Applied biosystems 7500 real time pcr system

The adipose tissues and cells were used to extract the total RNAs using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Next, the RNAs were reverse transcribed into first-strand cDNA by SuperScript II reverse transcriptase (Life Technologies, Gaithersburg, MD, USA) from 1 μg RNA. The primers were designed according to the sequences in GenBank (Table I). Semi-quantitative RT-PCR was performed on an Applied Biosystems 7500 real-time PCR system (Life Technologies). The reaction conditions were 1 cycle at 94°C for 4 min; 40 cycles at 94°C for 30 sec, 60°C for 30 sec and 72°C for 30 sec. The specifics of the primers were determined by dissociation curve. Following the reaction, the data were analyzed using SDS 1.3 software on the Applied Biosystems 7500 real-time PCR system.

Analyses of miR-431, Dkk1, Krm1, and Wnt in the brain were performed according to routine lab protocols (Wu et al., 2012 (link)). Real-time PCR reactions were carried out using EXPRESS SYBER® GreenER™ qPCR SuperMix Universal (Thermo Fisher Scientific) in triplicates for each cDNA sample on Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific). Primers specific for each miRNA and mRNA were obtained using Invitrogen (Thermo Fisher Scientific). As an internal control, primers for S12 were added for RNA template normalization, and the relative quantification of gene and miRNA expressions was calculated against S12 using a 2-ΔΔCT method. Gene-specific primer for miR-431: 5′-CAGGCCGTCATGCAAA-3′.

Reverse transcription was performed with TaqMan™ MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific) at the following conditions: 16 °C—30 min, 42 °C—30 min and 85 °C—5 min. Each 10 µL reaction volume contained 1 µL 10× Reverse Transcription Buffer, 0.67 µL Multiscribe Reverse Transriptase (50 U/µL), 0.13 µL RNAse inhibitor (20 U/µL), 0.1 µL dNTPs (100 Mm), 0.75 µL specific primers for 4 miRNA of interest, 5 µL (20 ng/µL) total RNA template and RNAse-free water. miR expression was measured in duplicates with quantitative real-time PCR (qPCR) analysis, using MicroAmp Optical 96-well reaction plates (Thermo Fisher) and Applied Biosystems 7500 Real time PCR System (Thermo Fisher). Each 10 µL reaction volume contained 5 µL 2× TaqMan Universal PCR Master Mix (Thermo Fisher Scientific), 0.5 µL TaqMan microRNA specific primer, 3.5 µL RNAse-free water and 1 µL (10 ng) cDNA template. PCR cycling conditions were: 95 °C—10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Data acquisition was performed at the end of each annealing/extension step. MiR primer assays are listed in Supplementary Table S4. Expression of the small nucleolar RNA (RNU48) was used as endogenous control to normalize the data. Data were analyzed with the comparative Ct method and presented as −ΔCt between the Ct of miR of interest and the Ct of endogenous control.

RNA from iPSCs was isolated using TRIzol™ LS Reagent (Thermo Fisher Scientific). Then, the RNA was transcribed to cDNA using the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific), as indicated by manufacturer. The RT-qPCR was performed with TaqMan^® Universal PCR Master Mix according to the manufacturer’s recommendations and the TaqMan™Gene Expression Assay (FAM) probes: TERT, TERC, TINF2, POT1, and TRF2 (Thermo Fisher Scientific). The q-RT-PCR was performed in an Applied Biosystems 7500 Real-Time PCR system (Thermo Fisher Scientific). ESCs (H1 cell line) were the reference group. Fold changes were calculated using the ddCT method, in which fold change data were represented as 2^−ddCT [27 (link)]. Results were normalized to GAPDH expression.