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.