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Fig. 3 | BMC Biotechnology

Fig. 3

From: A simple, accurate and universal method for quantification of PCR

Fig. 3

Quantification of CD40, IL7R and GAPDH in PBMCs stimulated with 0–1x PMA/ionomycin. a Absolute quantification of CD40, IL7R and GAPDH in PBMCs stimulated with 0, 0.25x, 0.5x and 1x PMA/ionomycin (20 ng ml−1 PMA, 500 ng ml−1 ionomycin; PMA/I) by RealCount software following qPCR using AccuCal-D calibrators. b Relative expression levels of CD40 and IL7R in PBMCs stimulated with 0, 0.25x, 0.5x and 1x PMA/ionomycin (20 ng ml−1 PMA, 500 ng ml−1 ionomycin). The hatched bars are relative expression levels determined by ΔΔCq using GAPDH as the reference gene and no PMA/ionomycin as the control sample, solid bars are relative expression levels determined by Pfaffl analysis, using GAPDH as reference gene, unstimulated cells as controls and individual efficiency values calculated by RealCount software, and the checkered bars are quantified by RealCount software following inclusion of AccuCal-D in the same PCR run, and expressed relative to the no PMA/ionomycin control. c Representative overlay graphs from flow cytometry showing relative measurement of CD40 and IL7R in the same population of PBMCs stimulated with 0 (red), 0.25x (blue), 0.5x (green) and 1x PMA/ionomycin (20 ng ml−1 PMA, 500 ng ml−1 ionomycin; orange) as in (a). **** p < 0.0001 relative to respective no PMA/ionomycin control, n = 4

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