Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans

Yang, H-C; Chen, T-L; Wu, Y-H; Cheng, K-P; Lin, Y-H; Cheng, M-L; Ho, H-Y; Lo, S J; Chiu, D T-Y

Quantitative real-time PCR. Quantitative real-time PCR was performed by using iQ5 real-time thermal cycler (Bio-Rad) and SYBR PCR Premix reagent (Yeastern Biotechnology, Taipei, Taiwan). Primers were designed using Beacon designer software (Bio-Rad) or Primer3. 49 In brief, reaction mixtures contained 1 mg of cDNA prepared from total mRNA extract, diluted primers, and SYBR PCR Premix in PCR microcentrifuge tube. The thermal cycle procedure was as followed: 95 1C for 10 min, 40 cycles of 95 1C for 15 s and 60 1C for 1 min. Relative gene expression (g6pd: forward primer, 5 0 -atgctcttgctgttgttcacatc-3 0 ; reverse primer, 5 0 -cgctttaattcaccagacggatag-3 0 ) was normalized against threshold cycle (Ct) values of the housekeeping gene (ef-1a : forward primer, 5 0 -acattgtcgtcatcggacatgtcgactc-3 0 ; reverse primer, 5 0 -cgagaacccaggcgtacttgaaggatc-3 0 ). The relative index (2 À DDCt ) was calculated by comparing the average expression levels for control samples with the index defined as 1.00.