Myiq

RNA was isolated from ciliary ganglia using the RNeasy micro kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. RNA was reverse transcribed to cDNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Schwerte, Germany). Quantitative RT-PCR analysis was performed with the MyiQ^TM (BIO-RAD, München, Germany) and the GoTaq^® qPCR Master Mix (Promega, Mannheim, Germany) with 5 ng of cDNA template in a 12.5 μl reaction mixture. BID and BCL-XL expression levels were assessed at three different time points during development (E7, E9, and E14), with three independent biological replicates for every time point and three technical replicates. The expression levels were normalized to chicken GAPDH and results were analyzed with the comparative CT method. Data are expressed as 2^-ΔΔC_T normalized to GAPDH and presented as fold change of expression values at E7 and E9 relative to the expression value at E14.

Total RNA was extracted from frozen tissue specimens and cells using TRIzol reagent (Life Technologies, Gaithersburg, MD, USA). cDNA was then synthesized with a PrimeScript RT reagent kit with gDNA Eraser (Takara Biotechnology (Dalian) Co., Ltd., Dalian, China). The sequences of the HURP primers were designed as follows: Sense, 5′-CAT GTGAAGAAGACTTTGTTTTTGA-3′; and antisense, 5′-GGTAATCCAGGACACTGAGCA-3′. The glyceraldehyde-3 phosphate dehydrogenase (GAPDH) gene served as an internal quality RNA reference control. The sequences of the GAPDH primers were as follows: Sense, 5′-ACCACAGTCCATGCCATCAC-3′; and antisense, 5′-TCCACCACCCTGTTGCTGTA-3′. qPCR was performed in MyiQTM and iQTM5 Real-Time PCR Detection Systems (Bio-Rad Laboratories, Hercules, CA, USA) using the SsoFast™EvaGreen^®Supermix (Bio-Rad Laboratories). qPCR was performed as follows: Enzyme activation at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing at 55°C for 5 sec. All mRNA copy numbers were calculated relative to the concentration of cDNA from Human Universal Reference total RNA (Takara Bio, Inc., Shiga, Japan). The HURP copy number was then divided by the copy number of the endogenous reference (GAPDH) to obtain normalized expression values.