The overall quality of an RNA preparation may be assessed by electrophoresis on a denaturing agarose gel; this will also give some information about RNA yield. A denaturing gel system is suggested because most RNA forms extensive secondary structure via intramolecular base pairing, and this prevents it from migrating strictly according to its size. Be sure to include a positive control RNA on the gel so that unusual results can be attributed to a problem with the gel or a problem with the RNA under analysis. RNA molecular weight markers, an RNA sample known to be intact, or both, can be used for this purpose.
Ambion's NorthernMax® reagents for Northern Blotting include everything needed for denaturing agarose gel electrophoresis. These products are optimized for ease of use, safety, and low background, and they include detailed instructions for use.
An alternative to using the NorthernMax reagents is to use the procedure described below. This denaturing agarose gel method for RNA electrophoresis is modified from "Current Protocols in Molecular Biology", Section 4.9 (Ausubel et al., eds.). It is more time-consuming than the NorthernMax method, but it gives similar results.
Gel Loading Buffer II - Denaturing PAGE
A 1-2X solution of 95% Formamide, 18mM EDTA, and 0.025% SDS, Xylene Cyanol, and Bromophenol Blue.
Gel Loading Solution - All purpose, native agarose
A 10X solution of 40% Sucrose, 0.17% Xylene Cyanol,and 0.17% Bromophenol Blue.
NorthernMax Formaldehyde Load Dye
This ready-to-use solution is added to RNA sample (3 parts solution : 1 part sample) and heated briefly. The samples are then ready to be loaded. Ethidium bromide can be added to the samples if desired.
NorthernMax-Gly Sample Loading Dye
An improved formulation used for RNA sample denaturation in any glyoxal gel protocol. The volume ratio of solution to sample is lower than in published protocols, so sample precipitation prior to gel loading is usually not required. Ethidium bromide is premixed into the solution.
- Prepare the gel.
- Heat 1 g agarose in 72 ml water until dissolved, then cool to 60°C.
- Add 10 ml 10X MOPS running buffer, and 18 ml 37% formaldehyde (12.3 M).
WARNING: Formaldehyde is toxic through skin contact and inhalation of vapors. Manipulations involving formaldehyde should be done in a chemical fume hood.
10X MOPS running buffer:
0.4 M MOPS, pH 7.0
0.1 M sodium acetate
0.01 M EDTA
- Pour the gel using a comb that will form wells large enough to accommodate at least 25 µl.
- Assemble the gel in the tank, and add enough 1X MOPS running buffer to cover the gel by a few millimeters. Then remove the comb.
- Prepare the RNA sample.
a. To 1-3 µg RNA, add 0.5-3X volumes Formaldehyde Load Dye.
- To simply check the RNA on a denaturing gel, as little as 0.5X Formaldehyde Load Dye can be used, but to completely denaturate the RNA, e.g. for Northern blots, use 3X volumes of Formaldehyde Load Dye.
- Ethidium bromide can be added to the Formaldehyde Load Dye at a final concentration of 10 µg/ml. Some size markers may require significantly more than 10 µg/ml ethidium bromide for visualization. To accurately size your RNA, however, it is important to use the same amount of ethidium bromide in all the samples (including the size marker) because ethidium bromide concentration affects RNA migration in agarose gels.
b. Heat denature samples at 65-70°C for 5-15 min.
- Denaturation for 5 min is typically sufficient for simply assessing RNA on a gel, but a 15 min denaturation is recommended when running RNA for a Northern blot. The longer incubation may be necessary to completely denature the RNA.
Load the gel and electrophorese at 5-6 V/cm until the bromophenol blue (the faster-migrating dye) has migrated at least 2-3 cm into the gel, or as far as 2/3 the length of the gel.
Visualize the gel on a UV transilluminator. (If ethidium bromide was not added to the Formaldehyde Load Dye, the gel will have to be post-stained and destained.)
Intact total RNA run on a denaturing gel will have sharp 28S and 18S rRNA bands (eukaryotic samples). The 28S rRNA band should be approximately twice as intense as the 18S rRNA band (Figure 1, lane 3). This 2:1 ratio (28S:18S) is a good indication that the RNA is intact. Partially degraded RNA will have a smeared appearance, will lack the sharp rRNA bands, or will not exhibit a 2:1 ratio. Completely degraded RNA will appear as a very low molecular weight smear (Figure 1, lane 2). Inclusion of RNA size markers on the gel will allow the size of any bands or smears to be determined and will also serve as a good control to ensure the gel was run properly (Figure 1, lane 1). Note: Poly(A) selected samples will not contain strong rRNA bands and will appear as a smear from approximately 6 kb to 0.5 kb (resulting from the population of mRNAs, and depending on exposure times and conditions), with the area between 1.5 and 2 kb being the most intense (this smear is sometimes apparent in total RNA samples as well).
Figure 1. Intact vs. Degraded RNA. Two µg of degraded total RNA and intact total RNA were run beside Ambion's RNA Millennium Markers™ on a 1.5% denaturing agarose gel. The 18S and 28S ribosomal RNA bands are clearly visible in the intact RNA sample. The degraded RNA appears as a lower molecular weight smear.
Generally, at least 200 ng of RNA must be loaded onto a denaturing agarose gel in order to be visualized with ethidium bromide. Some RNA preparations, such as those from needle biopsies or from laser capture microdissected samples, result in very low yields. In these cases, it may be impossible to spare 200 ng of RNA to assess integrity. Alternative nucleic acid stains, such as SYBR® Gold and SYBR® Green II RNA gel stain from Molecular Probes, offer a significant increase in sensitivity over ethidium bromide. Using a 300 nm transilluminator (6 x 15-watt bulbs) and a special filter, as little as 1 ng and 2 ng of RNA can be detected with SYBR Gold and SYBR Green II RNA gel stain, respectively.
- We generally load 1 µg and 2.5 µg samples on 1% agarose gels in TBE (89 mM Tris-HCl pH 7.8, 89 mM borate, 2 mM EDTA) with 0.5 µg/ml ethidium bromide added to the gel.
- Add 10X native agarose gel loading buffer (15% ficoll, 0.25% xylene cyanol, 0.25% bromophenol blue) to the RNA samples to a final concentration of 1X.
- On native gels, the samples can be loaded directly without heating.
- An aliquot of intact RNA should always be run as a positive control to rule out unusual results due to gel artifacts.
- Run the gel at 5-6 V/cm measured between the electrodes.