Real-time two-step RT-PCR analysis of ACTB, with and without gDNA removal step. Instead of using RNA, genomic DNA was purified from human whole blood and used as template. Different amounts of gDNA (100 ng, 10 ng, 1 ng) were spiked into the cDNA synthesis reactions using the QuantiNova Reverse Transcription Kit. Real-time PCR was performed in triplicate on an Applied Biosystems 7500 Fast Real-Time PCR System using a 1:10 cDNA dilution and the QuantiNova Probe PCR Kit. Without gDNA removal, genomic DNA contamination resulted in a strong signal. This signal was successfully eliminated by the integrated gDNA removal step (N.D. = not detected).
Real-time two-step RT-PCR analysis of various target genes and the QuantiNova Internal Control RNA (QN IC RNA). One microgram of total RNA was purified from human whole blood, and the QN IC RNA was added to the RT reaction. RNA samples with inhibitor (0.003% sodium dodecyl sulfate) and without were tested in parallel. Real-time PCR was performed in triplicate on a Bio-Rad CFX384 using a 1:100 cDNA dilution and the QuantiNova SYBR Green PCR Kit. Resulting Cq shifts for the internal control and endogenous target transcripts were comparable, demonstrating that the IC RNA can be used to detect the presence of inhibitors.
Real-time two-step RT-PCR analysis of RPS27A. Total RNA was purified from human whole blood. cDNA was then synthesized from a serial dilution of RNA (5 µg – 10 pg) using the QuantiNova Reverse Transcription Kit. Real-time PCR was performed in triplicate on a Bio-Rad CFX384 using a 1:100 cDNA dilution and the QuantiNova Probe PCR Kit. The amplification plot shows a high dynamic range of the cDNA synthesis for the whole tested range (5 µg – 10 pg) of total RNA.
