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An RNA template mix suitable for use in different assays was prepared by mixing total RNA from cultured human leukemia and cervix carcinoma cells with in vitro transcripts. Various multiplex assays were performed using the QuantiTect Multiplex RT-PCR Kit in combination with primers and TaqMan® probes. Triplicate reactions were run on the Applied Biosystems® 7500.
Results and DiscussionHigh PCR efficiencies and highly accurate standard curves were obtained in all the multiplex assays (Figure 1 shows data from one of the assays). Triplicate amplification plots closely overlapped each other, demonstrating highly reproducible CT values.
Conclusions
With the QuantiTect Multiplex RT-PCR Kit, no optimization is required to achieve high PCR efficiencies and highly reproducible CT values in multiplex assays, regardless of the combination of targets analyzed.
The high PCR efficiencies achieved with the kit allow precise relative quantification of gene expression by the ΔΔCT method.
QIAGEN News 2006, 49, which contains the additional triplex data described in this Application Forum, can be downloaded at www.qiagen.com/goto/QNews200649.
QuantiTect Multiplex RT-PCR Kits are intended for research use. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.
Trademarks: QuantiTect® (QIAGEN Group); Applied Biosystems® (Applera Corporation or its subsidiaries); TaqMan® (Roche Group).
