Superscript II versus Thermoscript in cDNA synthesis with random hexamers. Twenty narograms, 2 ng, 200 pg, and 20 pg of total tracheal RNA (i.e., RNA equivalent to ∼2000, 200, 20, and 2 cells) were used in cDNA synthesis with Superscript II or Thermoscript as described in Methods using random hexamers. Aliquots of RT reactions were PCR-amplified with the T4 Mix for 10, 15, 20, and 25 cycles as described in Methods, and 0.05% of each reaction was used in real-time PCR to quantify the levels of GAPDH. The number of preamplification cycles in the multiplex step was consistently and inversely related to cycle threshold for GAPDH in the real-time PCR step. Superscript II was more efficient than Thermoscript in random primed cDNA synthesis and this resulted in lower Cts for GAPDH. Multiplex preamplification with T4 Mix was also efficient and linear and produced slopes of −1.0 (at least for GAPDH).
