Tetro cdna strand cdna synthesis kit

The HuASCs cells were seeded onto the 6-well plastic plates and incubated with calystegines prior to hyperglycemia induction. After the incubation time, the cells were suspended in TRIzol reagent for RNA isolation following the manufacturer’s instructions. The quantity and the purity of the isolated RNA was assessed using a spectrophotometer at 260 nm wavelength (Epoch, Biotek, Bad Friedrichshall, Germany). A total of 150 ng RNA was used for cDNA synthesis using a Tetro cDNA Strand cDNA Synthesis Kit (Bioline, London, UK) based on oligo (dT) primers in a T100 Thermal Cycler (BioRad, Hercules, CA, USA). The detection of the target gene expression was performed using a SensiFAST SYBR Green Fluorescein Kit (Bioline, London, UK) in a CFX Connect™ Real-Time PCR Detection System (BioRad, Hercules, CA, USA). The relative expression of genes associated with apoptosis, oxidative stress, mitochondrial dysfunction inflammation and ER stress (Table 1) has been normalized to GAPDH (glyceraldehydes-3-phosphate, housekeeping gene) expression and calculated using the 2^−ΔΔcq method.

Total RNA was isolated from MC3T3‐E1 or 4B12 cells by using Trizol reagent (Sigma) as preconized by the supplier. RNA purity and concentration were established using a nanospectrophotometer (WPA, Biowave II). Genomic DNA (gDNA) digestion and cDNA synthesis were performed by reverse transcription reaction with oligo(dT) primers using a Tetro cDNA Strand cDNA Synthesis Kit (Bioline) by the mean of a T100 Thermal Cycler (Bio‐Rad) according to the provided kit instructions. Real‐time PCR analysis was used to quantify the transcripts of osteogenic and osteoclastogenic‐related genes (Table 1). SensiFAST SYBR Green Kit (Bioline) was used for detection of the target mRNA expressions in a CFX Connect™ Real‐Time PCR Detection System (Bio‐Rad). A total of 150 ng of cDNA were amplified in a total volume of 10 µL containing SYBR Green Master Mix, forward and reverse primers and tested samples. The thermal profile conditions were as follows: 95°C for 2 minutes, followed by 40 cycles at 95°C for 15 seconds, annealing for 15 seconds, and elongation at 72°C for 15 seconds. RT‐qPCR reactions were carried out in triplicate. The relative expression levels of all studied genes were normalized to the expression of house‐keeping gene glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH).