Scaling up in vitro transcription synthesis of RNA standards for competitive quantitative RT-PCR: looking for bigger yields.

Because the acquisition of in vitro transcription kits, for production of RNA standards, might not be affordable for some small laboratories, the current work describes some alternatives to the commercial Promega protocols. Yields of tested reactions were all higher than the ones reported for the standard Riboprobe kit (1mug RNA/mug template, 1x) and the optimized variant for high yields (5-10x). They were also as good as the ones described for the high RNA production kit RiboMAX (10-20x), exceeding them in approximately 70% of reactions. Our best results, achieved in a 500-mul format, were three to five times higher than the ones reported for the Promega kit.