Since RNA is a single-stranded nucleic acid and tends to form secondary structures, a standard agarose gel will not give an accurate size separation of your total RNA or mRNA sample. Therefore a denaturing agent like formaldehyde must to be added to the agarose gel and the RNA to ensure the molecules remain single-stranded. Because of the single-stranded nature of the molecule, RNA tends not to incorporate stains like ethidium bromide as well as DNA. We recommend including ethidium bromide in both the loading buffer and the gel to give better staining and visualization of the RNA. The following is a basic protocol for pouring a denaturing MOPS buffer/formaldehyde gel and performing electrophoresis of RNA samples to visually assess the quality of the purified RNA(1)
- Prepare a 1% agarose/formaldehyde gel containing 0.5µg/ml ethidium bromide as follows:
|5X MOPS buffer (see recipe below)
|DEPC-treated or Nuclease-Free Water (Cat.# P1193)
|Agarose, molecular biology grade
- Heat the mixture to boiling in a microwave oven, and cool to ~55°C. In a fume hood, add 17.6ml of 37% formaldehyde and 5µl of 10mg/ml ethidium bromide (Cat.# H5041) to the molten agarose. Gently mix. Pour the gel and allow to solidify in the fume hood. The final composition of the gel is 36.5mM MOPS, 9.1mM sodium acetate, 0.9mM EDTA, 2M formaldehyde, 0.5µg/ml ethidium bromide and 0.9% agarose in a final volume of 109.6ml.
- Prepare the RNA samples by mixing 1 part of your RNA sample with 2 parts of RNA Sample Buffer (see recipe below) to a total volume of 10–30µl, depending on the loading volume of the wells in your gel. Heat the RNA samples + buffer to 60°C for 5 minutes then cool on ice for 2 minutes. Add 2µl of RNA Loading Buffer (see recipe below).
- Prerun the gel for 10 minutes in 1X MOPS buffer prior to loading the samples. Load the RNA samples and run the gel at 4–5V/cm. Continue electrophoresis until the bromophenol blue dye has migrated 2/3 to 3/4 the length of the gel.
- Use shortwave UV (254nm) to view the RNA on the gel and capture an image. With mammalian RNA samples, the ribosomal RNA bands are 28S (~4,700 bases) and 18S (~1,900 bases) with the mRNA forming a smear from above the rRNA bands to below. For bacterial RNA samples, the rRNA bands are 23S (~2,900 bases) and 16S (~1,500 bases).
- Confirm the integrity of the RNA by examining the profile of molecules on the gel. If the larger rRNA band is not more intense than the smaller and the brightly stained portion of the gel is around the lower ribosomal band, your sample may be degraded.
Recipes of Buffers and Solutions
|Add Dowex® XG8 mixed-bed resin to formamide (Cat.# H5051) and stir at room temperature for 1 hour. Filter twice through Whatman® No. 1 filter paper. Store in small aliquots at –70°C.|
||MOPS (pH 7.0)|
||EDTA (pH 8.0)|
|For 2 liters of buffer, add 83.72g of MOPS (free acid) and 8.23g of sodium acetate to 1.6L of DEPC-treated water and stir until completely dissolved. Add 20ml of DEPC-treated 0.5M EDTA and adjust the pH to 7.0 with 10N NaOH. Bring the final volume to 2L with DEPC-treated water. Dispense into 200ml aliquots and autoclave. The solution will turn yellow, but this will not affect the quality of the buffer.|
|Dispense into single-use aliquots and store at –20°C in tightly sealed, screw-cap tubes. These can be stored for up to |
||glycerol (Cat.# H5433)|
||EDTA (Cat.# V4231)|
|Prepare in DEPC-treated or Nuclease-Free Water. Use high-grade glycerol to avoid ribonuclease activity. Dispense into single-use aliquots and store at –20°C.|