Northern blotting and narrow line hybridization analysis of RNA
Northern blotting and narrow line hybridization analysis of RNA
Northern blot is one of the most rigorous methods to study gene expression, which can quantitatively analyze the expression abundance of a specific mRNA in tissues, and also determine the size of gene expression transcripts based on their migration location. This technique is widely used to analyze the size and abundance of specific mRNAs in RNA samples, as well as for the study of gene expression regulation, gene structure and function, and genetic variation.
Operation method
Formaldehyde-agarose gel electrophoresis
Materials and Instruments
RNA Move 1. Dissolve 1 g of agarose in 72 ml of water and when cooled to 60°C in a water bath, transfer to a fume hood and add 10 x MOPS electrophoresis buffer and 18 ml of 12.3 mol/l formaldehyde. For more product details, please visit Aladdin Scientific website.
DEPC MOPS Formaldehyde Agarose Ammonium Acetate NaOH NaCl Tris-Cl Sodium Phosphate Ethidium Bromide SDS
Centrifuge Electrophoresis Incubator UV Transilluminator Hybridizer
2. Spread the gel to solidify, remove the comb, place the gel in the electrophoresis cell, and add enough 1 x MOPS electrophoresis buffer to submerge the gel surface by about 1 mm.
3. Adjust the volume of each sample to 11 μl with water and add:
(1) 5 μl 10×MOPS electrophoresis buffer
(2) 9 μl 12.3 mol/l formaldehyde
(3) 25 μl formamide
(4) Shake and mix in a vortex mixer and centrifuge briefly in a microcentrifuge for 5~10 s to recover droplets, then hold at 55°C for 15 min.4. Add 10 μl of Aldehyde Spiking Buffer, shake in a vortex mixer and centrifuge slightly in a microcentrifuge to recover the droplet.
5. Add 0.5 to 10 μg of RNA sample per split to each of the pair of spiking wells so that half of the gel can be taken for staining.
6. Electrophoresis is performed at a voltage drop of 5 V/cm until the bromophenol blue dye has swept half or two-thirds of the length of the gel.7. Staining with ethidium bromide
(1) Remove the gel and cut off the lanes to be stained.
(2) Place the gel in a glass flat blood without RNa enzyme, cover with a sufficient amount of 0.5 mol/l ammonium acetate, and soak for 20 min. Change the solution and soak for another 20 min to remove formaldehyde.
(3) Pour off the liquid, add 0.5 mol/l ammonium acetate containing 0.5 μl/ml ethidium bromide and stain for 40 min.
(4) Decolorize with 0.5 mol/l ammonium acetate for 1 h if necessary.
8. Staining with acridine orange
(1) Remove the gel and cut off the lanes to be stained.
(2) Stain in 1.1 mol/l formaldehyde/10 mmo/l sodium phosphate containing 10 μg/l acridine orange for 2 min.
(3) Decolorize in the same liquid without acridine orange for 20 min.
9. Visualize the RNA in the gel in a UV transilluminator, and take a photograph by placing a ruler along the edge of the gel so that the bands in the membrane can be subsequently identified.10. Place the non-stained portion of the gel in an RNAase-free glass dish and wash several times with enough deionized water to remove formaldehyde.11. The gel was immersed in 10x Hoven's 0.05 mol/l NaOH/1.5 mol/l NaCl for 30 min to partially hydrolyze the RNA.
12. The gel was then immersed in 10-fold volume of 0.5 mol/l Tris-Cl (pH 7.4)/1.5 mol/l NaCl buffer and neutralized for 30 min.
13. The solution was changed to 10-fold Hoven's 20× SDS and immersed for 45 min.14. Place a rectangular sponge slightly larger than the gel in a glass or plastic dish (two or more may be placed side by side, if necessary) and add enough 20 x SDS to half-submerge the sponge in the liquid.
15. Construct a transfer platform by cutting three pieces of Whatman 3 MM filter paper the same size as the sponge, placing them on the surface of the sponge, and moistening them with 20 × SDS.
16. Place the gel on the surface of the filter paper, remove air bubbles by rolling a glass pipette over the surface and cut 4 strips of plastic stave to cover the edges of the gel.
17. Cut a piece of nylon or nitrocellulose adhesive film the size of the exposed surface of the gel, add about 0.5 mm of deionized water to an RNAase-free glass dish, and wet the film by floating it on the water surface until it is submerged in the water.
18. If it is a nylon membrane, just let the membrane soak in the water for 5 min. If the light is a nitrocellulose membrane, change to 20× SDS and soak for another 10 min.
19. Place the wetted membrane on the surface of the gel, remove the air bubbles, and wash the membrane surface with 20×SDS.
20. Cut 5 pieces of Whatman 3 mm filter paper of the same size as the membrane and place them on the surface of the membrane.
21. Cut 5 pieces of Whatman 3 MM filter paper to the size of the membrane and place them on the surface of the membrane. 21. Then neatly stack the paper towels cut to the size of the membrane on the surface of the filter paper, about 4 cm high.
22. Place a glass plate on top of the pile, press a weight (0.2-0.4 kg), and leave overnight.
23. Dismantle the transfer device, recover the membrane and the flattened gel, and mark the position and direction of the spiking holes on the membrane with a pencil.
24. Wash the membrane with 2 x SDS and place on a piece of Whatman 3 MM filter paper and allow the membrane to dry completely.
25. Treatment of cellulose nitrate membranes: sandwich the membrane between two Whatman 3 MM filter papers and bake the membrane at 80°C for 2 h under vacuum.
26. Treatment of nylon film: Bake the film as described above or wrap the dry film in plastic cling film that can transmit ultraviolet rays, place the RNA side down and irradiate it for a suitable period of time with a UV transilluminator.27. If desired, the gel can be manipulated as in step 7 or 8, stained with ethidium bromide or acridine orange to check transfer efficiency, or stained in 0.3 mol/l pH 5.2 sodium acetate buffer containing 0.03% (w/v) methylene blue for 45 s and decolorized in water for 2 min.
28. Oligonucleotide probes with a radio specific activity of 108 dpm or more were prepared and unlabeled oligonucleotides were removed.
29. Wet the RNA-carrying membrane with 6× SSC solution.30. Place the membrane, RNA side up, in a hybridization tube and add 1 ml of formamide prehybridization solution/hybridization solution per 10 cm2 area. The tube is rotated in a hybridization oven for 3 h. The temperature is set at 42°C (DNA probes) or 60°C (RNA probes).31. In the case of double-stranded probes, heat to 100°C in a water bath or heater, hold for 10 min, and transfer to ice.
32. Pipette the desired volume of probe into a hybridization tube and rotate overnight in a hybridization oven at 42°C (DNA probes) or 60°C (RNA probes).33. Pour out the hybridization solution and wash the membrane sequentially according to the following procedure:
(1) 2× SSC/0.1% SDS rotary washed twice at room temperature for 5 min each time.
(2) 0.2×SSC/0.1% SDS rotary wash 2 times at room temperature, 5min each time (low rigor wash).
(3) Preheat 0.2 × SSC/0.1% SDS at 42°C and wash twice at this temperature for 15 min each (medium rigor wash, optional).
(4) Preheat 0.1× SSC/0.1% SDS at 68°C and wash twice at this temperature for 15 min each (high rigor wash, optional).
34. The film is washed at room temperature with 2 x SSC, excess liquid is blotted out, covered with UV-transparent plastic cling film and radiographically autoradiographed.