Each 20 µl PCR reaction contained 2 µl of DNA and 18 µl of mastermix containing FastStart DNA Master SybrGreen I, 4 mM MgCl2 (both from Roche), 0·5 µM of each primer and sterile dH2O. The PCR was performed as follows: one cycle of denaturation at 95°C for 10 min, 45 cycles of amplification consisting of denaturation at 95°C for 10 s, primer annealing at 72–62°C for 5 s (0·5°C drop in Selumetinib price each cycle for 20 cycles) and extension at 72°C for 6 s, followed by melting at 95°C for 0 s, 62°C for 10 s and 95° for 0 s (0·1°C/s temperature increase) and ending by cooling at 40°C for 30 s. Frozen mucosal tissue, preincubated
in RNAlater, as well as frozen thymic tissue obtained from human infants undergoing cardiac surgery, was homogenized with a mortar and pestle in liquid nitrogen and then added to RLT buffer (RNeasy mini kit, Qiagen). Frozen cells, preincubated in RNAlater, were placed in RLT buffer directly and both tissues and cells were homogenized by passing the lysate through a blunt 20-gauge needle. RNA was purified by the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. The RNA concentrations in all samples were determined by ultraviolet spectrophotometry at 260 and 280 nm and the purity and
integrity of extracted RNA was confirmed by electrophoresis in a 1% agarose gel. Fifty ng/µl RNA was reverse-transcribed to cDNA using QuantiTect reverse transcription kit (Qiagen) according to the manufacturer’s instructions Alpelisib ic50 and then stored at −20°C. The amount of RAG1 and pre-TCR-α mRNA relative to the amount of the reference gene CD3γ mRNA was determined by real-time PCR (LightCycler480; Roche Diagnostics GmbH), using specific primers and human universal probes as specified below for detection of specific products. The PCR primers were purchased from Invitrogen, Paisley, UK. The sequences were as follows: RAG1 forward: 5′-ATT GCA GAC ATC TCA ACA CTT TG-3′ and reverse: 5′-GAA AGA GGC TGC CAT GCT-3′; pre-TCR-α forward: 5′-TCC TGC CTC CTT CCG AGT-3′
and reverse: 5′-CCA GAG AAG GAA AGG GTG TG-3′; CD3γ forward: 5′-TTG GGG TCT ACT TCA TTG CTG-3′ and reverse: 5′-AAC AGA GTC Cediranib (AZD2171) TGC TTG TCT GAA GC-3′. These primers generated specific products of 74, 111 and 70 bp, respectively. Each 15 µl PCR reaction contained 80 ng of cDNA in a volume of 5 µl, 5 µl LightCycler480 Probes Master and 0·2 µl human universal probe number; 27 (RAG1-primer), 2 (pre-TCR-α-primer) or 58 (CD3γ-primer), 0·2 µl (20 µM) of each primer and 4·4 µl RNase free dH20. The PCR was performed as follows: denaturation at 95°C for 10 min, 45 cycles of amplification at 95°C for 10 s, annealing at 60°C for 30 s and extension at 72°C for 1 s, before the samples were cooled at 40°C for 30 s.