iScript™ Advanced cDNA Synthesis Kit for RT-qPCR

Total RNA (7.5 µg–1 pg) from HeLa cells was reverse transcribed using the iScript™ advanced cDNA synthesis kit for RT-qPCR in a 20 µl reaction. A tenfold dilution of generated cDNA was used as template to amplify α-tubulin in a 10 µl qPCR reaction with iQ™ SYBR^® Green supermix on a CFX384™ real-time PCR detection system. Efficiency = 90.7%, R² = 0.999, slope = –3.57. Cq, quantification cycle; RFU, relative fluorescence units.

Total RNA (7.5, 5, 2.5, and 1 µg) from human spleen was reverse transcribed using the iScript™ advanced cDNA synthesis kit for RT-qPCR. The generated cDNA samples were diluted to the equivalent of 50 ng/µl and used as template to amplify a low-expressed gene (IL-2) in a 10 µl qPCR reaction with SsoFast™ probes supermix on a CFX384™ real-time PCR detection system (FAM probe). There were no significant differences (<0.5 Cq) between diluted samples, which demonstrates accurate representation of mRNA transcripts for high and low input RNA. RFU, relative fluorescence units.

Six different input RNA amounts (7.5, 5, 2, 1, 0.1, and 0.01 µg) from HeLa cells were reverse transcribed using the iScript™ advanced cDNA synthesis kit for RT-qPCR in a 20 µl reaction. The generated cDNA was diluted 20-fold for qPCR. A 110 bp CBP amplicon (70% GC) was amplified in a 10 µl qPCR reaction with SsoFast™ EvaGreen^® supermix on a CFX96™ real-time PCR detection system. CBP efficiency = 99%, R² = 0.998, slope = –3.33. RFU, relative fluorescence units.

Total RNA (7.5, 2, 1, and 0.1 µg) from HeLa cells was reverse transcribed using the iScript™ advanced cDNA synthesis kit for RT-qPCR in a 20 µl reaction. One-tenth of the generated cDNA was used as template to amplify the APC gene in a 10 µl qPCR reaction with iQ™ SYBR^® Green supermix on a CFX384™ system. Primer pairs were designed at the 5' end () and 3' end () for 91 and 69 bp amplicons. There was no significant difference between the two primer pairs, which demonstrates superior coverage of the 5' and 3' regions of target mRNA. RFU, relative fluorescence units.