Denaturing Urea Polyacrylamide Gel Electrophoresis for RNA Analysis: A Technique to Separate Fluorescently Labeled Phosphorylated RNA Oligonucleotides from their Non-Phosphorylated Equivalents
Transcription
To begin, take a sample containing fluorescently labeled phosphorylated and non-phosphorylated RNA oligonucleotides mixed with the loading dye containing urea. Heat the sample.
Urea – a strong denaturant – in combination with the heat denatures secondary RNA structure, resulting in its linearized and unstructured conformation. Now, cast the gel by mixing acrylamide-bisacrylamide solution with urea.
Add tetramethylene diamine, a catalyst, and ammonium persulfate, an oxidizing agent, to facilitate subsequent gel polymerization.
Pour the solution into a pre-assembled cassette apparatus. Insert a comb, creating wells for sample loading. Allow the gel to polymerize. Place the cassette into the gel apparatus. Add a running buffer to the upper and lower reservoirs, maintaining current flow through the entire gel. Remove the comb.
Load the sample mixed with visualization dye into the well and connect the gel to a power supply at the desired voltage. The urea in the gel allows RNA molecules to migrate as linear species.
The gel acts as a sieve, permitting the negatively charged RNA molecules to move through pores toward the positive electrode. During the run, the phosphorylated RNA being inherently more negative moves faster than non-phosphorylated RNA.
Image the gel for the detection of fluorescently labeled RNA. Observe the mobility shift between the faster migrating phosphorylated RNA, which appears as a lower band compared with the unphosphorylated RNA.
