored at room temperature for hybridization.
(iv) Pre-hybridization
(2) Drain the air bubbles from the bag, then seal the bag and incubate it in a 42℃ water bath for 2~4 h. Discard the prehybridization solution.
(E) Hybridization
Add hybridization solution to the plastic bag (pre-hybridization solution added to the labeled probe is hybridization solution), and reseal it. Then put it in a water bath at 42℃ for 16~24 h.
(VI) Rinsing
(1) Discard the hybridization solution, take out the nitrocellulose membrane, and rinse it twice with 2xSSC/0.1% SDS solution at room temperature for 15 min each time.
(2) Rinse it twice with 0.1xSSC/0.1% SDS solution at room temperature for 15 min each time.
(3) Rinse it twice with 0.1xSSC/0.1% SDS solution at 55℃ for 15 min each. (4) Rinse it with 0.1xSSC/0.1% SDS solution at 55℃. 0.1xSSC/0.1% SDS solution twice at 55℃ for 30 min each time.
(vii) Radiation autoradiography
(H) Experimental results and analysis(1) After electrophoresis, carefully observe under ultraviolet light whether the RNA electrophoresis separation effect is good, whether there is any degradation of the RNA sample, whether the RNA band is clear, whether there is any trailing and whether the edges are blurred, etc. (2) After radiation autoradiography, the RNA bands can be seen on the X-ray film at -70 ℃.
(2) After radiographic autoradiography, observe the bands exposed on the X-ray film and compare the migration distance of the bands with the quality standard of the RNA molecules to find out the position of the corresponding RNA in the gel electrophoresis, and thus the size of the RNA for gene transcription. Scan the exposed bands with the "Automatic Optical Density Scanner", calculate the integrated optical density value of the bands, and use the integrated optical density value of the internal reference RNA bands as a correction value to determine the expression intensity of the gene transcripts in different samples.