Mrnaeasy

RNA was isolated from CD4⁺ T cells using miRNAeasy (for miRNAs) or mRNAeasy (for mRNAs) isolation kits (QIAGEN, Germany). For miRNA detection, cDNA synthesis and subsequent qPCR with locked nucleic acid primers for specific miRNAs was performed using miRCURY LNA^™ universal reverse transcriptase microRNA cDNA synthesis and qPCR kit (EXIQON, Denmark). For mRNA detection, cDNA was prepared using the miRscript cDNA synthesis kit (QIAGEN, Germany). qPCR was performed using IQ^™ BioRad SYBR green master mix (BioRad, USA) with the following primers for Rictor (Forward - 5′-ACCGGGCTTCTGACCATTAAA-3′ and Reverse - 5′-TTGTATGAACCGCCGACACT-3′). Measurements were performed using Chromo 4 BioRad PCR machines. The relative expression level of miRNAs was normalized to that of 5S rRNA and U6 snRNA, and of mRNAs to that of β-actin as described previously (24 (link)).