Goscript reverse transcriptase kit

Total RNA was extracted from murine liver tissue using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA quality and quantity were assessed using BioSpectrometer (Eppendorf). cDNA synthesis from 1 μg RNA was performed using the GoScript^™ Reverse Transcriptase Kit (Promega). Next, quantitative real-time PCR was performed in Rotor Gene 6000 (Corbett Research) using GoTagR qPCR Master Mix (Promega) according to manufacturer’s instructions with the following primers:
C_t values were normalized to housekeeping gene HPRT1 followed by normalization of ΔC_t values to average ΔC_t of db/+ control group.

RNA-extraction was performed with QIAGEN RNeasy Mini Kit, and cDNA was generated with GoScript Reverse Transcriptase kit (Promega) according to the manufacturers’ instructions. RT-PCR was performed with GoTaq G2 Green Master Mix (Promega) and gene-specific primers. Amplicons were fractionated on 2% TBE gel (Life Technologies) supplemented with 0.01% GelRed (Biotium). For qPCR, 2 μL of 1:10-diluted cDNA was added to 8 μl of 5 x HOT FIREPol Evagreen qPCR Supermix (SolisBiodyne). Raw gel images can be found in Source data 1. RT-qPCR was performed with a LightCycler480 II (Roche). Relative gene expression was determined with the comparative CT method. Genes with a median CT value of more than 33 cycles and a difference of less than 3.3 cycles to the template control (H₂O) were defined as not detectable. Sequences of all primers used in this study are listed in Supplementary file 3.

For miRNAs, the isolated total RNAs were reverse transcribed into complementary cDNAs and qRT-PCR analysis using Bulge-Loop miRNA qRT-PCR kit (Ribobio, Guangzhou, China) with U6 as an internal control. Commercially available primers for Has-miR-200a-3p (miRA0000682), Has-miR-200b-3p (miRA0000318), Has-miR-200c-3p (miRA0000617), Has-miR-141-3p (miRA0000432), Has-miR-429 (miRA1000755) and U6 (miRAN0002-1-100) were bought from the Ribobio (Guangzhou, China). For mRNAs, the isolated total RNAs were reversely transcribed into complementary cDNAs using GoScript™ Reverse Transcriptase Kit (Promega), and then qRT-PCR analysis was performed using SYBR Green PCR Master Mix Reagent Kit (TaKaRa), with GAPDH used as an internal control. The sequences of primers are as follows: Bcl2, 5′-GGTGGGGTCATGTGTGTGG-3′ and 5′-CGGTTCAGGTACTCAGTC ATCC-3′; Bax, 5′-CCCGAGAGGTCTTTTTCCGAG-3′ and 5′-CCAGCCCATGATGG TTCTGAT-3′; HDAC4, 5′-CTTGTGGGTTACCTGGCTCA-3′ and 5′-TCCAACGAGCTCC AAACTCC-3′; GAPDH, 5′-AAGCCTGCCGGTGACTAAC-3′ and 5′-GCGCCCAATACGA CCAAATC-3′. Data were presented as values calculated by 2^−△△Ct method.

For RNA extraction, cells were cultured in 6-well plates at 30–40% confluency. After 48 h of doxycycline induction, RNA purification was performed using TRIzol reagent (Invitrogen). 1.5 μg RNA with Oligo(dT)₁₈ and random hexamer primers were used for cDNA synthesis. cDNA synthesis was conducted by Maxima H Minus First Strand cDNA Synthesis Kit with DNase (Life Technology) or GoScript^™ Reverse Transcriptase kit (Promega) according to manufacturer’s instructions. cDNA samples were stored at −80 °C and used for real-time qPCR analysis within one week.

At optical densities of 0.1, 0.4, 1, and 3, further growth was halted and intracellular RNA was stabilized by the addition of a 0.4 volume of stop solution (5% [vol/vol] phenol [pH 4.3]–ethanol) and incubation on ice for 30 min. A volume of cells equivalent to a cell density of 1 ml of culture at an A₆₀₀ of 1 was harvested. Cells were pelleted by centrifugation at 3,220 × g for 10 min at 4°C. Cells were resuspended in Tris-EDTA buffer (TE; pH 8.0) containing 50 mg/ml lysozyme. RNA was extracted using an SV total RNA isolation kit (Promega, WI, USA). RNA was DNase treated using a Turbo DNase kit (Invitrogen). RNA integrity was assessed on a HT gel (77 (link)) and quantified using a DeNovix DS-11 spectrophotometer and A₂₆₀.
RNA (400 ng) was reverse transcribed into cDNA using random oligonucleotides and a GoScript reverse transcriptase kit (Promega). The cDNA was probed using primers specific to target genes in the StepOnePlus real-time PCR system as described above. The hemX gene was used as a control gene (with unchanged expression assumed), and the changes in expression of the other genes were calculated against hemX expression.
The oligonucleotide primer pairs used in qPCR are listed in Table S1.

Isolation of mRNA from HeLa cells was performed with NucleoSpin RNA kit (Macherey‐Nagel; 740,955.250) before generating cDNA with Promega GoScript Reverse Transcriptase kit (Cat# A5003) and adjusting to 10 μg/ml. PCR was performed with SYBR Green PCR mix (applied biosystems; Cat# 4309155), 0.6 μM of each primer, and 30 ng cDNA for 40 rounds at 95°C melting and 60°C elongation steps. Actin and GADPH were used as housekeeping genes for normalization. Primer sequences are listed in the Reagent Tools table.

Total RNA was extracted using TRI reagent (Euromedex) followed by RNAse free-RQ1 DNAse (Promega) and proteinase K (Sigma) treatments. 1 μg of whole cell RNA was reverse transcribed with random hexamer primers using Go Script Reverse Transcriptase kit (Promega) at 42°C for 60 min. mRNA expression was performed using the IQ Custom SYBR Green Supermix (Bio-Rad) qPCR. The relative quantification of gene expression was performed using the standard curve method with triplicates for each data point. For Northern blotting analysis, 5 µg of total RNA was fractionated by electrophoresis on a 6% polyacrylamide/7 M urea denaturing gel. Electro transferred onto a nylon membrane (Amersham Hybond-N, GE Healthcare) followed by UV crosslinking (Stratalinker). Hybridizations were carried out with 5′-end ³²P-labeled-DNA oligonucleotide probes. Membranes were incubated overnight at 50°C in 5X SSPE, 5X Denhardt's, 1% SDS, 150 μg/mL yeast tRNA and washed twice in 0.1% SSPE, 0.1% SDS for 15 min at room temperature. a Typhoon Biomolecular Imager (Amersham) and visualized using Multi Gauge V3.0 software. Radioactive signals were revealed using a Typhoon Biomolecular Imager (Amersham) and visualized using Multi Gauge V3.0 software. Primers are listed in Supplementary file 4.