Many factors will determine an appropriate number of PCR cycles, including but not limited to RNA input mass, quality and purity. The protocol conveniently asks you to only move half of the reaction product forward after 5’ ligation to reverse transcription and PCR amplification. This allows you to try a cycle number with your sample and then go back and use a more appropriate cycle number. While optimizing the kit, it was found that 12 cycles was more than enough cycles to amplify from 1ug of total purified RNA of high quality.
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