M mlv reverse transcriptase first strand kit

After being treated with different compounds for 24 h, cells were harvested and washed with ice-cold phosphate buffer. Total RNA was prepared using trizol reagent (TaKaRa, Japan) according to the manufacturer's instructions. The quantity and purity of the RNA were assessed by measuring the absorbance at 260 and 280 nm. The cDNA was synthesized from total RNA (2 μg) with oligo (dT) 20 primers using an M-MLV reverse transcriptase First-Strand Kit (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer's instructions. Quantitative real-time polymerase chain reaction (RT-PCR) was performed using SYBR Green master mix, and the detection of mRNA was analyzed using an ABI StepOne RT-PCR System (Applied Biosystems, Foster City, CA, USA). Primer sequences for the reference gene β-actin and the genes of interest were shown in Table 1. Typical profile times used were the initial step, 95°C for 10 min followed by a second step at 95°C for 15 s and 60°C for 30 s for 40 cycles with a melting curve analysis. The level of target mRNA was normalized to the level of the β-actin and compared with the control. Data were analyzed using the^ΔΔ CT method.

RNA was extracted from cells using an RNeasy Total RNA system (Qiagen, Inc, Valencia, CA, USA) according to the manufacturer's protocol. The quantity and purity of the RNA were assessed by measuring the absorbance at 260 and 280 nm. The cDNA was synthesized from total RNA (2 µg) with oligo (dT) primers using an M-MLV reverse transcriptase first strand kit (Invitrogen; Thermo Fisher Scientific, Inc.). A 25 µl PCR reaction contained 4 µl cDNA, 2.5 µl buffer, 1 µl forward primer, 1 µl reverse primer, 1 µl dNTP, 1 µl Taq DNA polymerase and 14.5 µl DEPC water. The primers used were as follows: Octamer-binding transcription factor-4 (OCT-4), forward 5′-TGGGCTAGAGAAGGATGTGG-3′ and reverse 5′-CTGGGAAAGGTGTCCCTGTA-3′; sex determining region Y-box 2 (SOX-2), forward 5′-GAACGCCTTCATGGTATGGT-3′ and reverse 5′-TCTCGGTCTCGGACAAAAGT-3′; GAPDH, forward 5′-GGTTGTCTCCTGCGACTTCA-3′ and reverse 5′-TGGTCCAGGGTTTCTTACTCC-3′. The PCR reaction conditions were as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 40 sec, 61°C for 30 sec and 72°C for 10 min. The PCR products were analyzed on a 2% agarose gel with ethidium bromide. The gel images were analyzed using Image-Pro plus 6.0 software (Media Cybernetics, Inc. Rockville, MD, USA).

Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. cDNA was synthesized using total RNA (1 µg) with random primers and the MMLV reverse transcriptase First Strand kit at 37°C for 15 min, followed by 85°C for 5 sec and storage at 4°C (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed using the SYBR^®Premix Ex Taq^™kit (Takara Bio, Inc., Otsu, Japan). The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. The following primers were used: GAPDH forward, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′; kidney injury molecule 1 (KIM-1) forward, 5′-GACAACGAGCATTCCAACAA-3′ and reverse, 5′-GCTGAGGTGAAGATGGTGAAG-3′; neutrophil gelatinase-associated lipocalin (NGAL) forward, 5′-ACAAAGACCCGCAAAAGATG-3′ and reverse, 5′-GCAACCTGGAACAAAAGTCC-3′. All data was normalized using the internal control GAPDH. Fold change were quantified using the 2^−∆∆Cq method (25 (link)).