Gene expression in sorted T conv cells and T reg cell populations was quantified using a custom nCounter probe set containing 301 probe pairs, including additional probes for positive and negative controls and housekeeping genes (NanoString Technologies; Yosef et al., 2013 (link)). In brief, total cellular RNA was isolated and quantitated as above, and 100 ng was hybridized with customized Reporter CodeSet and Capture ProbeSet (NanoString Technologies) by overnight incubation in a thermal cycler at 65°C. The next day, flow cell preparation and scanning were performed using an nCounter instrument (NanoString Technologies) according to the manufacturer’s instructions. Raw data were normalized using nSolver software (NanoString Technologies) and exported as raw transcript counts for presentation. To normalize data, average background was subtracted from raw count, and normalization was performed using the geometric mean count from four housekeeping genes (Gapdh, Hprt, Actb, and Tubb5). Heat maps were generated using GENE-E software.
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NanoString Analysis of T Cell Gene Expression
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Corresponding Organization :
Other organizations : Harvard University, Brigham and Women's Hospital, University of Oxford, Stanford University, Memorial Sloan Kettering Cancer Center, Howard Hughes Medical Institute, Dana-Farber Cancer Institute
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Variable analysis
independent variables
- Cell populations: T conv cells and T reg cells
- Gene expression in sorted T conv cells and T reg cell populations
- Total cellular RNA
- 100 ng of RNA used for hybridization
- Overnight incubation in a thermal cycler at 65°C
- Housekeeping genes (Gapdh, Hprt, Actb, and Tubb5) used for normalization
- Positive controls
- Negative controls
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