Stability of housekeeping genes in alveolar macrophages from COPD patients.
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 (n = 22) 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-1beta, lipopolysaccharide or tumour necrosis factor-alpha 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.
Ishii
T
T
James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St. Paul's Hospital, Vancouver BC, V6Z 1Y6, Canada.
Wallace
A M
AM
Zhang
X
X
Gosselink
J
J
Abboud
R T
RT
English
J C
JC
Paré
P D
PD
Sandford
A J
AJ
D000368:Aged; D018893:Bronchoalveolar Lavage; D005260:Female; D019204:GTP-Binding Proteins / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D007375:Interleukin-1 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000235:genetics*; D029424:Pulmonary Disease, Chronic Obstructive / Q000235:genetics* / Q000378:metabolism; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics*; D012129:Respiratory Function Tests; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D007375:Interleukin-1; D008070:Lipopolysaccharides; D009363:Neoplasm Proteins; C419368:RACK1 protein, human; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D012269:Ribosomal Proteins; D014409:Tumor Necrosis Factor-alpha; C041806:ribosomal protein L32; D007041:Hypoxanthine Phosphoribosyltransferase; D019204:GTP-Binding Proteins