7900t fast real time pcr system

Organs stored in RNAlater (Life Technologies) at −80 °C were thawed and lysed in TRIzol (Life Technologies) using 7 mm steel beads in a TissueLyser (Qiagen). RNA was isolated using the RNeasy Mini Kit II (Qiagen) and reverse transcribed with the SuperVilo cDNA synthesis kit (Life Technologies). Quantitative PCR (qPCR) was performed using TaqMan gene expression assays (see Supplementary Table 5 for assay details) on a 7900T Fast Real Time PCR system (Applied Biosystems, Thermo Scientific). Expression was normalized using 18 s rRNA as a housekeeping gene. Relative quantification of gene expression was analyzed using the 2^-ΔΔCT method.

NucleoSpin RNA II kit (Clontech) was used to purify total RNA. cDNAs were synthesized using High-Capacity cDNA reverse transcription kit (Applied Biosystems). cDNAs analyzed by qPCR utilizing Power SYBR Green and the 7900T Fast Real-Time PCR System (Applied Biosystems). The following primers were used to detect RNA levels of CFTR (1–2), GFP, (3–4), Actin (5–6), NAA10 (7–8), NAA15 (9–10), NAA20 (11–12), and NAA25 (13–14) (see Table 3). qPCR data was analyzed using the comparative C_T method [21 (link)].

Quantitative real-time PCR (qPCR) was used to validate microarray differential gene expression results. Firstly, the qScript cDNA Supermix (Quanta Bio, MA, USA) was used according to the manufacturer’s instructions to synthesise single stranded cDNA from each RNA sample obtained following LCM of plaque macrophages. The PCR reaction was run on a 7900T fast real time PCR system (Applied Biosystems, UK) using the GoTaq Probe qPCR Master Mix (Promega) in accordance with the manufacturer’s instructions. Data were normalised to cyclophilin A or 18S. Fold change was calculated using Delta Delta Ct values. The PCR primers used are listed in Supplementary Table 2.