Figure 4
The effect of different incubation conditions on RNA. Total RNA was extracted from a large number (2 × 106 cells) of formalin fixed paraffin embedded cells using Ambion RecoverAll™ kit. Five eluted RNA aliquots from one extraction were subjected to different incubation conditions on a hot rack (lanes 6 to 10). In parallel, five eluted RNA aliquots from one snap frozen extraction were incubated (lane 1 to 5). Each well contained 1 μl of 4 ng/μl RNA in Ambion Elution Solution. Incubation conditions were as follows: 0 = no treatment; 70-10 = 70°C for 10 min; 70-20 = 70°C for 20 min; 95-10 = 95°C for 10 min and 95-20 = 95°C for 20 min. RNA from snap frozen preparations was not degraded at 70°C for 20 min (lane 3), but was degraded at 95°C (lane 4 and 5). RNA from FFPE showed large fragments and cross-linked RNAs which are approximately 5 kb (lane 6 – arrow). It was found that incubation of eluted RNA at 70°C for 20 min is the optimal condition, among these tested, to break up cross-links while not compromising RNA integrity.