Fig. 4

Quantification of protein and mRNA levels of androgen receptor (AR, Ar) in mouse anterior prostate. a Representative immunohistochemistry showing AR protein expression (brown staining) and differential pathology in prostate of WT and pePTENKO mice. b Quantification of AR protein by Western blot in anterior prostates of WT (n = 2) and pePTENKO (n = 4) mice, using β-actin (ACTB) as a loading control to determine relative protein levels. c Relative quantification of Ar by ΔΔCq and Pfaffl analyses using Actb as the reference gene and WT as the control. d qPCR of Ar and Actb in WT (blue curves, n = 7 in duplicate) and pePTENKO (red curves, n = 5 in duplicate) mice. e Comparison of theoretical versus determined quantification of serial dilutions of plasmids containing Ar or Actb amplicons, by either traditional standard curves (blue diamonds and line) or use of AccuCal and RealCount (red squares and line). f Absolute mRNA copy number quantification of Ar and Actb reference gene in the anterior prostate of WT (n = 7) and pePTENKO (n = 5) mice as determined by RT-qPCR with AccuCal calibrators and RealCount software (AC) or standard curves (Std). g Absolute quantification of a number of reference genes using AccuCal and RealCount for both WT (n = 7) and pePTENKO (n = 5) mice. h Comparison of relative quantification methodologies for Ar. ΔΔCq and Pfaffl analyses used either Actb or Hmbs as the reference gene and WT as the control, and AccuCal (AC) and standard curve (Std) absolute values were expressed in a relative manner to WT as the control. All graphed data is displayed as mean ± SEM. **** p <0.0001, ** p = 0.001–0.01, * p = 0.01–0.05 by independent two sample t-test between expression of androgen receptor or β-actin mRNA or protein in pePTENKO mice compared to WT mice, or between relative expression methods compared with ΔΔCq and Pfaffl performed using Actb as reference gene