Principles, materials and procedures for denaturing agarose gel electrophoresis of RNA

This experiment is mainly used to determine the integrity of RNA extracts.

Operation method

RNA agarose gel electrophoresis

Principle

RNA can be detected using non-denaturing or denaturing gel electrophoresis. In nondenaturing electrophoresis, RNA molecules of different molecular weights in the mixture can be separated, where the molecular weight cannot be determined. Only in the case of denaturation, where the RNA molecules are fully stretched, is the mobility proportional to the molecular weight. Determining the integrity of an RNA extract is one of the main purposes of performing electrophoresis. The electrophoretic profile of an intact, undegraded RNA product should clearly visualize the three bands of 18s rRNA, 28s rRNA, and 5s rRNA, and the 28s rRNA should be twice as bright as the 18s rRNA.

Materials and Instruments

Total RNA extract
DEPC water Electrophoresis buffer Denatured agarose gel Sampling buffer Formamide
Electrophoresis System UV Transilluminator

Move

1. Rinse the glue making equipment with 70% ethanol and dry it.

2. Gel making:

Weigh 0.5g of agarose powder, add it to a conical flask with 36.5mL of DEPC water, and heat it to make the agarose dissolve completely. After slightly cooling, add 5mL of 10x electrophoresis buffer, 8.5mL of formaldehyde. Then the gel was filled in the gel tank, the comb was inserted and placed horizontally to be solidified and used.

3. Sample addition:

Mix the following reagents in a small clean centrifuge tube: 2μl of electrophoresis buffer (10x), 3.5mL of formaldehyde, 10mL of formamide, and 3.5μl of RNA samples. mix well, hold at 60°C for 10min, and speed cool on ice. Add 3μl of sampling buffer and mix well, take the appropriate amount of sample in the gel spotting well. At the same time, spot RNA standard samples.

4. Electrophoresis:

Turn on the electrophoresis instrument, stabilize the voltage 7.5V/cm electrophoresis.

5. Observe the end of electrophoresis by UV transilluminator.

Caveat

RNase contamination must be maintained in this experiment to avoid RNA degradation. All reagents were prepared in DEPC water and utensils were also rinsed and sterilized in DEPC water.

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