Favorprep total rna purification mini kit

Total RNA was obtained from the cultured HT22 neurons using FavorPrep^TM Total RNA purification mini kit according to the manufacturer’s protocol (Favorgen Biotech Corp., Pingtung, Taiwan). Reverse transcription and qPCR were performed using the SensiFAST Hi-ROX One-Step mastermix (BioLine, London, UK) in StepOnePlus Real-Time PCR instrument (Applied Biosystems, Foster City, CA, USA). The gene expression levels of Nuclear factor erythroid 2-related factor 2 (Nrf2), Heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax) were calculated according to the relative quantitative 2^−ΔΔCt method normalized with Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene. The PCR primer sequences used were as follows:
Nrf2:  Forward 5′-TCTCCTCGCTGGAAAAAGAA-3′  Reverse 5′-AATGTGCTGGCTGTGCTTTA-3′HO-1:  Forward 5′-AGG TGT CCA GAG AAG GCT T-3′  Reverse 5′-ATC TTG CAC CAG GCT AGC A-3′NQO1:  Forward 5′-ATC CTT CCG AGT CAT CTC TA-3′  Reverse 5′-CAA CGA ATC TTG AAT GGA GG-3′Bcl-2:  Forward 5′-TCGCAGAGATGTCCAGTCAG-3′  Reverse 5′-ATGCCGGTTCAGGTACTCAG-3′Bax:  Forward 5′-CACAGCGTGGTGGTACCTTA-3′  Reverse 5′-TCTTCTGTACGGCGGTCTCT-3′GAPDH:  Forward 5′-TCACCACCATGGAGAAGGC-3′  Reverse 5′-GCTAAGCAGTTGGTGGTGCA-3′

A375, B16F10, A2058, and MC38 cells were cultured in 12-well plates for 24 h, and total cellular RNA was extracted from the cells using a FavorPrepTM Total RNA Purification Mini Kit according to the manufacturer’s instructions (Favorgen, Ping Tung, Taiwan). The quantity and quality of the isolated RNA were determined using a NanoDrop^® ND-1000 Spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE, USA). Single-stranded cDNA was synthesized from 1 μg of total RNA using a Revert Aid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions (Thermo Scientific, Rockford, IL, USA). Quantitative real-time PCR was performed using the LightCycler^® 480II thermal cycler coupled with SYBR Green chemistry (Roche Applied Science, Penzberg, Germany). The amplification protocol consisted of an initial denaturation at 95 °C for 5 min, followed by 55 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 10 s. Amplification primers for EGFR included 5′-CCCACTCATGCTCTACAACCC-3′ (forward) and 5′-TCGCACTTCTTACACTTGCGG-3′ (reverse).

Total cellular RNA was extracted from the cells using a FavorPrepTM Total RNA Purification Mini Kit according to the manufacturer’s instructions (Favorgen, Ping-Tung, Taiwan). Following isolation, the quantity and quality of the RNA were determined using a NanoDrop^® ND-1000 Spectrophotometer (ND-1000, NanoDrop Technologies, Wilmington, DE, USA). Single-stranded cDNA was synthesized from 1 μg of total RNA using a Revert Aid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions (Thermo Scientific, Rockford, IL, USA). qRT-PCR was performed using a LightCycler^® 480II machine coupled with SYBR Green chemistry (Roche Applied Science, Penzberg, Germany). In terms of qRT-PCR settings, initial denaturation was performed at 95 °C for 5 min, followed by amplification at 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 10 s for 45 cycles. The cDNA obtained was amplified with the primers listed in Table 1.