Ready to go t primed first strand kit

RNA was extracted using RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacture's instruction. cDNA was synthesized using Ready-To-Go T-primed first strand kit (Invitrogen) or OmniScript Reverse Transcriptase Kit (Qiagen) or Biozym cDNA synthesis kit including random hexamers (Biozym Scientific GmbH, Oldendorf, Germany). Quantitative real-time PCR was performed with StepOne™ Real-Time PCR System (Applied Biosystem, Darmstadt, Germany). In each reaction, 2 μl cDNA, 0.4 μl reverse and forward primers, 5 μl SYBR Green mix (Bioline, Taunton, USA) and 2.2 μl DEPC-H₂O were mixed in one well in a 96-well plate and centrifuged briefly. After the initial denaturation step at 95 °C for 15 min, PCR reaction was cycled for 40 times with denaturation at 95 °C for 30 s and annealing-extension temperature at 65 for 30 s. Relative expression was calculated following 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)). Primers are listed in Table S1. Statistical analysis was done using REST software and if p < 0.05, it is reported as statistically different (Pfaffl et al., 2002 (link)). The graphs were generated using GraphPad Prism Software version 7 (GraphPad Software Inc., California/USA).

RNA was extracted using RNeasy mini kit (Qiagen, Germany) according to the manufacture's instruction. cDNA was synthesized using Ready-To-Go T-primed first strand kit (Invitrogen, Germany). qRT-PCR was performed with StepOne^TM Real-Time PCR System (Applied Biosystems, Germany). The primers were standardized and the efficiency was tested before performing real-time PCR; primers with an efficiency above 90% were used in this study. In each reaction, 1 μl cDNA, 1 μl reverse and forward primers (1:1), 4 μl EvaGreen mix and 14 μl DEPC-H₂O were mixed in one well in a 96-well plate and centrifuged briefly. After the initial denaturation step at 95°C for 15 min, PCR reaction was cycled for 40 times with denature at 95°C for 30 s and annealing-extension temperature at 65°C for 30 s. Relative expression was calculated following the 2(-Delta Delta C(T)) method (55 (link)). Primers are listed in Table 5.
Table 5.

Primers for real-time RT-PCR

Gene	Primer	Sequence (5′–> 3′)
Pax6	Pax6-qF	GTTCTTCGCAACCTGGCTA
	Pax6-qR	TGAGCTTCATCCGAGTCTTCT
TNF-α	TNFα_2F	CACCACGCTCTTCTGTCT
	TNFα_2R	GGCTACAGGCTTGTCACTC
IL-1β	IL-1β_FW	CAACCAACAAGTATTCTCCATG
	IL-1β_RV	GATCCACACTCTCCAGCTGCA
Tubea	TubeaF	CCAGATGCCAAGTGACAAGA
	TubeaR	GTGGGTTCCAGGTCTACGAA