RNA isolation and reverse transcription-quantitative PCR (RT-qPCR)
This protocol is extracted from research article:
Effects of miR-210-3p on the erythroid differentiation of K562 cells under hypoxia
Mol Med Rep, Jun 7, 2021; DOI: 10.3892/mmr.2021.12202

Total RNA was extracted from the cells harvested using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. RNA was quantified using an Ultra-microspectrophotometer (Nanodrop 2000; Thermo Fisher Scientific, Inc.) at 260 nm. cDNA was synthesized using a reverse transcriptase kit (60 min at 42°C and then 5 min at 70°C; cat. no. K1691; Thermo Fisher Scientific, Inc.) from 1 µg total RNA. For mRNAs, RT-qPCR was performed using the ABI 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and the QuantiNova SYBR Green PCR kit (Qiagen Sciences, Inc.), according to the manufacturer's instructions (95°C, 2 min for pre-degeneration; followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec). The relative quantification of the transcripts was performed using the 2−ΔΔCq method (18). The primer sequences used were as follows: γ-globin forward, 5′-GCAGCTTGTCACAGTGCAGTTC-3′ and reverse, 5′-TGGCAAGAAGGTGCTGACTTC-3′; and β-actin forward, 5′-CCTGGCACCCAGCACAAT-3′ and reverse, 5′-GCTGATCCACATCTGCTGGAA-3′.

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