Running glyoxalated RNA gels
For the midi gel tray, (12cmX15cm; 1.0% gel) weigh out 0.5g of agarose
into a clean dry 100ml conical flask. Add 2.0ml 25X MOPS buffer (1M MOPS/NaOH,
pH 7.0, 250mM sodium acetate, 25mM EDTA) and 50ml of MilliQ water. Invert
a small conical flask in the neck of the 100ml flask and zap in microwave
at hi power 2mins. Give the flask a swirl on the shaking platform to cool
down. Addition of 2µl of 10mg/ml ethidium bromide is optional at
Meanwhile clean the gel tray thoroughly and rub with ethanol. Assemble
the tray and place on a level surface. Up to 4 combs may be placed in a
typical midi gel tray. This allows many samples to be run simultaneously.
Allow the melted agarose solution to cool to about 60°C, swirling continuously,
and pour into the gel tray. Position the comb(s) immediately.
Samples are prepared in 10% glyoxal and 5M DMSO. Both of these substances
are harmful and gloves should be worn. 40% glyoxal (Sigma) is deionised
by stirring with Amberlite for a couple of hours and filtered. This solution
is stored in 122µl aliquots in microtubes in the freezer. Each tube
is used only once. 190µl of DMSO and 13µl of 25X MOPs buffer
are added to 122µl of glyoxal. The RNA is diluted with 2 volumes
of the glyoxal/DMSO solution. This mixture is incubated at 55°C for
1 hour before adding a one twentieth volume of dye buffer (40% Ficoll 400,
0.4% bromophenol blue, 0.4% xylene cyanole). It is important not to add
too much RNA to a well as resolution is compromised- the less the better.
20µg of RNA in the gel described above is the maximum- this means
that 10µl of 2µg/µl denatured RNA can be added. Clearly
a gel with deeper wells ie more agarose can be run and will accommodate
proportionately more RNA. This depends on the concentrations of the final
RNA preparations and the abundance of the target sequence- ideally RNA
concentration should be >5µg/µl before denaturation.
The gel is run in 1X MOPS buffer (ie diluted 25X buffer; ethidium bromide,
0.5ug/ml- optional) at 60-100V.
For direct visualisation of RNA, ethidium bromide is included during the
run. If RNA is very faint or not seen then the gel should be stained by
first incubating with 50mM NaOH, 0.5ug/ml ethidium bromide, followed by
20mM Tris/HCl, pH 7.6, 0.5µg/ml ethidium bromide and the gel viewed
on a UV transilluminator. For blotting the gel can be run in ethidium bromide
but should not be treated afterwards at all, and is blotted directly with
10-20X SSC onto nylon filters which are then baked at 80°C for 1hour.
This fixes the RNA and drives off the glyoxal.
Glyoxal and DMSO are irritant and harmful. Ethidium bromide is
a suspected carcinogen and gloves should be worn whenever handling any
of these compounds. Dilute ethidium bromide solutions and gels (0.5µg/ml)
should be disposed of carefully according to local regulations, and all
containers thoroughly washed.
For hybridisation experiments it is recommended that gels be run in
ethidium bromide. This allows direct visualisation of the RNA and hence
verification of the loading consistency and a check of the RNA integrity.
The 28S (upper) rRNA band should be about twice as intense as the lower
18S band. If not the result will be of poor quality. To use ethidium bromide
in these gels the buffer pH is very important and care should be taken
during preparation. The gels can then be used directly for blotting.
If using heterologous probes it is vital to include a control sample
known, or at least thought to, contain homologous sequence eg rat sample
for rat probe. This provides evidence that the probe works and has been
efficiently labelled. It is claimed that glyoxalated DNA runs at the same
rate as RNA and therefore DNA size markers can be used. Treat in the same
way as RNA samples and load about four times as much as in a DNA (ie nondenaturing)
gel. DNA markers can also be simply end-labelled with 32P, glyoxalated
and transferred with RNA thus providing markers for blots.
Care should be taken to minimise RNAase contamination, but glyoxalated
RNA appears to be very stable so some sloppiness is possible. The stability
of glyoxalated RNA also means that glyoxalated samples can be stored indefinitely
at -40°C (or better -80°C). If the samples are only to be used
for Northern analysis then the whole lot can be glyoxalated and stored-
a better option than constantly thawing and refreezing native RNA.