Identification of suitable reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) in the fibrotic phase of the bleomycin mouse model of pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a severe lung disease with a poor prognosis and few treatment options. In the most widely used experimental model for this disease, bleomycin is administered into the lungs of mice, causing a reaction of inflammation and consequent fibrosis that resembles the progression of human IPF. The inflammation and fibrosis together induce changes in gene expression that can be analyzed with reverse transcription quantitative real-time PCR (RT-qPCR), in which accurate normalization with a set of stably expressed reference genes is critical for obtaining reliable results. This work compares ten commonly used candidate reference genes in the late, fibrotic phase of bleomycin-induced pulmonary fibrosis and ranks them from the most to the least stable using NormFinder and geNorm. Sdha, Polr2a and Hprt were identified as the best performing and least variable reference genes when alternating between normal and fibrotic conditions. In order to validate the findings, we investigated the expression of Tnf and Col1a1, representing the hallmarks of inflammation and fibrotic changes, respectively. With the best three genes as references, both were found to be upregulated relative to untreated controls, unlike the situation when analyzed solely with Gapdh, a commonly used reference gene. We therefore recommend Sdha, Polr2a and Hprt as reference genes for RT-qPCR in the 4-week bleomycin challenge that represents the late fibrotic phase.