spotted hybridization experiment
spotted hybridization experiment
Summary
Spot hybridization (dotblot) is a technique for detecting, analyzing, and characterizing DNA, RNA, and proteins, and determines whether or not there is hybridization and the intensity of hybridization by the presence or absence of a developed spot and the shade of its color. As one of the most abundant post-transcriptional modifications of RNA in higher eukaryotes and plants, m6A modification can regulate the efficiency of RNA splicing and translocation, stability, nucleation, and translation into protein. Therefore, spot hybridization is often used in the study of m6A modification of RNA to qualitatively or semi-quantitatively analyze the level of m6A modification of total intracellular RNA.
Principle
m6A spot hybridization refers to the denaturation of the RNA to be measured and then spotted on a nitrocellulose membrane or NC membrane, UV cross-linking and fixation, then hybridization with m6A antibody, washing the membrane, and radiographic autoradiography, and the presence or absence of spots or the color of the spots represents the level of m6A modification.
Operation method
m6A spot hybridization
Principle
m6A spot hybridization refers to the denaturation of the RNA to be measured and then spotted on a nitrocellulose membrane or NC membrane, UV cross-linking and fixation, then hybridization with m6A antibody, washing the membrane, and radiographic autoradiography, and the presence or absence of spots or the color of the spots represents the level of m6A modification.
Materials and Instruments
m Move 1, use Trizol reagent to isolate the total RNA from the cells, and try to adjust the RNA concentration of different treatment groups to be consistent. 2、Configure the denaturing solution (RNA denaturing solution is made of 20 × SSC buffer: 37% deionized formaldehyde=3:2); mix the denaturing solution with RNA=1:1, and then react at 95 ℃ for 5 min in the PCR instrument, so as to denature the nucleic acid. After denaturation, the sample was quickly spotted on the nitrocellulose membrane, and then irradiated and cross-linked for 6 min under 302 nm UV light. After cross-linking, the membrane was washed with PBST buffer for 5 min, then closed with 5% skimmed milk powder for 2 h at room temperature, washed with PBST buffer for 5 min, and then incubated with m6A antibody at 4 ℃ overnight. 5. Wash the membrane with PBST buffer 4 times, each time for 5 min, and then incubate the membrane with the corresponding secondary antibody IgG at room temperature for 2 h. Wash the membrane with PBST buffer 4 times, each time for 5 min. 6. ECL development was performed using ECL color development solution. 7, Methylene blue quantification of nucleic acids: take a piece of the same treated membrane, add the same volume of sample dropwise, after UV crosslinking, methylene blue staining, put the membrane in the solution of staining for 10 min, wash with PBST for 10 min, change the PBST several times during the period, and then take pictures. Caveat 1. Methylene blue is very difficult to clean, remember to wear a mask and a white coat during the experimental operation. In the methylene blue staining, pay attention to try not to absorb the bottom precipitation. 2、Nucleic acid denaturing solution should be prepared and used now. 3、Denatured RNA can be put in the refrigerator at 4 ℃, and the experiment can be finished within one week. 4、When spotting samples on the nitrocellulose membrane, use a water-based pen to mark the membrane so as not to remember the wrong order. 5、When counting samples, the speed should be fast, and use the tip without RNA enzyme for counting samples, and the tip should be changed for each sample. 6， when spotting, the volume of the sample should not be more than 1 μL, so as not to cause diffusion, and can not form a good spot. 7， the choice of chemiluminescence imager also has a slight difference, it is best to choose a chemiluminescence imager that can adjust the exposure time by itself. Common Problems 1、It is easy to overexpose when developing? A: Reduce the amount of sample, before doing the formal test, it is best to find the best concentration range. Generally, a range from 500 ng~100 ng is fine. 2、After staining with methylene blue, the spots are not obvious? A: Generally speaking, 10 min of methylene blue staining is enough. However, you need to adjust the staining time according to your specific sample volume. You can use tweezers in the staining process (tweezers are preferably flat-tipped, not easy to damage the nitrocellulose membrane) to clip out and observe, when you see clearly visible blue dots, you can stop staining. The same decolorization time also need to be observed at all times, generally when the observation of the background color and the color of the spots can be clearly distinguishable time. For more product details, please visit Aladdin Scientific website.
6
A antibody (antibody capable of spot hybridization), 20 × SSC buffer, mixed-bed resin PCR instrument, nitrocellulose membrane, medium-wave 302 nm wavelength UV lamp, PBST, secondary IgG, methylene blue powder, sodium acetate (M=136), ECL developer, and chemiluminescence imager.
Reagents were prepared:
(1) PBST buffer preparation: commercial PBS powder was dissolved in 2 L of distilled water, followed by the addition of 1 mL of Tween-20, mixing can be used, stored at room temperature.
(2) 37% deionized formaldehyde preparation: 0.3 g of mixed-bed resin add 3 mL of formaldehyde, mix back and forth, so that the resin part of the blue color into golden yellow, briefly centrifuged and carefully sucked up the supernatant loading, room temperature storage.
(3) Preparation of methylene blue solution: weigh 20 mg of methylene blue powder dissolved in 10 mL of 0.3 mol/L sodium acetate solution, fully dissolved to form a dark blue solution, stored at room temperature.
(4) Preparation of 0.3 mol/L sodium acetate solution: weigh 20.4 g of sodium acetate dissolved in 500 mL of deionized water, add hydrochloric acid and adjust pH=5.2.