In vitro transcription of labeled RNA: synthesis, capping, and substitution

All in vitro RNA transcription reactions involve the use of common components: a template, phage RNA polymerase, and ribonucleoside triphosphates (rNTPs). The concentration of rNTPs is a crucial variable that is manipulated to determine the specific activities of labeled RNAs. This protocol describes methods used to synthesize RNAs of low specific activity (trace labeled), medium specific activity, and high specific activity. Also described is how the same principles that determine the level of incorporation of labeled rNTPs are applicable to the synthesis of transcripts containing modified nucleotides, including 5' cap and internal (body) modifications such as biotinylated uridine, 4-thiouridine, and phosphorothiotated nucleotides. Careful attention to detail in setting up the transcription reaction will permit the synthesis of any number of "designer RNAs."