Figure 2

RT PCR Analyses of pure in vitro sense and antisense RNA. (a) Schematic diagram depicting the relative overlap between complementary in vitro RNA, Sp6 and T7 RNA. Position of the RT primer (P1 and P2) and of the PCR targets (PCR-1, PCR-2, and PCR-3) are also depicted. (b) Representative ethidium bromide stained gel results of RT-PCR. Sp6 and T7 RNA (5 ng) were reverse transcribed in 20 μl reaction either each one in pure form or mixed together. Reverse transcription was carried out in absence of any primers (-p), or in presence of Sp6-specific primer (+p); P1 for PCR1 and PCR-2; and P2 for PCR-3. PCR was performed on 100 nl cDNA for 22 cycles. (c) Sp6 RNA (5 ng) was reverse transcribed in 20 μl reaction mixed together with increasing amounts of T7 RNA as shown (from 1 to 10 ng). Reverse transcription was carried out in absence of any primers (-p), or in presence of Sp6-specific primer (+p, P1) followed by PCR using PCR-3 on 100 nl cDNA for 22 cycles. A representative ethidium bromide stained gel is shown, as well as a bar graph of the net signal [(+p)-(-p)] in arbitrary units (AU) as means+SE of 3 independent RT-PCR reactions. See Additional file 4 for primer information.