Beyofasttm sybr green qpcr mix

Total RNA was isolated from cells or human tissues using Trizol reagent (Invitrogen, Carlsbad, CA). The cDNA was generated from each 2 ug RNA sample using All-in-One™ First-Strand cDNA Synthesis Kit (GeneCopoeia, Cat. No QP006). The temperature protocol for reverse transcription was: 28 ºC for 2 min, 42 ºC for 30 min, and 85 ºC for 5 min. cDNA was subjected to initial denaturation at 94˚C for 2 min, followed by 40 cycles at 95˚C for 15 s and 60–68 ºC for 30 s, after the end, it starts from 65˚C to 95 ºC, rising by 0.5 ºC per second, using the BeyoFastTM SYBR Green qPCR Mix (Bio-Rad, Cat. No 1708882AP). The primers used in qRT-PCR are shown in Additional file 3: Table S2. The method was used for relative quantification and the quantifications were normalized by taking GAPDH as an internal reference. All experiments were repeated in three times.

According to the manufacturer’s fractions, the Cytoplasmic & Nuclear RNA Purification Kit (Norgen, Cat. No 21000, Canada) was used to prepare the nuclear and Cytoplasmic RNA fraction from 1 × 10⁶. After using the manufacturer’s instructions to isolate RNAs, RNA concentrations of nuclear and cytoplasmic were measured at OD260, and their purity was calculated with OD260/OD280. Following the manufacturer’s guidelines, a All-in-One™ First-Strand cDNA Synthesis Kit (GeneCopoeia, Cat. No QP006) and BeyoFastTM SYBR Green qPCR Mix (Bio-Rad, Cat. No 1708882AP) were applied to determine the expression level of DIO3OS in the nucleus (U6 as an internal reference) and cytoplasm (GAPDH as an internal reference). The primer sequences are performed in Additional file 3: Table S2.