Quantification of Keap1, Nrf2, GPX1, NQO1, HO1, GCLM, and GAPDH mRNA Expression Using Quantitative Real-Time PCR

Total RNA was extracted from the jejunum sample stored at −80 °C with RNAiso Plus (D9108B; Applied TaKaRa, DaLian, P.R. China) according to the manufacturer’s instructions. The purity and concentration of the RNA was assessed using an Eppendorf Biophotometer (RS323C; Eppendorf Aktien Gesellschaft, Hamburg, Germany) at an absorbance ratio of 260:280 nm (a range of 1.8 to 2.0 indicates a pure RNA sample). The RNA integrity was verified using agarose gel electrophoresis. Total RNA was reverse transcribed to cDNA using a Reverse Transcription System kit (Prime-Script RT Master Mix, RR036A; Applied TaKaRa).

A total volume of 20 µL of the PCR mixture containing 10-µL SYBRY Premix Ex Taq II, 0.4-µL DyeII (SYBRY Premix Ex Taq-TIi RNaseH Plus, DRR420A; Applied TaKaRa), 0.4 µL of both forward and reverse primers, and 2-µL cDNA (<100 ng) was used for the quantitative real-time PCR (qRT-PCR) analysis. The optimized qRT-PCR protocol included an initial denaturation step at 95 °C for 30 s followed by 43 cycles at 95 °C for 5 s, 60 °C for 34 s, 95 °C for 15 s, 60 °C for 60 s, and 95 °C for 15 s. The qRT-PCR reactions were conducted in an AB 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). The amounts of relative expression of Keap1, Nrf2, GPX1, NQO1, HO1, GCLM, and GAPDH mRNA were expressed and calculated as being equal to 2^−ΔΔCt (Livak and Schmittgen, 2001). The analysis was repeated 3 times for each sample. The primer sequences and production lengths are presented in Table 2.

Sequence of primers for real-time PCR

¹ Nrf2 = nuclear factor erythroid 2–related factor 2; Keap1 = Kelch-like erythroid cell-derived protein with CNC homology (ECH)–associated protein 1; GPX1 = glutathione peroxidase 1; HO1 = hemeoxygenase 1; NQO1 = quinone oxidoreductase 1; GCLM = modifier subunit of glutamate-cysteine ligase; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

²F = forward primer; R = reverse primer.