Rt reagent kit gdna eraser

Trizol Reagent (Invitrogen, Carlsbad, CA, United States) was used to extract total RNA from OV cell lines, which was then reverse transcribed using the RT Reagent Kit gDNA Eraser (TaKaRa). Then the expression of cDNA was detected by SYBR-Green (TaKaRa) and qRT-PCR, with β-ACTIN acting as an internal reference. Primers are depicted below:
hUSP1 Forward (F): 5′-GCC​AAT​GAG​AGC​GGA​AGG​AG-3′;
hUSP1 Reverse(R): 5′-CAA​TTA​GAT​GGG​CGG​GAG​CA-3′.
hβ-ACTIN Forward (F): 5′-AGT​TGC​GTT​ACA​CCC​TTT​CTT​G-3′;
hβ-ACTIN Reverse(R): 5′-CAC​CTT​CAC​CGT​TCC​AGT​TTT-3′.

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The concentration and quality of RNA were determined using a NanoDrop one spectrophotometer (Thermo Fisher Scientific). An amount of 1 μg of RNA was used to synthesize cDNA using the RT reagent Kit gDNA Eraser (RR047A, Takara, Shiga, Japan). The corresponding cDNAs were subjected to quantitative PCR analysis using TB Green (RR820A, Takara). The used primers for qRT-PCR assays are listed in Supplementary Table S2.

Total RNA was extracted from 293T cells with Trizol Reagent (Invitrogen) according to the manufacturer’s instructions, and was reversely transcribed with RT reagent Kit gDNA Eraser (Takara, Kusatsu-shi, Japan). TB Green® Premix Ex TaqTM II (Takara) was applied to detect cDNA expression levels with β-ACTIN as the internal reference. In addition, total miRNA was isolated using the EasyPure® miRNA Kit (TransGen, Beijing, China) following the manufacturer’s protocol. The miRNA expression analysis was performed using TransScript^® Green miRNA Two-Step qRT-PCR SuperMix (TransGen) with U6 as the internal reference. Real-time PCR was performed on the LightCycler System 2.0 (Roche, Mannheim, Germany) at a temperature of 95 °C for 30 s, followed by 40 cycles with a temperature of 95 °C for 5 s, and 60 °C for 30 s. The primers are shown in Supplementary Table S1. All experiments were repeated three times and the relative gene expression was calculated by the 2^-ΔΔCt method.

Total RNA extracted from transfected cells was reverse transcribed with RT reagent Kit gDNA Eraser (TaKaRa) and detected by SYBR-Green (TaKaRa). The PCR primers were listed in Supplementary Table S2.

Total RNA extracted from MCF-7 and MDA-MB-231 cells with Trizol Reagent (Invitrogen, Carlsbad, CA, USA) were reverse transcribed with RT reagent Kit gDNA Eraser (TaKaRa). Next, SYBR-Green (TaKaRa) and qRT-PCR analysis were used for detecting cDNA expression levels and β-ACTIN was used as internal reference. Primers were shown as follows: β-ACTIN, Forward (F): 5′-TGGCACCCAGCACAATGAA-3′, Reverse(R): 5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′; hFBXO1, Forward (F): 5′-ATGGCTCACGGACAACACTT-3′, Reverse (R): 5′-TGGGGACTCGAATCTTCCCT-3′.