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Table 1 RNase If -qPCR assay RTL (Relative transcript level) and % recovery for 10-point dilution series of dsRNA and ssRNA

From: RNase If -treated quantitative PCR for dsRNA quantitation of RNAi trait in genetically modified crops

dsRNA ssRNA
Standard point dsRNAamount (pg) R9S RTL R70RTL 70 RTL MeasureddsRNAamount % recovery ssRNAamount (Pg} R95 RTL R70RTL 70 RTL MeasuredssRNAamount % recovery
I 6.67 138.64 0.03 2.44 8 08 121% 6.67 0.08 0.00 79.82 7.89 118%
2 3.33 S9.26 0.01 0.83 3.57 107% 3.33 0.06 0.00 32.71 3.32 100%
3 1.67 26.95 0.01 0.40 1.67 100% 1.67 0.00 0.00 15.67 1.62 97%
4 0.83 12.68 0.00 0.21 0.81 97% 0.83 0.00 0.00 7.03 0.75 89%
5 0.42 4.97 0.00 0.08 0.33 19% 0.42 0.05 0.00 4.07 0.44 105%
6 0.21 2.28 0.00 0.05 0.16 75% 0.21 0./9 0.00 1./8 0.20 95%
/ 0.10 1.44 0.00 0.03 0.10 96% 0.10 0.00 0.00 0.83 0.09 90%
8 o.os 0.81 0.00 0.01 006 110% O.OS 0.10 0.00 0.43 0.05 95%
9 0.03 0.35 0.00 0.01 003 99% 0.03 0.06 0.00 0.23 0.03 105%
10 0.01 0.20 0.00 0.00 0.02 117% 0.01 0.05 0.00 0.12 0.01 110%
  1. 10-point standard synthetic dsRNAs and ssRNAs were prepared as the concentration shown. dsRNA/ssRNA amount (pg) is represented in a 10 μL qPCR reaction. The synthetic RNAs were analyzed using the RNase If -qPCR protocol described above. The relative transcript level (RTL) from different steps of RNase If -qPCR protocol is denoted as R95 RTL (95 °C RTL with RNase If treatment). R70 RTL (70 °C RTL with RNase If treatment) and 70RTL (70 °C RTL without RNase If treatment). The dsRNA was analyzed via R95 RTL, and R70 RTL and 70 RTL were assayed as negative control. The ssRNAs were analyzed by 70RTL, and R95 RTL and R70 RTL were used as negative control. Each standard point was analyzed via three technical replicates and the data was presented. The calculated dsRNA and ssRNA amount were back calculated via the equation of trend lines. The percent recovery is calculated from the measured RNA amount divided by the theoretical RNA amount