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.