T100 thermal cycler

cDNA was prepared from one microgram of pure RNA according to the manufacture’s instruction (NEB) with random primers and Moloney Murine Leukemia Virus reverse transcriptase (NEB) using a T100 Thermalcycler (BioRad, Hercules, CA, USA). Reverse transcription polymerase chain reactions (RT-PCRs) were performed using a 2.0X Taq RED Master Mix Kit (Genesse Scientific, San Diego, CA, USA) with cDNA as a template and probes for 23S RNA and rnj (Table S3).
With cDNA prepared above, real-time qPCR was performed accordingly [39 (link)], using iTAQ SYBR green supermix (Bio-Rad) and a T100 Thermalcycler (Bio-Rad). The coding sequence of 23S RNA of C. diphtheriae was used as a control. Primers for qRT-PCR are shown in Table S3. Results were analyzed by GraphPad Prism 5.0 (La Jolla, CA, USA).

Expression levels of SIRT1 and PGC-1α genes were determined using the housekeeping gene-independent ACTB (normalizer) by Droplet Digital polymerase chain reaction (ddPCR) using the ddPCR Supermix for gene expression (Bio-Rad). PCR was completed in a T100 thermal cycler and analyzed with a QX200 ddPCR (Bio-Rad). We first isolated total RNA from the globular fraction samples using the TRIzol^® method, and we proceeded to generate the cDNA using the RevertAid First-Strand cDNA Synthesis Kit (Thermo Scientific). ddPCR reactions were carried out in triplicate and following the manufacturer’s directions, using specific primers to amplify SIRT1 and PGC-1α. PCR reactions were performed in a T100 thermal cycler (Bio-Rad). After PCR samples were analyzed with a QX200 droplet, data analysis was performed with Quanta Soft analysis software (Bio-Rad).

Total RNA was obtained from differentiated MC3T3-E1 and osteoinduced huBM-MSCs with or without PFF-A treatment using Trizol reagent (Invitrogen, CA, USA) at day 12 of differentiation. Also, a non-induced untreated blank group which was fed growth medium instead of differentiation medium was analyzed after 12 days of incubation. Synthesis of cDNA was started with addition of total RNA (2 μg) to RNase-free water containing oligo (dT) and followed by denaturation at 70 °C for 5 min. Next, the mixture was reverse transcribed in a master mix (1 X RT buffer, 1 mM dNTPs, 500 ng oligo (dT), 140 U M-MLV reserve transcriptase and 40 U RNase inhibitor) using an automatic T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) with a cycle of 42 °C for 60 min and 72 °C for 5 min. Sense and antisense primers previously detailed [38 (link)] were used for the amplification of the target cDNA. The cDNA amplification was carried out using T100 Thermal Cycler (Bio-Rad) with cycle settings at 95 °C for 45 s, 60 °C for 1 min and 72 °C for 45 s for 30 cycles Final PCR products were separated by gel electrophoresis on 1.5% agarose gel for 30 min at 100 V. Bands were then observed following the staining with 1 mg/mL ethidium bromide under UV light using CAS-400SM Davinch-Chemi imager^TM (Seoul, Korea).