Abi prism 7700 sequence system

Sorted lung CD11c⁺ cells from CD11c-Cre-PPARγ^fl/fl and PPARγ^fl/fl mice were lysed using RLT plus Buffer (Qiagen) and RNA was extracted using an RNeasy Plus Micro kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using a High Capacity cDNA Reverse Transcription Kit (Life Technologies) and was used as a template for quantitative PCR according to the manufacturer’s instructions (Life Technologies). Primer and probe sets for assaying individual gene expression were purchased from Life Technologies (Taqman Gene Expression Assays). qRT-PCR reactions were carried out using the ABI PRISM 7700 Sequence System (Applied Biosystems) at the Genomics Research Core at the University of Pittsburgh. Results were analyzed using the SDS 2.2.2 software. mRNA expression was calculated using the 2^−ΔCt method using hprt1 as internal reference control.

Mouse lung mRNA isolation and cDNA synthesis were as described previously.^{56 , 57} qPCR was performed using TaqMan 2× Master Mix reagent (Applied Biosystems, catalog 4304437) and validated Taqman gene expression primer/probe sets (Life Technologies) that included: mouse Hprt1 (Mm03024075_m1), mouse Ifng (Mm01168134_m1), mouse Il13 (Mm00434204_m1), mouse Il17a (Mm00439618_m1), mouse Irf4 (Mm00516431_m1), mouse Irf8 (Mm00492567_m1) and mouse S100a4 (Mm00803372_g1). The PCR reactions were performed at the University of Pittsburgh Genomics Research Core using ABI PRISM 7700 Sequence System (Applied Biosystems). Results were analyzed using SDS 2.2.2 software. Relative gene expression was determined by calculating 2^−ΔCt normalized to Hprt1.