Denaturing Agarose Gel Electrophoresis
The isolated RNA samples were separated by denaturing 1.5% agarose gel containing 1xMOPS buffer and 2% formaldehyde. 5 μg of RNA (no more than 6.6 μL in final volume) was mixed with 5.4 μL of formaldehyde, 3 μL of 10x MOPS buffer and 15 μL of formamide. The samples and RNA markers from Thermo Scientific (RiboRuler High Range, #SM1821 and Low Range RNA ladder, # SM1831) were denatured at 55°C for 15 minutes. The RNA mixes were then cooled on ice. Sample loading dye (5μL, 1:6) (0.25% bromophenol blue, 40% sucrose) was added to the samples, and the samples were loaded. The electrophoresis buffer was the same as the buffer used to prepare the gel, 1 x MOPS. During the first hour, 60V was applied, and the voltage was increased to 85V.
After 5 hours, when the run ended, the ladder region was cut off and stained for 30 min in the running buffer containing 5000x diluted Diamond™ Nucleic acid dye (Promega). The transfer of the RNA from the agarose gel to the nylon membrane (Amersham Hyband™-N+, GE Healthcare, #RPN303B) was done via capillary transfer of RNA from the denaturing agarose gel to the nylon membrane (Sambrook, 2001). RNA crosslinking to the nylon membrane was done via UV (Stratagene crosslinker).