RNA electrophoresis after denaturation treatment with glyoxal and dimethyl sulfoxide
This method originated from McMaster and Carmichael (1977). Gels containing glyoxal-DMSO are more difficult to perform electrophoresis than gels containing formaldehyde, because the former has a slower swimming rate and the electrophoresis solution needs to be cycled through to avoid excessive H + gradients during electrophoresis. Although the above two gels have nearly equal resolution (Miller, 1987), RNA fractionation using gels containing pendialdehyde-DMSO usually shows sharper RNA bands by Northern hybridization.
1) In a sterilized micro-centrifuge tube, mix the following liquids:
6mol / L glyoxal 5.4Î¼l
0.1mol / L phosphoric acid (pH7.0) 3.0Î¼l
RNA (up to 10Î¼l) 5.4Î¼l of commercially available glyoxal is usually a 40% solution (6mol / L). Because glyoxal is easily oxidized after contact with air, the mixed bed resin (Bio-Rad AG 501-X8) should be used to deionize the glyoxal solution until the pH value of the solution is greater than 5.0. Store small portions in tightly capped tubes at -20 Â° C. Use only 1 portion of each glyoxal solution, and the remaining liquid should be discarded. The mixing method of 0.1mol / L sodium phosphate (pH 7.0) is as follows: 3.9 ml 1mol / L sodium dihydrogen phosphate, 6.1ml 1mol / L disodium hydrogen phosphate and 90ml water are mixed, treated with DEPC after the above solution is autoclaved. Each lane can analyze up to 10Î¼g RNA, usually 10-20 Î¼g total cells Northern hybridization of RNA can detect high-abundance mRNA (accounting for more than 0.1% of the total mRNA). For example, if the RNA content is extremely small, each lane should be added with 0.5-3.0Î¼g poly (A) + RNA.
2) Cap the microcentrifuge tube tightly, incubate the RNA solution at% 0 â„ƒ and 50 â„ƒ for 60 minutes, cool the sample with an ice water bath, and centrifuge for 5 seconds to allow all the liquid in the tube to settle to the bottom of the tube.
3) While incubating the RNA solution at 50 Â° C, agarose horizontal gel was poured, RNA samples under 1 kb were analyzed with 1.4% agarose, and RNA samples over 1 kb were analyzed with 1% agarose. After using 0mmol / L sodium phosphate (pH 7.0), cool down to 70 â„ƒ, add sodium iodoacetate solid to a final concentration of 10 mmol / L (inactivate RNase), then cool down to 50 â„ƒ, make gel, add RNA sample Let it set for at least 30 minutes before setting. The electrophoresis tank used for RNA electrophoresis needs to be washed with a detergent solution, rinsed with water, dried with ethanol, and then filled with 3% H2O2. After being left at room temperature for 10 minutes, the electrophoresis tank is thoroughly rinsed with DEPC-treated water. Glyoxal can react chemically with ethidium bromide, so during the gel-making and electrophoresis processes, ethidium bromide is avoided.
4) Cool the RNA sample to 0 Â° C, add 4 Î¼l of sterilized glutaraldehyde-DMSO gel sample buffer treated with DEPC, and then immediately add the above sample to the gel sample well. Using glyoxyl RNA of known size as a molecular weight standard reference, such as 18S and 28S rRNA or 9S rabbit Î²-globin mRNA, the length of the above-mentioned RNA is 6322, 2366 and 710 bases, respectively. RNA mixtures of known sizes can also be purchased from BRL as molecular weight standards. Usually the molecular weight standard reference lane is located at the edge of the gel, which is easy to cut off after electrophoresis for ethidium bromide staining, if possible between the molecular weight standard reference and the sample to be transferred to the nitrocellulose filter or nylon membrane Leave a swim lane blank.
Glyoxal-DMSO gel loading buffer
10mmol / L sodium phosphate (pH7.0)
0.25% bromophenol blue
0.25% xylene green FF
5) Immerse the gel in 10mmol / L sodium phosphate electrophoresis solution and conduct electrophoresis at a voltage drop of 3-4V / cm. At the same time, continuously recycle the sodium phosphate solution according to Figure 7.1 to keep the pH of the solution acceptable Within the limits (glyoxal will dissociate from PNA molecules at pH> 8.0). Another method is to change the sodium phosphate buffer every 30 minutes during electrophoresis.
6) After electrophoresis (bromophenol blue migration area 8cm), cut the gel strip of the molecular weight standard reference material and immerse it in the ammonium bromide solution (0.5Î¼g / ml,
Dye with 0.1mol / L ammonium acetate for 30-45 minutes. Place a transparent late on the gel, the distance from each RNA band to the sample well on the photo under the UV lamp, draw the logarithm value of the RPN fragment size to the migration distance of the RNA band, and use the obtained curve to calculate the The size of the RNA molecule detected by hybridization after the gel is transferred to the solid support.
7) RNA is transferred from the gel to the nitrocellulose filter, and the RNA is transferred to the nylon membrane.
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