Denaturing Urea Polyacrylamide Gel Electrophoresis for RNA Analysis: A Technique to Separate Fluorescently Labeled Phosphorylated RNA Oligonucleotides from their Non-Phosphorylated Equivalents

Abstract

Source: Pillon, M. C., et al., Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates. J. Vis. Exp. (2020)

In this video, we demonstrate a denaturing polyacrylamide gel electrophoresis for the separation of phosphorylated RNA species from their non-phosphorylated counterparts. The RNA species are fluorescently labeled for laser detection on the gel.

Protocol

1. Gel electrophoresis

1. Prepare 15% acrylamide/8 M urea gel solution.
   CAUTION: Acrylamide must be handled with care, because it is a neurotoxin. Always wear gloves, a lab coat, and goggles when handling it.
   1. In a 150 mL glass beaker, combine 22.5 mL of premixed 40% acrylamide/bis-acrylamide 29:1 solution, 6 mL of 10x TBE, 28.8 g of urea, and RNase-free water to a total volume of 59 mL. Gently stir the solution.
      NOTE: Depending on the RNA substrate length, altering the percentage of polyacrylamide may improve the resolution between unphosphorylated and phosphorylated RNA.

Results

Figure 1: Example of denaturing gel analysis of phosphorylated RNA. In vitro RNA kinase assay of Las1-Grc3 (110 nM) incubated with 500 nM SC-ITS2 RN.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.4 mm 34-well comb	BioRad	1653848
0.4 mm spacer	BioRad	1653812
0.5 M EDTA ph 8.0	KD Medical	RGF-3130
1M Magnesium Chloride	KD Medical	CAC-5290
1M Tris pH 8.0	KD Medical	RGF-3360
40% Acrylamide/Bis Solution 29:1	BioRad	1610146
5M Sodium Chloride	KD Medical	RGF-3720
ammonium persulfate (APS)	BioRad	161-0700
ATP	Sigma	A2383-1G
boric acid	Sigma	B0394
bromophenol blue sodium salt	Sigma	B5525-5G
Glass Plates	Thomas Scientific	1188K51
Hoefer SQ3 Sequencer	Hoefer	N/A
Image J Software	N/A	N/A	<a href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a>
Labeled RNA oligonucleotides	IDT	Custom Order
Pharmacia EPS 3500 Power Supply	Pharmacia	N/A
Steriflip 0. 22 um Filter	Millipore	5FCP00525
TEMED	BioRad	161-0800
tris base	Sigma	TRIS-RO
Typhoon FLA 9500 gel imager	GE Healthcare	N/A
Ultra Pure DEPC Water	Invitrogen	750023
Ultra Pure Glycerol	Invitrogen	19E1056865
urea	Fisher Chemical	U15-500