Rna extraction mini kit

Total RNA was extracted using the RNA extraction mini kit (FAVORGEN, Taiwan) according to the manufacturer recommendations. Then, qRT-PCR was performed by stem-loop TaqMan real-time PCR assay, using unique sequence index (USI) barcodes and probe described by Fattahi et al. [33 (link), 36 (link)]. Gene amplification was carried out using a StepOnePlus™ real-time PCR system (Applied Biosystems, CA, USA), and the results were expressed as the fold change calculated using the 2^-ΔΔCt method relative to the control sample. GAPDH was used as a housekeeping gene to normalize gene expression. Primer sequences were designed by AlleleID 6.0 software and showed in Table 1. To amplify the genes, we used the following thermal profile: initial denaturation at 95°C for 5 minutes and then 40 repetitions at 95°C for 15 seconds and 60°C for 60 seconds.

Total RNA was extracted from 40 mg of the tumor tissue using a RNA extraction mini kit (Cat No: FATRK 001, Favorgen Biotech Corp) and TRIzol following the manufacturer's protocol. The quality of the extracted RNA was determined by electrophoresis on 2% agarose gel, and its concentration and purity were assessed using NanoDrop spectrophotometer (BioTeK) by reading A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ nm absorbance ratio. The extracted RNA (1,000 ng) was converted to cDNA using a cDNA synthesis kit (BioFact™ cDNA synthesis kit, Cat.No. BR123‐R10k, BioFact™ RT Series). The reaction mixture included 10 µl 5X RT Reaction Buffer, 1 µl Random Hexamer Primer, 1 µl oligo‐d (T), and 1 µg of total RNA, which was reached to the final volume of 20 µl by adding DEPC water. The reverse transcription was performed at 95°C for 2 min, and then 60°C for 30 s. The enzymatic reaction was stopped by heating at 74°C for 4 min. The cDNA template was stored at −20°C until use (Akbari Bazm et al., 2020).