Transfer and immobilization of denatured RNA on membranes
Transfer and immobilization of denatured RNA on membranes
In most cases, to detect a specific target mRNA, the RNA is separated by agarose electrophoresis and then transferred from the gel to a two-dimensional support, usually a nylon membrane, which is then hybridized to a differentially labeled probe. As discussed in Nonhem hybridization, a variety of reagents and membranes can be used for RNA transfer to achieve tight binding of the RNA to the membrane. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Peitang Huang et al.
Operation method
Transfer and immobilization of denatured RNA on membranes
Principle
In most cases, to detect a specific target mRNA, the RNA is separated by agarose electrophoresis and then transferred from the gel to a two-dimensional support, usually a nylon membrane, which is then hybridized to a differentially labeled probe. As was discussed in the case of Nonhem hybridization, the experimenter can employ a variety of reagents and membranes for RNA transfer to achieve tight binding of the RNA to the membrane. We believe that optimal Nonhern blotting can be achieved by transferring RNA from the gel to the nylon membrane under neutral or alkaline conditions.
Materials and Instruments
RNA Sample Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonium acetate Methylene blue solution Infiltration solution SSC transfer buffer
Blotting filter paper Cross-linking equipment Glass dishes Nylon film Plexiglas or glass plates Paper cutter blades Thick blotting filter paper Visible cassettes Weights Yellow filters
1. Buffers and solutions
0.1 mol/L ammonium acetate containing 0.5 μg/ml ethidium bromide
Methylene blue solution (0.02% methylene blue (Sigma, 89% pure) in 0.3 mol/L sodium acetate (pH 5.5))
Infiltration solution
0.2XSSC with 1% SDS (m/V)
20X SSC
Transfer Buffer
2. Nucleic acids and ribosomes
RNA samples, separated by agarose electrophoresis
3. Specialized equipment
Blotting filter paper (Schleicher and Schuell GB002 or Sigma P 9039)
Cross-linking equipment (e.g. Stratalinker, Stratagene; GS Gene Linker, Bio-Rad) or microwave or vacuum ovens
Bake-dried glass dishes
Positively charged or uncharged nylon membranes
Plexiglass or glass plates used to support the adhesive during the transfer process
Paper cutter blades
Thick blotting filter paper (Whatman 3 MM/L, Schleicher and Schuell GB004, or Sigma Quick-Daaaraw)
Visible light cartridge
400 g weight
Photographic yellow filters
II. Methods
Preparation of transfer gel
1. (Optional) Partial hydrolysis of agarose-isolated RNA samples by immersing the gel in the appropriate soaking solution is performed as follows. Partial hydrolysis of electrophoresed gel by NaOH can improve the transfer efficiency of RNA to positively charged or uncharged nylon membrane. This treatment is especially suitable for agarose concentration >1%, gel thickness >0.5 cm, or RNA to be analyzed >2.5kb.
(1) If transferring to an uncharged nylon membrane
① Wash the gel with DEPC-treated water.
② Immerse the gel in 5 times the volume of 0.05 mol/L NaOH for 20 min.
③ Transfer the gel to 10 times the volume of 20x SSC for 40 min.
③ Transfer the gel into 10 times the volume of 20x SSC for 40 min. ④ Go directly to step 2 and quickly transfer the partially hydrolyzed RNA to the uncharged nylon membrane through a capillary tube.
(2) If transferring to a charged nylon membrane, transfer the partially hydrolyzed RNA through a capillary tube.
① Wash the gel with DEPC-treated water.
② Immerse the gel in 5 times the volume of 0.01 mol/L NaOH-3 mol/L NaCl for 20 min.
③ Go directly to step 2 and quickly transfer the partially hydrolyzed RNA to the positively charged nylon membrane through a capillary tube.
2. Transfer the gel to a glass dry baking dish, trim off the unwanted parts of the gel with a sharp blade to ensure the alignment of the gel with the membrane during the transfer process, and cut a corner of the gel at the upper left corner to serve as a marker for the orientation of the gel during the following operations.
3. Use a piece of Plexiglas or glass plate larger than the gel in both length and width as a platform and place it in a large dry baking dish with a piece of filter paper on top.
