Mj research dna engine opticon 2 pcr cycler

Real-time PCR using the SYBR Green I method [42 (link), 43 (link)] was used to examine the CD3ζ, CD28, CTLA-4, and Cbl-b genes with cDNA obtained from the PBMCs of AA patients and healthy individuals. The CD3ζ and β₂M primer sequences and PCR conditions were previously described [26 (link)]. The other primer sequences were as follows: CD28: 5′-GAAACACCTTTGTCCAA GTC-3′ and 5′-GGGGAGTCATGTTCATGTAG-3′, CTLA-4: 5′-GTCAGCCTGCCGAAGC ACT-3′ and 5′-GTCAGCCTGCCGAAGCACT-3′, and Cbl-b: 5′-CCCTGGAATTGACCATTGGG-3′ and 5′-ACTTGCCCAACTCAGTGAGAA-3′. The real-time PCR reactions were performed in a total volume of 25 μl containing approximately 1 μl cDNA, 0.5 μM of each primer pair, and 11.25 μl 2.5× RealMasterMix (Tiangen, Beijing). After an initial denaturation at 95 °C for 15 min, 40 cycles of the following procedure were performed using an MJ Research DNA Engine Opticon 2 PCR cycler (BIO-RAD, Hercules, CA, USA): 15 s at 95 °C and 30 s at 60 °C followed by 1 s at 80 °C for plate reading. The 2(^–∆CT) method was used to analyze the genes of interest relative to an internal control gene.