RNA electrophoresis

From Molecular Biology Wiki

Due to the nature of RNA, the separation of RNA on gels also called RNA electrophoresis is a bit different than that of DNA.

RNA that is usually single stranded can fold upon itself to form strong and stable secondary structures. This is a problem when you need to separate RNA on a gel.

Also, double stranded RNA (dsRNA) such as RNA:RNA hybrids are quite stable. DsRNA and RNA:RNA hybrids have hairpin elements and other secondary structures that are resistant to denaturing conditions able to denature DNA.

Gel Electrophoresis Separation Methods for RNA

Gels used to separate DNA such as agarose run with TAE or TBE buffer are useful for RNA analysis, however RNA is usually denatured before electrophoresis using denaturing gels and denaturing conditions.

MOPS or Formaldehyde conditions are usually used for analysis and RNA hybridization protocols.

MOPS RNA Gel Electrophoresis Protocol

MOPS gel electrophoresis employs the MOPS buffer as a running buffer to separate [RNA] molecules in an agarose gel.

10X MOPS buffer

6X Loading Buffer: 0.25% (w/v) Orange G, 30% Glycerol, 1.2% SDS, 1X MOPS

Electrophoresis Buffer: 1X MOPS

Gel: 1% Agarose, 1X MOPS

• Denature RNA samples in loading buffer for 5-10 minutes at 75-80°C prior to electrophoresis.

Formaldehyde RNA Gel Electrophoresis Protocol

Prepare and assemble the following:

10X Running Buffer Recipe: Add the following:

• 0.4 M MOPS
• 0.1 M Sodium Acetate
• 10 mM EDTA (disodium)
• Adjust pH to 7.0 using NaOH or Glacial acetic acid CH3COOH

10X Loading RNA Buffer Recipe

To prepare 10X Loading RNA Buffer add the following:

• 0.35% (w/v) Orange G
• 30% (w/v) Ficoll 400
• 1 mM EDTA (disodium)

For electrophoresis buffer dilute 10X running buffer to make 1X running buffer.

Agarose gel

Prepare 1% RNA Agarose gel. This is prepared with 1X Running Buffer and 7% formaldehyde

Recipe for 1% RNA Agarose Gel

For a 20 ml gel add the following:

• 0.2 g agarose
• 2 ml 10X running buffer
• 14.2ml ultrapure water

Microwave for 45 seconds, then allow to cool. Add 3.8 ml of 37% formaldehyde and then dump into casting tray with combs.

To prepare RNA for loading onto wells combine the following:

• 3.5 µl of each RNA sample with:
• 10 µl formamide
• 3.5 µl 37% formaldehyde
• 1 µl 10X Running buffer
• 2 µl 10X loading buffer

Denature RNA in buffers for 10 minutes at 65-75°C. Then load onto cooled and well formed gel wells.

References for RNA Electrophoresis

1. Sambrook, J., et al. (1989). Molecular Cloning: A Laboratory Manual. 2nd ed, Cold Spring Harbor Laboratory Press.
2. Brown, T.A. (1991). Molecular Biology LabFax. Bios Scientific Publishers Limited.
3. Anermuller, S.A. (1990). Rapid visualization of genomic DNA and total RNA in agarose gels. BioTechnology 8(1), 36-37.
4. Almeida, T.A., et al. (2000). Size-fractionation of RNA by hot agarose electrophoresis. BioTechniques 28(3), 414-6.
5. Gerard, G. and Miller, K. (1997). Comparison of glyoxal and formaldehyde gels for sizing rRNAs. Focus 19(1), 17-8.
6. Pelle'R et al. (1993). Northern hybridization: rapid and simple electrophoretic conditions. NAR 21(11), 2783.