Specificity and sensitivity of different stem–loop primers with overlapping binding sites for a distinct RNA 3′ end and impact of forward primer position. The universal probe was applied to detect a chemically synthesized siRNA. (A) Positioning of stem–loop primers relative to the 3′ end of the siRNA. (B) Threshold cycles (CT) in PCR with template (siRNA) and without (NTC) for each stem–loop primer. CT was defined as 40 if not detectable. Error bars represent value range from mean of two independent experiments. (C) Impact of forward primer design CT, siRNA mimicking forward primer with 105 siRNA target molecules with (gray bars) or without template RNA (black bars) and conventional forward primer without template RNA (diagonal pattern).
