Trireagent

The level of p53 mRNA in the colon tissue was determined by real time PCR. Total RNA was extracted by using Tri Reagent according to the manufacturer’s instructions (MP Bio). according to the manufacturer’s protocol. The yield and purity of RNA was quantified by nano spectrophotometry at 260 and 280 nm. Total RNA (1 μg/ml) were reverse transcribed to to cDNA by using First Strand cDNA Synthesis (Gbioscience Cat. 786–814) according to the manufacturers. A real time assay was performed according to Light Cycler-Fast Start DNA Master^PLUS SYBR Green I (Roche Applied Science Light Cycler® Diagnostic, USA) Primer sequences were: p53 Forward 5′CCG ACC TAT CCT TAC CAT CATC 3′, Reverse 5′TTC TTC TGT ACG GCG GTC TTC TC 3′. β-actin Forward 5′TGG AAT CCT GTG GCA TCC ATG AAA C 3′, Reverse 5′TAA AAC GCA GCT CAG TAA CAG TCC G 3′.
The amplification program consisted of the following steps: pre-incubation at 95 °C was 10 min, followed by 40 cycles of denaturation was 95 °C was 10 s, annealing was 50 °C for 20 s and extension was 25 s. Each gene expression was normalized with β-actin mRNA content [22 (link)]. The relative induction of mRNA expression comparative Ct was calculated using ^ΔΔCt was calculated using following formula: (Ct target - Ct β-actin) treatment - (Ct target - Ct β-actin) non treatment.

Total RNA extraction from skelatal muscle tissue or C2C12 mouse myoblasts was done using TRI Reagent (Millipore-Sigma #T9424). Muscle tissue samples were flash-frozen in liquid nitrogen until further processing. Tissues were resuspended in 600 μL of TRI Reagent, then homogenized on Lysing Matrix D 2 mL tubes (MP Biomedicals) on a BeadBug homogenizer (Benchmark Scientific). For both skeletal muscle and C2C12 cells, total RNA was purified using the Direct-zol RNA MiniPrep (Zymo Research #R2052).

Total RNA was extracted from the tissues using TRI Reagent (Sigma-Aldrich) as per the manufacturer’s instructions. Tissue sample of 30–50 mg was homogenized in 1 ml of TRI Reagent using lysing matrix D beads (1.4 mm) in FastPrep homogenization system (MP Biomedicals). RNA pellet was dissolved in diethyl pyrocarbonate (DEPC) treated nuclease-free water and stored at –80 °C until further use. RNA concentration and purity were analysed in Denovix UV-V is spectrophotometer (Denovix). To remove the residual genomic DNA, 1 µg total RNA was treated with 1U of DNase I, RNase free (Thermo Scientific) following manufacturer’s protocol. DNase-treated total RNA (1 µg) was reverse-transcribed using RevertAid First Strand cDNA Synthesis kit with oligo-dT primer (Thermo Scientific) as per the manufacturer’s instructions.

Total RNA was extracted using TriReagent^® according to the manufacturer’s protocol following the homogenization of the samples in the FastPrep-24 5G Homogenizer (MP Biomedicals, France). RNA concentrations and purity were determined spectrophotometrically using the NanoDrop ND-1000 UV–Vis Spectrophotometer (Thermo Fisher Scientific, MA, USA) at a wavelength of 260 and 280 nm. The samples were analyzed by the Agilent 2100 Bioanalyzer on RNA Nano chips (Agilent Technologies, CA, USA). Four µg of RNA were treated with DNase I (NEB, UK) and diluted to a concentration of 0.1 µg/µL. One half microgram of the total RNA, random hexamers and Protoscript^® II Reverse Transcriptase (NEB, UK) were used for reverse transcription (in 20 µL reaction mixture) according to the manufacturer’s protocol, following which the obtained cDNA was diluted 10× and stored at − 20 °C.