Caspase 3 rn00563902 m1

From retinal tissue harvested 24 h after ischemia, total RNA from ¼ of retina was extracted using a column-purification based kit (RNeasy Micro Kit, Qiagen, Hilden, Germany) according to the manufacturer's instructions. Reverse transcription was performed with 50 ng of total RNA using random primers (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Darmstadt, Germany). Real time polymerase chain reactions (RT-PCR) were done with TaqMan probe-based detection kit (TaqMan PCR universal mastermix, Applied Biosystems, Darmstadt, Germany). Following primers were used: Bax #Rn02532082_g1, Bcl-2 #Rn99999125_m1, Caspase-3 #Rn00563902_m1, NF-κB #Rn01399565_m1 (all from Applied Biosystems, Darmstadt, Germany). The PCR assays were then performed on a RT-PCR System (ABI Prism 7000, Applied Biosystems, Darmstadt, Germany) with the following cycling conditions: 95°C for 10 min, 40 cycles of 95°C for 10 sec and 60°C for 1 min. Reaction specificity was confirmed by running appropriate negative controls. Cycle threshold (CT) values for each gene of interest were normalized to the corresponding CT values for GAPDH (ΔCT). Relative gene expression in I/R injured retinal tissue either with Argon or room air was calculated in relation to the corresponding gene expression in the non-injured retinal tissue of each individual animal (ΔΔCT).

From retinal tissue harvested 24 h and 48 h after ischemia, total RNA from ¼ of retina was extracted using a column-purification based kit (RNeasy Micro Kit, Qiagen, Hilden, Germany) according to the manufacturer´s instructions. Reverse transcription was performed with 50 ng of total RNA using random primers (High Capacity cDNA Reverse Transcription Kit, Applied Biosystems, Darmstadt, Germany). Real time polymerase chain reactions (RT-PCR) were done with TaqMan^® probe-based detection kit (TaqMan^® PCR universal mastermix, Applied Biosystems, Darmstadt, Germany). Following primers were used: Caspase-3 #Rn00563902_m1, Bax #Rn02532082_g1 and Bcl-2 #Rn99999125_m1 (all from Applied Biosystems, Darmstadt, Germany). The PCR assays were then performed on a RT-PCR System (ABI Prism 7000, Applied Biosystems, Darmstadt, Germany) with the following cycling conditions: 95°C for 10 min, 40 cycles of 95°C for 10 sec and 60°C for 1 min. Reaction specificity was confirmed by running appropriate negative controls. Cycle threshold (CT) values for each gene of interest were normalized to the corresponding CT values for GAPDH (ΔCT). Relative gene expression in IR injured retinal tissue either with injection of ALF-186 or PBS was calculated in relation to the corresponding gene expression in the non-injured retinal tissue of each individual animal (ΔΔCT).