Real-time, two-step RT-PCR analysis of β-actin with (+RT) or without (-RT) reverse transcriptase. cDNA was synthesized from 100 ng total RNA, and real-time PCR was performed in duplicate on the LightCycler 2.0 using the QuantiTect SYBR® Green PCR Kit. The β-actin-specific primers detected both mRNA and genomic DNA sequences. Control reactions with no template were also performed (green). [A] The RT step was carried out using the QuantiTect Reverse Transcription Kit. The red, flat –RT plot indicates efficient removal of residual genomic DNA. [B] The RT step was carried out using a kit from Supplier I (Enzyme SIII). The purple –RT plot indicates amplification of residual genomic DNA.
Real-time, two-step RT-PCR of a target located at the 5' region of the mouse dystrophin gene (about 12.5 kb upstream of the poly-A site). Total RNA purified from mouse testis was reverse transcribed with the QuantiTect Reverse Transcription Kit as well as with reverse transcriptases from Supplier I and Supplier R. Identical volumes of triplicate reverse-transcription reactions were analyzed by real-time PCR on the LightCycler system. The error bars show the standard deviation for each set of triplicates. Compared with the other two kits, the QuantiTect Reverse Transcription Kit generated much higher amounts of cDNA (indicated by the lower CT values) and provided greater reproducibility in real-time RT-PCR (indicated by the smaller error bars). RFU: relative fluorescence units.
(Data kindly provided by Dr. Andrej-Nikolai Spiess, Department of Molecular Andrology, University Hospital Hamburg, Germany).
Real-time, two-step RT-PCR analysis of [A] TGFB2 (low expression) and [B] IL8 (higher expression). Total RNA was purified from human whole blood using the PAXgene Blood RNA system. cDNA was then synthesized from 1 µg RNA using the QuantiTect Reverse Transcription Kit, a kit from Supplier AII, or a kit from Supplier I. Real-time PCR was performed in duplicate on the ABI PRISM 7900 using the QuantiTect Probe PCR Kit and QuantiTect Gene Expression Assays for TGFB2 or IL8. The CT values for TGFB2 were lowest with the QuantiTect Reverse Transcription Kit, demonstrating that even low-abundance genes can be efficiently reverse transcribed and sensitively detected in real-time PCR.
Genomic DNA removal and cDNA synthesis take only 20 minutes with the QuantiTect Reverse Transcription Kit. The procedure is fast and convenient since both reactions are run using the same incubation temperature and are set up using master mixes. In contrast, the procedure for the kit from Supplier I is much longer and requires more "hands-on time" due to additional pipetting steps and frequent changes in incubation temperature.