4. Pour the appropriate transfer buffer (0.01 moI/L NaOH-3 mol/L NaCl for positively charged membranes, 20X SSC for uncharged membranes) into the dry baking dish until the level of the liquid is slightly below the surface of the platform, and when the filter paper on top of the platform is completely wetted, remove any air bubbles with a glass rod or pipette.
Preparation of transfer membrane
5. Use a new scalpel or paper cutter to cut a piece of nylon membrane 1 mm longer and wider than the adhesive.
6. Float the nylon membrane on the surface of deionized water until the membrane is completely wet from the bottom up, then immerse the membrane in 10X SSC for at least 5 min, and use a clean scalpel blade to cut off a corner of the membrane so that it corresponds to the cut corner of the gel.
Installation of the transfer system and transfer of RNA
7. Invert the gel and place it in the center of the filter paper on the platform, making sure that no air bubbles are trapped between the thick filter paper and the gel.
8. Place Saran Cling Film or Parafilm around the perimeter of the gel, not over the gel.
9. Wet the gel with Transfer Buffer (see Step 4) and place a moist nylon membrane over the gel so that the tangents overlap and one edge of the membrane is just beyond the edge of the line of the upper gel spiking well.
10. Wet two pieces of filter paper of the same size as the gel with the appropriate transfer buffer, place them on top of the wet nylon membrane, and drive away any air bubbles with a glass rod.
11. cut a stack of 5 ~ 8 cm thick, slightly smaller than the filter paper paper, placed on the filter paper, in the paper towel on a piece of glass plate, and then pressed on the 400 g weight.
12. The transfer time for upstream RNA transfer should not exceed 4 h in neutral transfer buffer and 1 h in basic transfer buffer.
13. Remove the capillary transfer system, mark the location of the gel spiking wells on the membrane with a ballpoint pen, transfer the membrane to a glass dish containing 300 ml of 6X SSC, and then place the dish on a shaker and gently shake it for 5 min at room temperature of 23℃.
14. Remove the gel from the 6X SSC solution, drain off the excess liquid, and place the membrane RNA side up on dry filter paper for a few minutes.
Staining of RNA and immobilization of RNA on membranes
The steps for staining and fixation vary depending on the type of transfer, the type of membrane, and the method of fixation. Since RNA in alkaline buffer covalently binds to positively charged nylon membranes, it is not necessary to immobilize RNA on membranes before staining, while RNA transferred to uncharged nylon membranes in neutral buffer can be immobilized on the membranes after staining by dry baking under vacuum or heating in a microwave oven. If RNA is crosslinked to the membrane by UV irradiation, staining of the RNA should follow fixation. 
RNA transferred to nylon membranes can be identified by staining with methylene blue (Herrin and Schmidt 1988), a simple method that can be used for RNA concentration monitoring, estimation of transfer efficiency, and estimation of the location of the primary RNA (usually rRNA) on the membrane. If the RNA is immobilized by UV cross-linking, go to step 16.
15. Membrane staining.
(1) Transfer the wet membrane to glassware containing methylene blue solution for staining for as long as it takes to identify the rRNA (3-5 min).
(2) Photograph the stained membrane under visible light with a yellow filter.
(3) After imaging, the membrane was decolorized in 0.2X SSC and 1% (m/V) SDS for 15 min at room temperature.
16. RNA was immobilized on an uncharged nylon membrane.
(1) Dry fixation
① After the membrane was dried in air, the dried membrane was sandwiched between two pieces of filter paper and dry baked in a vacuum oven at 80°C for 2 h. The membrane was then dried in a vacuum oven at 80°C for 2 h.
② Or place the wet membrane on the dry filter paper and heat it in microwave oven (750~900 W) at maximum power for 2~3 min.
(2) Crosslinking fixation by UV irradiation
① Place the wet, unstained nylon film on a piece of dry filter paper and irradiate it at a dose of 1.5 J/cm2 at a wavelength of 254 nm for 1 min 45s.
② After UV irradiation, stain with methylene blue as described in step 15. 
