The total RNA isolated from conjunctival tissues and conjunctival fibroblasts of control subjects and patients with OCP was used to detect relative expression of HSP47, TGF-β1, and type I collagen mRNA. The principle of quantitative real-time PCR has been described elsewhere.
16 17 The quantification of transcription of real-time PCR takes advantage of the 5′ nuclease activity of
Taq DNA polymerase. Total RNA was extracted from conjunctival tissues and conjunctival fibroblasts using a RNA isolation kit (Qiagen, Valencia, CA). The primers and probe used for detection of HSP47, TGF-β1, and type I collagen are listed in
Table 1 . Each PCR reaction contained equivalent amounts of total RNA. Real-time PCR was always performed in duplicate with a PCR reagent kit (
TaqMan; Applied Biosystems, Foster City, CA). All the reactions were controlled by standards (nontemplate control and standard positive control). The quantity of mRNA was calculated by normalizing the cycle threshold (C
T) of HSP47, TGF-β1, or type I collagen to the C
T of the housekeeping gene 18S or GAPDH of the same RNA sample, according to the following formula: the average 18S or GAPDH C
T (each multiplex PCR was performed in duplicate) was subtracted from the average HSP47, TGF-β1, or type I collagen C
T. This result represents the ΔC
T, which is specific and can be compared with the ΔC
T of a calibration sample (for example control conjunctival tissues or control conjunctival fibroblasts). The subtraction of control ΔC
T from the ΔC
T of OCP samples or fibroblasts of OCP samples was referred as ΔΔC
T. The relative expression of HSP47, TGF-β1, or type I collagen (in comparison to the control) in tissues and fibroblasts obtained from conjunctiva of patients with OCP was determined by 2
−ΔΔCT. For all the probes the quencher dye was 6-carboxy-tetramethyrhodamine (TAMRA), the reporter dye was 6-carboxy fluorescein (FAM) for HSP47, TGF-β1, and type I collagen, and VIC for 18S or GAPDH.