Quantitative Polymerase Chain Reaction

Quantitative real-time PCR was performed using TaqMan probes specific for the following: Ras homolog family member A (RHOA, Hs00357608_m1), Unconventional myosin-IXb (MYO9B, Hs00188109_m1), Rho-associated coiled-coil-containing protein kinase 1 (ROCK1, Hs01127701_m1), BH3 interacting domain death agonist (BID, Hs00609632_m1), Direct inhibitor of apoptosis-binding protein with low pI (DIABLO, Hs00219876_m1), B-cell lymphoma 2 (Bcl-2, Hs04986394_s1), myeloid cell leukemia 1 (Mcl-1, Hs06626047_g1), tumor necrosis factor receptor 1 (TNFR1, Hs01042313_m1), tumor necrosis factor receptor 2 (TNFR2, Hs00961750_m1), ACTB (β-actin) (Hs01060665_g1), and 18S (18S ribosomal RNA gene) (Hs03928990_g1). Single reactions were prepared with the Maxima Probe/ROX qPCR Master Mix (Thermo Fisher Scientific, Waltham, USA), and all amplifications were run in duplicate under the following thermal conditions: 95°C for 10 min followed by 40 cycles of 60°C for 1 min and 95°C for 15 s, with the StepOnePlus™ Real-Time PCR Systems (Applied Biosystems). The relative expression of transcripts was quantified using the ΔΔCT method. The n-fold change was calculated for each gene, normalizing to the endogenous controls ACTB and 18S and relative to a control group without stimulus during the culture of T cells from four healthy donors (=1). Then, the log2 (fold change) in CLF and IPF was determined, where log2 (fold change) of control condition = 0.