A method for detecting 5-hydroxymethylcytosine in RNA and its kit
A technology of hydroxymethylcytosine and detection kits, which is applied in the field of biochemistry, can solve the problems of cumbersome operation, long time-consuming, expensive and other problems, and achieve the effect of simple sample preparation, high sensitivity and easy operation
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Embodiment 1
[0075] Example 1 RNA 5hmC detection method
[0076] 1. Extract total RNA and dilute to a concentration of 100ng / μL.
[0077] 2. Add an equal volume of 2M NaOH solution and react at 4°C for 30 minutes.
[0078] 3. Take a 4×8 nitrocellulose membrane, drop 2 μL of samples onto the membrane sequentially, and place it at room temperature for 15 minutes.
[0079] 4. Place the film in an oven at 80°C and bake for 30 minutes.
[0080] 5. After taking it out from 80°C, block it with 5% milk, react at room temperature for 1 hour, and then wash it with PBST solution 3 times, 10 minutes each time.
[0081] 6. Incubate the NC membrane with the 5hmC antibody diluted in PBS solution, react overnight at 4°C, and then wash with PBST solution 3 times, 10 minutes each time.
[0082] 7. Incubate the NC membrane with a specific secondary antibody diluted with 5% milk, react at room temperature for 1 hour, and then wash with PBST solution 3 times, 10 minutes each time.
[0083] 8. After develop...
Embodiment 2
[0086] Example 2 Effects of Different Denaturing Agents and Concentrations on RNA 5hmC Detection
[0087] (1) The influence of different denaturing reagents on the detection effect of RNA 5hmC
[0088] Denaturing reagent type setting: NaOH, formaldehyde, Tris-HCl;
[0089] The test method is:
[0090] 1. Extract total RNA and dilute to a concentration of 100ng / μL.
[0091] 2. Add an equal volume of denaturing reagent solution (NaOH, formaldehyde or Tris-HCl) with a concentration of 2M, and react at 4°C for 30 minutes.
[0092] 3. Take a 4×8 nitrocellulose membrane, drop 2 μL of samples onto the membrane sequentially, and place it at room temperature for 15 minutes.
[0093] 4. Place the film in an oven at 80°C and bake for 30 minutes.
[0094] 5. After taking it out from 80°C, block it with 5% milk, react at room temperature for 1 hour, and then wash it with PBST solution 3 times, 10 minutes each time.
[0095] 6. Incubate the NC membrane with the 5hmC antibody diluted in...
Embodiment 3
[0115] Example 3 Effects of different denaturation times on the detection effect of RNA 5hmC
[0116] Denaturation time setting: 30min, 1h, 3h, 6h, 12h, 24h, 48h;
[0117] The test method is:
[0118] 1. Extract total RNA and dilute to a concentration of 100ng / μL.
[0119] 2. Add an equal volume of NaOH with a concentration of 2M, and react at 4°C for a certain period of time (respectively 30min, 1h, 3h, 6h, 12h, 24h, 48h).
[0120] 3. Take a 4×8 nitrocellulose membrane, drop 2 μL of samples onto the membrane sequentially, and place it at room temperature for 15 minutes.
[0121] 4. Place the film in an oven at 80°C and bake for 30 minutes.
[0122] 5. After taking it out from 80°C, block it with 5% milk, react at room temperature for 1 hour, and then wash it with PBST solution 3 times, 10 minutes each time.
[0123] 6. Incubate the NC membrane with the 5hmC antibody diluted in PBS solution, react overnight at 4°C, and then wash with PBST solution 3 times, 10 minutes each ...
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