13

Formaldehyde Gel Electrophoresis of Total RNA

Sian Bryant and David L. Manning

1. Introduction
RNA has the tendency to form both secondary and tertiary structures that
can Impede its separation by electrophoresis. As such, identical speciesof RNA
exhibiting varying degrees of intramolecular base-pairing, migrate at different
rates and result in the smearing of distinct RNA molecules. Consequently, the
electrophoresis of RNA needs to be performed under denaturing conditions.
Heat denaturing the RNA sample prior to electrophoresis is insufficient, as
secondary structures will simply reform unless a denaturing system is used.
Successful electrophoresis of RNA is therefore accomplished in two steps:
RNA is heat denatured prior to electrophoresis; and during electrophoresis,
conditions are established that maintain the RNA in a denatured state.
The methodology described in this chapter involves the use of formaldehyde
as a denaturant within the agarose gel. In addition, both formaldehyde and
formamide are added to the sample before electrophoresis to aid the denaturation
of the RNA sample. For procedures such as Northern analysis, in which RNA
is transferred or blotted from the gel to a solid matrix for subsequent
hybridization (see Chapters 15 and 17), the optimal balance between electro-
phoretic resolution and efficiency of transfer is achieved with a l-l -2% agar-
ose gel (see refs. I-3).
2. Materials
It is very important that all reagents used are of molecular biology-grade
and free from RNase contamination. As in all molecular brological procedures,
gloves should be worn throughout.
1. Formaldehyde+ Formaldehyde IS a suspectednose, nasopharynx, and liver
carcinogen; it is toxic both through inhalation and ingestion. Its use should
From Methods m Molecular Biology, Vol 86 RNA /so/at/on and Charactematron Protocols
E&ted by R Rapley and 0 L Manntng 0 Humana Press Inc , Totowa, NJ

69
70 Bryant and Manning

therefore be restricted to a ventilated fumehood. Formaldehyde is routinely

supplied as a 37% (v/v) stock solution. It should be stored at room temperature
and out of drrect sunlight to prevent oxidation
2. Formamide: The use of this chemical should be restricted to the fumehood, as it
is a suspected teratogen and is Irritating to the eyes, skin, and respiratory system.
Formamide should be stored m the dark and at room temperature to prevent
oxidation.
3. RNase-free water. This can be achieved by treating the water first with dlethyl
pyrocarbonate (DEPC). Add DEPC to water to a final concentration of 0.1%.
Incubate overnight at 37°C and autoclave to destroy any residual DEPC DEPC
is an efficient, nonspecific inhibitor of RNase, however it 1s carcinogemc and
should be handled in a fume hood with extreme care.
4. 10X MOPS buffer: (0 2M MOPS (3-(N-Morpholino)propanesulfonic acid) pH
7.0; 50 mA4 sodium acetate, pH 6 0; 10 mM EDTA, pH 8.0 This solution is
prepared usmg RNase-free water. When autoclaved it assumes a characteristic
golden color and can be stored at room temperature
5 Agarose (molecular biology-grade): Store at room temperature.
6. Ethidium bromide solution (10 mg/mL): This is a powerful mutagen, extreme
care must be taken when usmg this substance. Store at 4°C.
7. Horizontal electrophoresis tank and a 11 x 14 cm casting tray.
8. RNA-loading buffer: 50% glycerol, 1 mMEDTA and 0.4% bromophenol blue, made
up to the required volume with DEPC-treated water.

3. Method
1. Denature the RNA in a stenle RNase-free microcentrifuge tube by mixing the
following:
10 pg of RNA (in a final volume of 5 yL with DEPC-treated water),
2 pL of 10X MOPS solution,
3 5 pL of formaldehyde,
10 pL of formamlde.
Incubate the RNA solution at 65°C for 15 min m a fumehood and transfer
immediately to ice.
2. For a 1% agarose gel using an 11 x 14 cm casting tray, mix 0.8 g of agarose with
57.5 mL of sterile RNase-free water and boil to dissolve agarose Cool to 6O”C,
then add 8 mL of 10X MOPS buffer, 14.5 mL of 37% (12.3A4) formaldehyde and
finally 8 FL of ethidium bromide solution (at a concentration of 10 mg/mL). Mix
the components of the gel by gently rotatmg the bottle, being careful not to
introduce any air bubbles. Pour the gel into the prepared casting tray (m a fume
hood), msert the comb and let it set for a least 60 min at room temperature.
3 After cooling the denatured RNA solution in ice, add 2 pL of sterile RNA
loading buffer.
4. Fill the buffer reservoirs and cover the gel with 1X MOPS buffer.
5. Pre-electrophorese the gel at 50 V for 10 min.
6. Load the heat denatured samples into the wells and run the gel at 50 V until the
Formaldehyde Gel Electrophoresis 71

bromophenol blue has moved approximately half to three quarters of the way
along the gel (see Note 1).
7. Visualize RNA using a 254 nm short wave ultra-violet transilluminator (see Notes
2-7). Gels can be photographed using a Polaroid land camera with 66s Polaroid film.

