Running glyoxalated RNA gels


Notes

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.