2.3 Total RNA extraction and cDNA synthesis
Total RNA (including miRNAs) was isolated from CD4+CD25- T cells according to the manufacturer’s instructions using the miRNeasy Mini Kit (Qiagen, Germany). The integrity of the isolated total RNA was confirmed by an average OD260/OD280 nm absorption ratio of 1.98. cDNA was synthesized with oligo-dT primers and RevertAidTM H Minus reverse transcriptase (Fermentas, Lithuania). The miRNA cDNA was synthesized with the miScript II RT Kit from Qiagen. Negative control samples (no first-strand synthesis) were prepared by performing reverse transcription in the absence of reverse transcriptase.