Total RNA isolated from conjunctival fibroblasts was used to determine the relative induction of MIF in IL-1–, TNF-α–, and TGF-β1–treated fibroblasts by real-time PCR, as described in earlier studies.
11 12 25 26 The primers and probe used for detecting mRNA for MIF are as follows: forward, AGC CCG GAC AGG GTC TAC A; reverse, GGA GTT GTT CCA GCC CAC A; probe (
TaqMan; Applied Biosystems, Foster City, CA), FAM-CAA CTA TTA CGA CAT GAA CGC GGC CA-TAMRA. Each PCR reaction contained equivalent amounts of total RNA. Real-time PCR was performed in duplicate with a kit used according to the manufacturer’s recommendation (
TaqMan One-step RT-PCR Master Mix Reagents kit; Applied Biosystems). All the reactions were controlled by standards (nontemplate control and standard positive control). While extracting total RNA from conjunctival fibroblasts, we routinely used DNase to prevent DNA contamination. When real-time PCR was performed without adding reverse transcriptase, no PCR product was detected either for the target gene or the housekeeping gene, eliminating any DNA contamination. The quantity of mRNA was calculated by normalizing the C
T (threshold cycle) of MIF to the C
T of the 18S housekeeping gene’s ribosomal RNA in the same sample, according to the following formula: The average 18S C
T (each multiplex PCR was performed in duplicate) was subtracted from the average MIF C
T; the result represents the ΔC
T. This ΔC
T is specific and can be compared with the ΔC
T of a calibration sample (for example, control conjunctival fibroblasts). The subtraction of control ΔC
T from the ΔC
T of OCP fibroblasts is referred as ΔΔC
T. The relative quantification of expression of MIF (in comparison to control) was determined by using 2
−ΔΔCT . For all the probes, the quencher dye was 6 carboxy-tetramethyrhodamine (TAMRA). The reporter dye was 6-carboxyfluorescein (FAM) for MIF and VIC for 18S. The relative expression of MIF was also detected as described earlier, using total RNA extracted from control conjunctival tissues and from selected conjunctival tissues of patients with OCP.