Stability of housekeeping genes in alveolar macrophages from COPD patients

Abstract

The stability of housekeeping genes is critical when performing gene expression studies. To date, there have been no studies that look at the stability of commonly used housekeeping genes in alveolar macrophages. Expression levels may be affected by culture, stimulation or disease severity.The present study investigated the expression level of 10 housekeeping genes and analysed the stability of their expression in alveolar macrophages from chronic obstructive pulmonary disease patients (n522) who were classified according to disease severity.Guanine nucleotide-binding protein, beta polypeptide 2-like 1 (GNB2L1), hypoxanthine phosphoribosyl transferase 1 (HPRT1) and ribosomal protein L32 (RPL32) were the most stably expressed in alveolar macrophages, irrespective of disease severity. There was no difference in the expression levels of 10 housekeeping genes between mild and moderate/severe patients. GNB2L1, HPRT1 and RPL32 were also stably expressed in alveolar macrophages cultured with no stimulation, or with interleukin-1b, lipopolysaccharide or tumour necrosis factor-a stimulation.In conclusion, as fluctuations in the expression of some housekeeping genes were observed, including glyceraldehyde-3-phosphate dehydrogenase, it is recommended that guanine nucleotide binding protein, beta polypeptide 2-like 1 be used as a reference gene for alveolar macrophages in similar study designs, or that the stability of housekeeping genes be validated in alveolar macrophages prior to expression studies.

Long noncoding RNA are aberrantly expressed in vivo in the cystic fibrosis bronchial epithelium

“…As regards qRT-PCR, "traditional" reference genes such as GAPDH and ACTB have previously been found to be unsuitable for normalisation of mRNA levels in the airways of asthmatics (Glare et al, 2002), for gene expression studies employing alveolar macrophages from people with chronic obstructive pulmonary disease (Ishii et al, 2006b) and in bronchoalveolar cells from patients with other pulmonary diseases (Kriegova et al, 2008). A study to determine the reference genes with the most stable mRNA expression in bronchoalveolar cells from horses with inflammatory airway disease identified an optimal four gene reference panel (Beekman et al, 2011).…”

Housekeeping gene stability in pristane-induced arthritis and antigen-induced pulmonary inflammation of rats

“…The mRNA expression of a target gene relative to the reference is calculated, and the final result is represented as a target/reference ratio [1]. It is well known that the most commonly used endogenous references are housekeeping genes which are critical to cell viability and traditionally defined as constitutively and stably expressed.…”

The effect of peroxisome proliferator-activated receptor- ligands on in vitro and in vivo models of COPD

“…PPAR-c gene expression levels were compared between macrophages from COPD patients and controls as described in the online supplementary material; the endogenous housekeeping controls glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and gene b-peptide 2-like 1 (GNB2L1) [29] were used.…”