4. Notes
1. To avoid overheating and the generation of a smile effect on the migration of RNA
and bromophenol blue dye, we routinely run gels at 4 V per cm length of gel (i.e.,
for a 14-cm long gel, electophoresis is performed at 50-60 V for approx 4 h).
2. RNA bands very broad or trailing. This problem usually occurs as a result of a
fault in the sample preparation (RNA isolation) or in the loading of the gel. The
salt composition (introduced with the sample) of the loading solution may be too
high, too much RNA may have been loaded into the well (slight band broadening
may occur with volumes above 30 yL or more than 50 pg of RNA per well); and
applying higher voltages when running the gel may cause broadening and trailing
probably due to excess heat. Electrophoresls may be carried out quite successfully
at room temperature.
3. Little or no RNA detected m the gel after electrophoreas. RNA tends to aggregate
at the top of the gel, close to the well, if it is contaminated with proteins.
Normally, however, this is prevented since most procedures for RNA extraction
and purification involve the use of proteolytic enzymes or deproteinizmg mix-
tures such as phenol:chloroform. RNA may be degraded into fragments that are
so small they pass straight through the gel, if this is the case run the RNA on a gel
containing a higher concentration of agarose or preferably run the gel for a shorter
period of time. If there is a problem with RNA degradation, then the extraction
and purification methods need to be examined. Degradation of the RNA could be
a result of nbonucleases m the electrophoresls buffer or tank, therefore care must
be taken to ensure that the equipment is clean and both this and the buffer are
nuclease-free. The gel concentration may be too high, preventing the RNA from
entering it, although this is very unlikely as the concentration used in this
methodology is not very high.
4. There may be problems with ethidium bromide-staining in formaldehyde gels. It
can retard the running of the nucleic acid on the gel by up to 15%, but more
importantly it can reduce the transfer efficiency of the RNA to a solid support.
Ethidium bromide can be removed by soaking the gel in runnmg buffer or transfer
buffer, with several changes, for 1 h.
5 An aliquot of total cellular RNA should electrophoretically resolve only two very
distinct bands, the 28s and 18s rRNAs. The appearance of these two bands gives
an idea of the integrity of the RNA. The mRNA component manifests itself as a
significantly lighter stained smear above, between and below the rRNA bands.
The 5s rRNA, 5.8s rRNA, and the tRNAs all migrate close to the leading edge of
the gel. Totally degraded samples appear as heavily localized smears below the
level at which the 18s rRNA appears in intact samples. Heavy smears that appear
along the length of the lane may be indicative of degradation or may simply mean
72 Bryant and Manning

the sample was incompletely denatured. Fluorescence wtthin the well suggests
that genomic DNA is present wtthin the sample
6. The positioning of the 18s and 28s bands should be noted as this mformatton may
be required to estimate the size of a particular RNA of interest (18s rRNAs range
m size from 1.8 to 2 Kb, 28s rRNAs range in size from 4.6 to 5 2 Kb)
7. Prolonged exposure to UV light can damage the RNA Formaldehyde can be
removed from the gel by placing m either 1X MOPS buffer or DEPC-treated
water for about 15 mm, with several changes of buffer.

References
1. Boedtker, H. (197 1) Conformation-independent molecular determmations of RNA
by gel electrophoresis. Biochim. Bzophys Acta. 240,448
2 Rave, N., Crkvenjakov, R , and Boedtker, H. (1979) Identification of procollagen
mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde gels
Nucleic Acids Res. 6,3559.
3. Manning, D. L., McClelland, R. A., Gee, J. M , Chan, C. M., Green, C. D.,
Blarney, R. W , and Nicholson, R. I (1993) The role of four estrogen responsive
genes pLIV1, pS2, PSYD3, and pSYD8 m predictmg responsiveness to therapy in
primary breast cancer. Eur J Cancer 29(10), 1462-1468.