Sensitivity of prestaining RNA with ethidium bromide before electrophoresis and performance of subsequent northern blots using heterologous DNA probes.

Molecular Biotechnology
Yun ZhaoNianhui Zhang

Abstract

Adding ethidium bromide (EtBr) at low concentrations to RNA samples before running formaldehyde-agarose gels affords the advantages of checking RNA integrity and evaluating the quality of size-separation at any time during electrophoresis or immediately after either electrophoresis or blotted the separated RNA onto the membrane without significantly compromising mobility, transfer, or hybridization. In this study, we systematically examined the factors that affect the sensitivity of RNA prestaining by heating RNA samples that include EtBr before electrophoresis under different denaturation conditions. We also examined the efficiency of the hybridization of EtBr-prestained RNA with heterologous DNA probes. The results showed that the fluorescent intensity of EtBr-prestained RNA was affected not only by the EtBr concentration as previously reported but also by the RNA amount, denaturation time, and denaturation temperature. Prior staining of RNA with 40 μg/mL EtBr significantly decreased the efficiency of Northern blot hybridization with heterologous DNA probes. We propose that to best combine staining sensitivity and the efficiency of Northern blot hybridization with heterologous DNA probes, the concentration of EtBr used to pre...Continue Reading

References

Nov 25, 1989·Nucleic Acids Research·R A Kroczek
May 1, 1984·Analytical Biochemistry·J Meinkoth, G Wahl
Jun 25, 2005·Biotechnology Letters·Tao WangLingfang Du
Dec 1, 1981·Proceedings of the National Academy of Sciences of the United States of America·R BroglieA R Cashmore
May 14, 2008·BioTechniques·Heather D VanGuilderWillard M Freeman

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Citations

Dec 30, 2017·Biochemistry and Molecular Biology Education : a Bimonthly Publication of the International Union of Biochemistry and Molecular Biology·Nianhui ZhangXiaofeng Zhu

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