Corbett 65h0 machine

The SYBR Green-based RT-PCR technique was used to measure the expression of p19 and p40. The specific primers for real-time PCR were as follows: for p19, sense 5′-CTC­TGC­TCC­CTG­ATA­GCC­CT-3′ and antisense 5′-TGC­GAA­GGA­TTT­TGA­AGC­GG-3′; for p40, sense 5′-GGA­GAG­TCT­GCC­CAT­TGA­GG-3′ and antisense 5′-TCT­TGG­GTG­GGT­CAG­GTT­TG-3′; and for EF1α, sense 5′-ATA­TGG­TTC­CTG­GCA­AGC­CC-3′ and antisense 5′-GTG­GGG­TGG­CAG­GTA­TTA­GG-3′. RT-PCR was performed on a Corbett 65H0 machine (Corbett Research, Sydney, NSW, Australia), and the cycling conditions used were 95°C for 15 s and 60°C for 1 min, for 40 cycles. The expression of target gene was normalized to the housekeeping gene EF1α messenger RNA from the same sample.

To assess the expression of CTGF mRNA in the bleb area and compare its expression at different time intervals after trabeculectomy, total RNA was extracted from five different rabbit eye groups which included one intact eye without any surgery, one eye 5 hr after trabeculectomy, one eye five days after trabeculectomy with no injection, one eye five days after trabeculectomy with the administration of a BSS injection, and finally one eye five days after trabeculectomy with MMC injection. The globe sections including the trabeculectomy areas in the operated eyes were homogenized and the AccuZol total RNA extraction kit (K-3090, Bioneer, Korea) was used to extract the total RNA. The concentration/purity and the integrity of the isolated RNA were determined using a NanoDrop instrument (Thermo Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. cDNA was then generated by reverse transcription of the total RNA using a Revert Aid First Strand cDNA Synthesis Kit (#K1621, Thermo Scientific, Waltham, MA, USA). Subsequently, real-time PCR was performed utilizing a Corbett 65H0 machine (Corbett Research, Sidney, Australia) using the SinaSYBR Blue HS-qPCR Mix (#MM2171, Sinaclon, Tehran, Iran). B2M (beta-2 microglobulin) gene expression was quantified as the reference gene. Real-time PCR primers sequences used in this study are listed in Table 1.