An average of ∼ 500 ng of total RNA input per reaction was diluted in water to a total volume of 25 µl, and 25 µl of master mix that contained EZ buffer, rTth enzyme, dNTPs, Rox reference dye, MnCl2 and primer / probe was added to obtain a final reaction volume of 50 µl. (journals.plos.org)
For the GAPDH RT - PCR assay, 1.25 µl of TaqMan pre-developed human GAPDH primers (Applied Biosystems) was used in a final reaction volume of 25 µl. (journals.plos.org)
The reaction mix per sample consisted of 6.33 µl H2O, 1 µl Buffer 1, 1 µl Cresol Red 10x, 0.2 µl dNTP mix (10 mM), 0.07 µl polymerase mix, 0.2 µl each forward / reverse primer (25 µM stock) and 1 µl template gDNA for a total reaction volume of 10 µl. (journals.plos.org)