Real time pcr cycler rotor gene q 2plex system

RNA extraction and qPCR were performed as previously described [21 (link)]. Briefly, total RNA from BMDMs or lung tissues were isolated for using TRIzol reagent (Thermo Fisher Scientific). The cDNA synthesis was performed using Superscript II reverse transcriptase (Invitrogen, 18064). QPCRs were carried out with cDNA, primers, and SYBR Green PCR Kits (Qiagen, 204074) using a Real-time PCR Cycler Rotor-Gene Q 2plex system (Qiagen GmbH, 9001620, Hilden, Germany). The samples were amplified for 50 cycles as follows: 95°C for 5 s and 60°C for 10 s. To analyze qPCR data, we performed relative quantification using the 2^ΔΔCt method with Gapdh as an internal control gene; data were expressed as relative fold changes. The primer sequences are shown in Table S1.

Total RNA from mouse lung tissue homogenates was extracted using TRIzol reagent (15596026; Invitrogen) in accordance with the manufacturer’s instructions, followed by RNA quantification and assessment using QIAxpert (Qiagen). cDNA was synthesized from total RNA using reverse transcription master premix (EBT-1543; ELPIS Biotech) in accordance with the manufacturer’s instructions. Real-time PCR was performed using SYBR Green reagent (204074; Qiagen) in a real-time PCR Cycler Rotor-Gene Q 2plex system (9001620; Qiagen). The mRNA expression was calculated with the 2^ΔΔ threshold cycle (Ct) method, with normalization relative to Gapdh. The sequences of primers used in this study are shown in Table S2.