Oligo dt 18 primer and reverse transcriptase

Total RNA from HeLa, HL60, U937, and THP-1 cell lines and from different subpopulations of PBMCs (pDC, NK, MO, CD40+, and B) was extracted using the TRIzol reagent (Invitrogen). For experiments with interferon stimulation, HL60 and U937 cells were either mock treated or incubated with 250 units/mL of IFN-α2a (Miltenyi Biotec) and harvested after 48 h. The RNA was converted to cDNA using an oligo(dT)18 primer and reverse transcriptase (Invitrogen). Reverse transcriptase quantitative PCR (RT-qPCR) reactions were performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using SYBR Green I (Eurogentec). All reactions were performed in triplicate and experiments were repeated at least twice. Relative amount of mRNA was calculated using the comparative Ct method (Applied Biosystems): 2^−ΔΔCt = 2^{−[(Ct_target−Ct_housekeeping)  sample−(Ct_target−Ct_housekeeping)  control]}, where Ct is the threshold cycle and HPRT or β-microglobulins are the housekeeping genes.

Total RNA was extracted using TRIzol^® reagent (Invitrogen) according to the manufacturer’s instructions, and then treated with RNase-free DNase I for 30min at 37°C (New England BioLabs, Beverly, MA, USA) to remove residual DNA.
First-strand cDNA was synthesized using Oligo(dT)18 primer and reverse transcriptase (Invitrogen). Before cDNA synthesis, 5 μg total RNA was treated with RQ1 RNase-free DNase (Promega), according to the manufacturer’s instructions, to ensure no DNA contamination. cDNA synthesis was then carried out with the purified RNA using the SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturer’s instructions. The RT reaction was performed using Mastercycler Gradient (Eppendorf).