RT-PCR for MAGT1, miR-199a-5p, and miR-199b-5p quantification

Total RNA was extracted from CD8⁺ T cells using TRIzol reagent (Invitrogen, CA, USA). The first-strand cDNA synthesis was carried out in a 20-µL reaction mixture containing 1 µg DNase-treated total RNA using the superscript III reverse transcriptase (Promega, WI, USA, http://www.promega.com) and random primers (Promega) according to the manufacturer’s instructions, and 2 ng cDNA products were used in each RT-PCR reaction. Primer information is as follows: MAGT1, forward: 5′-AGACACTGGAG TACTGGAAAT-3′ and reverse: 5′-TGACAGGAGAATCGCTTAAAC -3′. GAPDH served as the internal control, forward: 5′-TTCTATA AATTGAGCCCG CAG-3′ and reverse: 5′-CGATACCAA AGTTGTCATGGA-3′. The sequence of the miR-199a-5p, RT primer: 5′-GTCGTATCCAGTGCGTGTCGTG GAGTCGGCAATTGCACTGG ATACGACGAACAG-3′, forward: 5′-GGGCCCAGTGTT CAGACTAC-3′, reverse: 5′-CAGTGCGTGTCGTGGAGT-3′. The sequence of the miR-199b-5p, RT primer: 5′-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTG GATACGAC GAACAG-3′, forward: 5′-GGGCCCAGTGTTTAGACTAT-3′, reverse: 5′-CAGT GCGTGTCGTGGAGT-3′. U6 served as the internal control: RT primer: 5′-AACGC TTCACGAATTTGCGT-3′, forward: 5′-CTCGCTTCGGCAGCACA-3′, reverse: 5′-AACGCT TCACGAATTTGCGT-3′. The relative expression ratio of MAGT1, miR-199a-5p, and miR-199b-5p was calculated by the 2^−△△CT method.