Total RNA was purified from human breast, liver, or kidney FFPE samples with the RNeasy FFPE Kit. Duplex, real-time RT-PCR was performed with (+RT) or without (–RT) the reverse transcription step for a range of different targets with different expression levels using QuantiFast Probe Assays (FAM labeled) and [A] the QuantiFast Probe RT-PCR Plus Kit or [B] an alternative kit for multiplex PCR which does not include an integrated gDNA removal step. All reactions were performed on the ABI StepOnePlus cycler. [A] High CT values in the –RT samples indicate no amplification and the absence of genomic DNA contamination. Therefore the CT values obtained in the +RT samples reflect reliable and accurate detection of RNA. Targets 12 (TUSC2) and 13 (EZH2) are not expressed. These results show that the QuantiFast Probe RT-PCR Plus Kit efficiently removes genomic DNA. [B] Similar CT values in both the –RT and +RT samples indicate that contaminating genomic DNA is being detected, making these results unreliable.
Total RNA (10 ng to 100 pg) from HeLa cells with a RIN value of 4.7 was amplified and detected in one-step RT-PCR on the ABI 7500 Fast cycler using [A] a QuantiFast Probe Assay for the human CENPM gene in combination with the QuantiFast Probe RT-PCR Plus Kit (amplicon size: 76 bp) or [B] a predesigned assay and kit from Supplier AII (amplicon size: 111 bp). Results from QuantiFast Reagents showed lower CT values and higher sensitivity compared to results using reagents from Supplier AII.
With the QuantiFast Probe RT-PCR Plus Kit procedure, genomic DNA contamination is effectively eliminated through an optimized incubation step. After addition of the master mix for real-time RT-PCR, reverse transcription and, subsequently real-time PCR, take place in the same tube.