Quantification of Selenium-Binding Protein RNA

According to the manufacturer’s instruction, total RNAs were extracted from the hLE cells using Trizol reagent (Invitrogen). cDNA was obtained by incubation of the mRNA with M-MLV reverse transcriptase (Toyobo, Osaka, Japan), oligo (dT) (Toyobo) and dNTPs (Toyobo) at 42 °C for 40 min in the buffer (Toyobo). After inactivation of the enzyme by incubation at 95 °C for 5 min, polymerase chain reaction (PCR) was carried. The primer sequences are as follow: SelR: 5′-ATGTCGTTCTGCAGCTTCTTC-3′ (forward) and 5′-CACACTTGCCACAGGACAC-3′ (reverse) [50 (link)]; GAPDH: 5′-CCATGTTCGTCATGGGTGTGAACCA-3′ (forward) and 5′-GCCAGTAGAGGCAGGGATGATGTTC-3′ (reverse) [51 (link)]; Real-time PCR was performed in DNA Engine Opticon 2 (MJ Research, Watertown, MA, USA). PCR conditions for SelR were 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min; for GAPDH, the conditions were referred [51 (link)].