Electrophoresis is a common lab technique used to identify, quantify, and purify nucleic acid fragments and assess quality. RNA molecules are negatively charged, and during gel electrophoresis they migrate toward the anode in the presence of an electric current.
The length of RNA generally determines its migration in the gel, since longer RNA molecules move slower than shorter fragments. However, RNA molecules are subject to extensive secondary structure via intramolecular base pairing, and this can affect their gel migration. For most applications, RNA is run using denaturing conditions to disrupt secondary structure. For applications such as resolving different conformations of RNA, native (or nondenaturing) gels are recommended.
Nondenaturing RNA Electrophoresis
Gel electrophoresis of RNA under nondenaturing conditions maintains the secondary structure of RNA molecules. Agarose is generally preferred to acrylamide because it has lower toxicity and, at the concentrations needed to resolve typical RNA molecules, it is easier to handle. We offer convenient reagents for nondenaturing agarose gel electrophoresis, including hassle-free precast E-Gel™ agarose gels and UltraPure™ reagents to pour your own agarose gels.
Products for Nondenaturing RNA Electrophoresis:
Denaturing RNA Electrophoresis
To accurately determine the molecular weight of RNA molecules, it is essential to run RNA gels using denaturing conditions. Since northern blots typically seek to characterize RNA molecules based on their size, denaturing electrophoresis is commonly used prior to RNA analysis by northern blotting.. Denaturing conditions disrupt hydrogen bonding so that RNA runs without secondary structure, as single-stranded molecules. There are a number of denaturing agents available, including glyoxal, formamide, and methyl mercury. The disadvantage of these compounds is that they are toxic and therefore should be treated with caution.
Traditionally, formaldehyde has been used as a denaturant for RNA electrophoresis. The NorthernMax™ Kit contains a complete set of RNase-free reagents for running formaldehyde-containing agarose gels. These gels must be poured and run in a fume hood. With the NorthernMax™-Gly Kit, RNA samples are denatured in glyoxal/DMSO loading buffer prior to electrophoresis and run in a formaldehyde-free agarose gel.
For determination of RNA size, a wide range of RNA ladders are available for accurate size and mass estimations, including the Millennium™ Markers.
- Learn more about RNA Ladders
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