Abi 7500 system

The MPX assay has been detailed elsewhere
^{24 (link)}. Briefly, 140 µl of respiratory specimen was employed to extract RNA by Qiacube HT using an RNeasy extraction kit (Qiagen, Germany). The extracted RNA was subsequently tested for RSV (A and B) by a multiplex real-time PCR assay in triplex sets (Qiagen, ABI 7500 system). Samples yielding cycle threshold (Ct) values ≤35 were deemed positive for RSV.

Total RNA was purified using the RNeasy Plus micro kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol and cDNA was prepared using the RT² first strand kit (Qiagen). For the analysis of receptor and transcription factor gene expression in ILCs, the following primers were purchased from Qiagen: Gapdh (PPM02946E), Gpr43 (PPM04863A), Gpr41 (PPM59038A), and Rorc (PPM25095A). The Gpr109a (NM_030701) gene was amplified using the following primer set: forward primer, 5′-ACC CTA GGA CGA AGA GCC AT-3′ and reverse primer, 5′-TTT GAC TCC CAG ATG CAC CC-3′. cDNA was analyzed using an ABI 7500 system (Life Technology, Carlsbad, CA) and the expression level of each gene was normalized to the level of Gapdh expression using a relative quantitation method.
For the analysis of cytokine and chemokine gene expression in ILCs, the RT² Profiler PCR Array kit for mouse cytokines and chemokines (PAMM-150ZA, Qiagen) was used with the ABI 7500 system. Heat map data were analyzed using the MeV software.