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RNA-specific adenosine deaminase (ADAR) was amplified from log-fold dilutions of total HeLa cell cDNA (100 ng to 1 pg) using PerfeCTa™ SYBR Green FastMix or the SYBR GreenER qPCR SuperMix (Invitrogen) according to each manufacturers protocol. Averaged plots for quadruplicate reactions for each input quantity are shown. Replicate CT values are shown on the standard curve (Panel A, inset). Cycling conditions: Invitrogenn: 95°C, 10 min followed by 40 cycles of 95°C, 10s; 60°C, 60s; PerfeCTa™ SYBR Green FastMix: 95°C, 20s followed by 40 cycles of 95°C, 1s; 60°C, 20s. PerfeCTa SYBR Green FastMix amplified the ADAR gene with higher efficiency and greater sensitivity. All replicate reactions for SYBR GreenER qPCR SuperMix failed to amplify ADAR from 1 pg of cDNA.
RNA-specific adenosine deaminase (ADAR) was amplified from log-fold dilutions of total HeLa cell cDNA (100 ng to 10 pg) using PerfeCTa™ SYBR Green FastMix or SYBR PreMix Ex Taq™ (Takara) according to each manufacturers protocol. Averaged plots for quadruplicate reactions for each input quantity are shown. PerfeCTa™ SYBR Green FastMix produces higher fluorescent signal and detection of equal target amounts at earlier Cts. Cycling conditions for both kits: 95°C, 20s followed by 40 cycles of 95°C, 1s; 60°C, 20s
RNA-specific adenosine deaminase (ADAR) was amplified from log-fold dilutions of total HeLa cell cDNA (100 ng to 10 pg) using PerfeCTa SYBR Green FastMix or the DyNAmo Flash SYBR Green PCR Kit (Finnzymes) according to each manufacturers protocol. Averaged plots for quadruplicate reactions for each input quantity are shown. Fusion of DNA-binding peptide to Tbr DNA pol results in lower specificity of the DyNAmo kit which is evident in false positive results for no template control (NTC) reactions. Chemically modified polymerase produces delayed Cts and lower signal strength compared to AccuStart™ Taq. Cycling conditions: Finnzymes: 95°C, 7 min followed by 40 cycles of 95°C, 10s; 60°C, 20s PerfeCTa™ SYBR Green FastMix: 95°C, 20s followed by 40 cycles of 95°C, 1s; 60°C, 20s
