Linearity and sensitivity of two-step RT-PCR for quantification of expression of multiple genes using 2.0 ng of total RNA from human trachea. For each of 11 representative genes, 0.05% of the multiplex PCR product, that is, an equivalent of 1 pg of starting RNA was used in the real-time PCR step. For genes of varying abundance, the number of preamplification cycles in the multiplex step was consistently and inversely related to the corresponding cycle thresholds in the real-time PCR step. At 25 cycles of amplification, 2.0 ng of starting RNA would allow reliable quantification of at least 1000 genes in an optimal cycle threshold range for detection.