FIG. 4.
Comparison between standard single-round and nested RT-PCR and one-step and two-step real-time Q-RT-PCR. All cDNA reactions were programmed with 1 μl of total RNA and were analyzed side-by-side in the indicated RT-PCR assays. (A) Summary chart of RNA dilutions and the subsequent classification (+ or −) by each assay. For the Q-RT-PCR assays, the corresponding triplicate Ct values associated with each RNA dilution are shown. For a result in the Q-RT-PCR assay to be considered positive, at least two of the three triplicate samples had to register a Ct of <40. (B) One percent agarose gel showing the amplification products present in 5 μl of reaction mix for each of the RNA dilutions after standard single-round and nested RT-PCR. The corresponding sizes of the expected DNA amplicons are 185 and 150 nucleotides, respectively.