Genejet rna purification

Hydrogels’ treated cells lysate was used for RNA isolation by using a commercially available kit (Gene JET RNA Purification) according to the manufacturer protocol (Thermo Scientific, # K0731, USA). RNA was extracted in triplicates [34 (link)]. For reverse transcription, cDNA was synthesized by using a commercially available kit (RevertAid™ H Minus First Strand) by following the manufacturer’s instructions (Thermo Scientific, # K1622, USA). Amplification was done by real-time PCR (Applied Biosystems 7900HT Fast Real-Time PCR System) using kit (Maxima SYBR Green/ROX qPCR Master Mix (2X), Thermo Scientific, USA), following manufacturer’s guidelines (Thermo Scientific, # K0221, USA). The relative expression levels of each target gene were calculated using the Ct method by using Beta-actin (β-actin), and Relative mRNA expressions were analyzed using the ΔΔCt equation.

Nucleic acids were isolated by means of Genejet RNA purification and Genejet PCR purification kits (Thermo Fisher, Waltham, MA, USA). The quantity of DNA and RNA was quantified using a Nanodrop 2000 (Thermo Fisher, Waltham, MA, USA). Regarding the RNA samples, cDNA was synthesised using oligo dT as a primer in order to utilise only the mRNA in the synthesis.

To measure the mRNA expression level of p53 and β-Catenin genes in the renal tissues of negative control and treated mice; whole RNA was extracted and reversely transcribed into complementary DNA (cDNA) using the GeneJET RNA Purification and Revert Aid First Strand cDNA Synthesis Kits (Thermo Scientific, USA), respectively. For cDNA synthesis each a µg of template RNA and 1 µl of primer were added to a sterile nuclease free water to complete the final volume to 12 µl in nuclease free tube on ice. Then added reaction buffer, RNase Inhibitor, nucleotides and Revert Aid M-MuLV RT completed the final volume to 20 µl. Samples were mixed gently, incubated 42 °C for 60 min and terminated the reaction by heating at 70 °C for 5 min. Amplification of p53 and β-Catenin genes was then conducted using the primers' sequences listed in Table 1 and previously designed by^{32 (link),33 (link)}. The results obtained from RT-PCR were then normalized to the housekeeping β-actin gene and the comparative Ct (DDCt) method was used to calculate the fold changes in the expression level of p53 β-Catenin genes.