For relative quantitation normalized to GAPDH, an endogenous control, standard curves were prepared for both MMPs and GAPDHs and TIMPs and GAPDHs. Standard curves for MMP-1, -2, -14, -15, -16, -19, and -24 and their counterpart GAPDHs were constructed using a serial dilution of total RNA with a range of 0.2, 0.5, 1, 2, 5, 10, and 20 ng
(Fig. 1A) . Standard curves for MMP-3, -11, and -17 and their counterpart GAPDHs were constructed with a serial dilution of total RNA with a range of 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 ng
(Fig. 1B) . Standard curves for MMP-12 and its counterpart GAPDH were constructed with a serial dilution of total RNA of the cultured CBSM cells, using a range of 5, 10, 20, 40, and 80 ng
(Fig. 1C) . Standard curves for TIMPs and their counterpart GAPDHs were constructed with a serial dilution of total RNA of the cultured trabecular meshwork endothelial cells, with a range of 0.2, 0.5, 1, 2, 5, 10, and 20 ng for TIMP-1, -2, and -3 and with a range of 2.5, 5, 10, 20, 40, and 80 ng for TIMP-4
(Fig. 1D) . Using three C
T levels (i.e., threshold cycle, defined as the fractional cycle number at which the amount of amplified target reaches a fixed threshold) for each input amount of dilution, standard curve equations for MMPs, TIMPs, and their counterpart GAPDHs were generated
(Table 3) . The comparative ΔC
T method was used for relative quantitation of those genes whose absolute value of the slope of log input versus ΔC
T was less than 0.1
(Table 3) . The relative standard curve method was used for genes that did not satisfy the criteria. The coefficient of variation (CV) was used for data reproducibility. Reproducibility of standard curves of GAPDHs obtained from those cell lines used for different genes was as follows: 4.45 to 5.57 of CV (%) for HT-1080, 2.27 to 3.61 of CV (%) for CCF-STTG1, and 0.98 to 2.38 of CV (%) for cultured TM endothelial cells. All triplicates of C
Ts used for the experiment showed much less than 1% CV
(Table 3) . According to the manufacturer’s protocol, triplicates showing less than 2% CV are accepted for this assay (ABI).