Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels Separation of RNAs according to size is the first stage in northern blotting and hybridization. The method described in this protocol uses glyoxal to denature the RNA, ethidium bromide to stain it, and agarose gel electrophoresis to separate the resulting glyoxal-RNA-ethidium adducts.
Separation of RNA According to Size: Electrophoresis of RNA through Agarose Gels Containing Formalde Separation of RNAs according to size is the first stage in northern blotting and hybridization. The method described in this protocol uses formaldehyde to denature the RNA, ethidium bromide to stain it, and electrophoresis through agarose gels containing 2.2 M formamide to separate the resulting formaldehyde-RNA-ethidium adducts.
Transfer and Fixation of Denatured RNA to Membranes Protocol Protocol describes the transfer of RNA from agarose gels to neutral or positively charged nylon membranes, using upward capillary flow of neutral or alkaline buffers. RNA becomes covalently fixed to positively charged nylon membranes during transfer in alkaline buffers. However, treatment by UV irradiation or heating is required to fix RNA to neutral membranes.
Visualization of RNA Preparations on 1% Agarose Gels Protocol Method is used to assess (roughly) the integrity of total RNA samples by visualization of discreet 18S and 28S ribosomal RNAs. Total RNA is separated by electrophoresis through a 1% agarose gel containing 1.3 ìM ethidium bromide. Binding of the ethidium bromide to the RNA allows visualization of the separated RNA molecules when the gel is exposed to ultraviolet (UV) light.
