Sep 25, 2020
Denaturing PAGE for resolving RNA
Forked from Denaturing RNA Urea-PAGE
- 1University of Pennsylvania;
- 2Institute for Synthetic Microbiology
- Pogson Group
Protocol Citation: Diep R Ganguly, Anna Behle 2020. Denaturing PAGE for resolving RNA. protocols.io https://dx.doi.org/10.17504/protocols.io.bj8rkrv6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 24, 2020
Last Modified: September 25, 2020
Protocol Integer ID: 40945
Keywords: RNA, Electrophoresis, Denaturing-PAGE, PAGE,
Abstract
Protocol denaturing PAGE separation and identification of total RNA extracts using Urea-PAGE with the BioRad Mini-PROTEAN II / Tetra Cell electrophoresis system. Modified from dx.doi.org/10.17504/protocols.io.gszbwf6.
Guidelines
Use filter tips and ensure you are using RNase free components and surfaces.
This is a fantastic resource to help planning and troubleshooting: RNA: A Laboratory Manual (https://www.cshlpress.com/default.tpl?cart=1598856781901971730&fromlink=T&linkaction=full&linksortby=oop_title&--eqSKUdatarq=725).
Materials
MATERIALS
ssRNA Ladder - 25 gel lanesNew England BiolabsCatalog #N0362S
dsRNA Ladder - 25 gel lanesNew England BiolabsCatalog #N0363S
Low Range ssRNA Ladder - 25 gel lanesNew England BiolabsCatalog #N0364S
SYBR GoldThermo ScientificCatalog #S-11494
TEMEDBio-rad LaboratoriesCatalog #1610801
Mini Protean tetra cell Bio-rad Laboratories
SYBR GreenThermo Fisher Scientific
Ethidium bromide [EB, EtBr]Bio Basic Inc.Catalog #EB0195.SIZE.1g
DEPC WaterBio Basic Inc.Catalog #DD1005(D0121).SIZE.4x500ML
Axygen 0.5 mL PCR tubes, 0.5 mL, thin wall, clear, flat caps CorningCatalog #PCR-05-C
Thermal cycler
RNaseZap® RNAse Decontamination Solution Life TechnologiesCatalog #AM9780
Filter tips
Depc (Diethyl Pyrocarbonate)VWR international LtdCatalog #80730-888
40% Acrylamide-bisacrylamide (19:1)
30% Acrylamide / Bis. solutionBio-rad LaboratoriesCatalog #161-0158
0.1% SDS solution
10% Ammonium persulfate (APS)Sigma Aldrich
Gel Loading Buffer II (Denaturing PAGE)Thermo FisherCatalog #AM8546G
TBE (10X), RNase-freeThermo FisherCatalog #AM9865
Mini-PROTEAN® Spacer Plates with 0.75 mm Integrated SpacersBioRad SciencesCatalog #1653310
Mini-PROTEAN® Short PlatesBioRad SciencesCatalog #1653308
Safety warnings
Wear vinyl or nitrile gloves when handling PAA (neurotoxic).
Make sure to thoroughly wash wells before loading samples.
2x RNA loading buffer contains formamide.
Before start
Thoroughly clean all components with 70% ethanol and RNase away (or 0.05 - 0.1 % SDS). Ensure RNA samples of interest have been normalized to a known concentration with nuclease-free/DEPC-treated water.
Gel preparation
Gel preparation
Before preparing the gel, clean all components, gel tank, and surfances with 70 % ethanol (can use 0.1% SDS or RNase Zap to be extra thorough, we do this periodically).
Assemble gel plates in the green brackets and place on casting unit. Ensure glass plates are not cracked and are sitting flush against the separator plate and on the rubber mats (to prevent gel leakage).
Use the following table to prepare the required gel recipe using either 30 % or 40 % PAA (~4 mL=1x0.75 mm gel).
| PAA percentage | 8 % | 10 % | 12 % | 15 % | |
| 30 % / 40 % PAA | 2 / 2.67 mL | 2.5 / 3.33 mL | 3 / 4 mL | 3.75 / 5 mL | |
| 8 M Urea | 5 g | 5 g | 5 g | 5 g | |
| 10x TBE | 1 mL | 1 mL | 1 mL | 1 mL | |
| 10 % APS | 80 µL | 80 µL | 80 µL | 80 µL | |
| TEMED | 10 µL | 10 µL | 10 µL | 10 µL | |
| DEPC-treated H2O | ad 10 mL | ad 10 mL | ad 10 mL | ad 10 mL |
Urea-PAGE recipe
Dissolve urea in 10x TBE, PAA, and 1/2 volume DEPC-treated H2O.
Warming solution (e.g. 37˚C water bath) with gentle agitation will help to dissolve urea.
Safety information
Wear vinyl or nitrile gloves and work in fumehood when handling PAA (neurotoxic!)
Once dissolved, allow to cool to room temperature and add DEPC-treated H2O up to 10 mL.
Add 80 µL APS (10 %) and 10 µL TEMED to the gel solution and gently swirl once. Immediately pipette solution into gel apparatus ( ~4.5 mL, fill until just before it overflows to prevent bubbles when inserting comb). Retain some gel mixture to check for complete polymerisation.
Immediately insert comb tilted at an angle (to minimise trapping air bubbles). Make sure you are wearing appropriate gloves. Lower completely and allow gel to polymerise for >15 mins. Gels can be stored at 4˚C wrapped in plastic (use with 3-5 days).
RNA preparation
RNA preparation
Determine desired loading amount of RNA per well (e.g. 0.5 - 2 µg RNA). Normalize RNA concentrations accordingly (e.g. 100 - 200 ng/µL).
Add required RNA to equal volume 2x gel loading buffer II (Ambion). Up to to 50% can be RNA sample although less is better. Final volumes for loading may vary, however, less than 5 µL results in poor banding when using the Mini protean system (10 µL / gel works well). Keep prepared samples @ 4 °C until ready for denaturation and loading.
Safety information
RNA loading dye contains formamide. Wear goggles/lab coat/ gloves!
RNA loading
RNA loading
Once remaining gel solution has polymerised, remove from casting bracket and insert into running bracket (keep combs). Fill interior with 1x TBE and ensure there are no leaks (~5 mins). If leaking, re-adjust and check again. Place bracket into gel tank and fill with 1x TBE.
Remove combs and rinse individual wells thoroughly.
Connect electrodes, and pre-run gel @ 10 mA / gel for ~20-30 minutes to remove residual urea.
During the pre-run, denature RNA samples and ladder, in gel loading buffer II, at 72˚C for 5 min. Once finished, immediately keep at 4˚C until ready to load (minimise time kept at 4˚C).
Turn power supply off and rinse the well a second time.
Pipette denatured samples into wells slowly while ensuring there are no air bubbles trapped in the pipette tip (Ambion recommends loading while samples are still hot).
Close the lid and plug electrodes into power supply. Separate RNA @ 10 mA / gel for ~1-2 hours (make sure you have a reliable power supply that holds current, particularly as urea has poor conductivity). Allowing the upper marker, from the Ambion loading buffer II, to run off the gel is a reasonable indictor for running time.
Visualization
Visualization
After sufficient running time switch off power supply, pour out buffer, disassemble gel and clean components. Transfer gel to clean container and rinse once with deionized water.
Incubate gel in 0.5 µg ethidium bromide / mL 1x TBE for ~15 minutes (or use SYBR gold/green safe stains). Rinse 3x with water and allow to destain for ~2 minutes. Visualize.