Quantitative real-time PCR (RT-qPCR)
Total RNA was extracted from approximately 10 mg of whole lung tissue using a RNeasy® Mini Kit (Qiagen, Germany). Isolated mRNA was then reverse transcribed with a High Capacity RNA-to-cDNA kit (Thermo Fisher Scientific, USA). Real time PCR reactions were performed using Thermo Fisher Scientific pre-developed Taqman assay reagents in triplicate using GAPDH as the internal housekeeping control. Through utilisation of the threshold cycle (Ct) value which is the PCR cycle number out of 40 at which the fluorescence signal measured exceeds the calculated background threshold, indicative of amplification of the target sequence value, which is proportional to the number of target copies within the sample and converting this result to the threshold cycle time (∆∆CT). mRNA expression levels can then be quantified and referenced against GAPDH, allowing comparisons between treatment groups to be made.