In vitro synthesis of double stranded RNA and measurement of thermal stability: effect of base composition, formamide and ionic strength.

Double stranded RNA was prepared by transcription of complementary strands followed by annealing. RNA duplexes prepared in this way showed cooperative thermal transition. Thermal denaturation of RNA duplexes of 33%-70% G+C content shows that Tm increases more sharply [0.93 degrees C/(%G+C)] with increase in G+C than that of DNA. Formamide (up to 80%) reduces Tm of RNA duplex linearly, and the rate of reduction is much less than that of DNA. The relation between salt concentration and Tm of double stranded RNA was also determined. The following relationship has been derived based on the above observations: Tm (ds RNA) = 14.88 logM + 0.93 (%G+C) + 67.62 - 0.4 (%Formamide).