RNA gel electrophoresis separates RNA molecules by size and charge using a gelatinous sheet. RNA must be purified and denatured before being added to the gel. Separating fragments by size can be useful for comparing samples.
In biochemistry, RNA gel electrophoresis is a common method used to analyze the biological molecule called ribonucleic acid (RNA). Gel electrophoresis separates molecules by size and charge as they are “sieved” through a sheet made of a gelatinous chemical. This process is most often employed in laboratories to analyze fragments of deoxyribonucleic acid (DNA) or RNA, molecules that contain an organism’s genetic information. RNA gel electrophoresis differs slightly from DNA gel electrophoresis in procedure, as RNA molecules, unlike DNA, are single-stranded chains that tend to fold into structures. This makes size separation more difficult for RNA fragments.
The first step in RNA gel electrophoresis is the isolation of RNA molecules from the biological cells of the sample. The sample tissue is dissolved in a specific mixture of chemicals and purified to remove the RNase enzyme, proteins and DNA. It is especially important that the sample is not contaminated with RNase at any point in the process, as RNase catalyzes the degradation of RNA molecules, causing them to break down. After the sample is purified, it is cooled and another chemical is added to precipitate the RNA or cause it to fall as a solid out of solution. The sample is then placed in a centrifuge, which spins it at high speed and isolates the solid precipitate at the bottom of the tube.
In a DNA or RNA gel electrophoresis procedure, purified biological molecules are added to wells in one end of a flat gel sheet. An electric current is then passed through the gel. Since the DNA and RNA molecules are negatively charged, they are attracted to the positive electrode at the end of the gel and migrate through the pores of the gel to that end. The pores in the gel are of a fixed size, so smaller fragments of DNA or RNA migrate more quickly than larger fragments, which have a harder time navigating through the porous matrix.
After the gel has been used for a specified amount of time, the electric current is turned off and the results are examined. The DNA or RNA molecules can be colored and made visible at this point. Each fragment will appear as a band in a different spot on the gel, depending on how far it has been able to migrate given its size. Separating fragments by size can be useful for comparing samples, as in forensics, to determine if there is a match: identical samples will have identical banding patterns.
In RNA gel electrophoresis, the additional denaturation step is required before the gel can be run. Under normal conditions, RNA molecules aggregate or fold into secondary structures, affecting the mobility of RNA fragments through the gel. To prevent this from happening, the RNA sample must be denatured by adding a chemical, such as formaldehyde, to the sample and gel.
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