Rt2 profiler pcr array software

For the qPCR array experiments depicted in Supplemental Figure S8A and Supplemental Table S3, DP CD71⁻ cells were purified from thymocytes that were first enriched for CD4⁺ cells by MACS (miltenyi) and then FACS purified for CD4⁺, CD8⁺, and CD71⁺ using the scheme outlined in Supplemental Figure S6C. Cell purity was typically 85%–95%. Total RNA was extracted from cells using TRIzol reagent (Life technologies) followed by purification through an RNAeasy colum (Qiagen). RNA quality was assessed using Bioanalyzer (Agilent). From each of two biological replicates per sample, 100 ng of RNA was reverse-transcribed using the RT² first strand kit (Qiagen). qRT–PCR was performed on technical quadruplicates using the mouse P53 pathway qRT–PCR array (Qiagen, PAMM-027Z). Normalization and all downstream analysis steps were performed according to the manufacturer's instructions, and data analysis was conducted using the RT² profiler PCR array software (Qiagen). For the experiment in Supplemental Figure S8B, total thymocytes were incubated with Hoechst for 30 min in Hank's balanced salts solution, sorted according to DNA content, and processed as above using primers designed in house. All qRT–PCR data in Supplemental Figure S8, A and B, and Supplemental Table S3 represent mean values from two biological replicate experiments, each comprising two technical replicate PCRs.