TY - JOUR T1 - Identification of stable housekeeping genes for real-time PCR in human pulmonary fibroblasts JF - European Respiratory Journal JO - Eur Respir J VL - 38 IS - Suppl 55 SP - p3805 AU - Carmel Stock AU - Patricia Leoni AU - Xu Shi-Wen AU - David Abraham AU - Andrew Nicholson AU - Athol Wells AU - Elizabeth Renzoni AU - Gisela Lindahl Y1 - 2011/09/01 UR - http://erj.ersjournals.com/content/38/Suppl_55/p3805.abstract N2 - Background: Quantitative real time PCR is an important tool in investigating gene transcription levels under different biological conditions. Accurate results rely on controlling for differences in mRNA quantity and quality between samples, commonly achieved by normalisation to expression of housekeeping genes (HKGs). Expression stability of the HKGs used is critical, but to date no systematic study has been performed in pulmonary fibroblasts.Methods: Microarray data of gene expression in explanted fibroblasts from SSc-ILD (systemic sclerosis lung biopsies, n=8) and control (normal periphery of resected tumors, n=10) lung tissue was used to assess variation in expression of commonly used HKGs; ACTB, GAPDH, HPRT1, RPL32, TBP, and YWHAZ (HUGO nomenclature). The four most stable genes (<15% variability) were tested by qRT-PCR in an independent experiment in which SSc-ILD (n=3) and control (n=3) fibroblasts were cultured in 0.1% BSA for 24hrs, and a further 24hrs with or without IFNγ (10ng/mL). A measure of expression stability (M), the average pairwise variation with each of the other studied genes (with a recommended maximum value of 1.5) for each HKG, was calculated using the program geNorm (Vandesompele, J. et al. Genome Biology 2002;3:34.1-12).Results: While ACTB and GAPDH were relatively unstable, the four HKGs tested further in geNorm all had an expression stability of M<1.0; the most stable gene was YWHAZ (M=0.56), followed by HPRT1 (M=0.60), RPL32 (M=0.67), and TBP (M=0.70).Conclusion: We have identified four HKGs suitable for normalisation of mRNA expression levels in human lung fibroblasts under the conditions tested, and show the necessity for empirical identification of HKGs. ER -