Semi-Quantitative RT-PCR and Quantitative RT-PCR

RNA was extracted by TRIzol (15596-018, Invitrogen) according to the manufacturer’s instruction. 2 μg RNA of each sample was reversed to complementary DNA by First-strand cDNA Synthesis System (Thermo Fisher Scientific, catalog No. K1622), and 0.2 μg cDNA was used as a template to perform PCR. Quantitative PCR were performed on CFX96 Real-Time PCR System (Bio-Rad, United States) and the amplifications were done using the SYBR Green PCR Master Mix (Gene Star, China). The primer pairs were: CREB1, 5′-ATTCACAGGAGTCAGTGGATAGT-3′ (forward) and 5′-CACCGTTACAGTGGTGATGG-3′ (reverse); ATF1, 5′-AGGACTCATCCGACAGCATAG-3′ (forward) and 5′-TTCTGCCCCGTGTATCTTCAG-3′ (reverse); CBS, 5′-GTCA GACCAAGTTGGCAAAGT-3′ (forward) and 5′-CACCCCGA ACACCATCTGC-3′ (reverse); CTH, 5′-AAAGACGCCTCCT CACAAGG-3′ (forward) and 5′-AAGGCAATTCCTAGTG GGATTTC-3′ (reverse); CDO1, 5′-TCCATTGGCTTACATCG AGTAGA-3′ (forward) and 5′-CCCGAAGTTGCATTTGGAGT-3′ (reverse); CSD, 5′-AGAAGCGGGAAGGGTTTGAG-3′ (forward) and 5′-CCTTTCGTGGTAATCTGGACTC-3′ (re verse); GCLC, 5′-GGAGACCAGAGTATGGGAGTT-3′ (fo rward) and 5′-CCGGCGTTTTCGCATGTTG-3′ (reverse); GCLM, 5′-CATTTACAGCCTTACTGGGAGG-3′ (forward) and 5′-ATGCAGTCAAATCTGGTGGCA-3′ (reverse); GSS, 5′-GGGAGCCTCTTGCAGGATAAA-3′ (forward) and 5′-GAATGGGGCATAGCTCACCAC-3′ (reverse); β-actin, 5′GAC CTGACTGACTACCTCATGAAGAT-3′ (forward) and 5′-GTCACACTTCATGATGGAGTTGAAGG-3′ (reverse). The fold changes of gene expression are normalized to β-actin.