ArticleThorax2002
beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels.
Article in Thorax, 2002. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 198 papers.
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198 citing papers in PubMed, 411 citations in OpenAlex.
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- Exploring heterogeneous expression of beta-actin (ACTB) in bladder cancer by producing a monoclonal antibody 6D6.BMC urology · 2024Article
- Selection of suitable reference genes for normalization of RT-qPCR in three tissues of Northern bobwhite (Colinus virginianus) infected with eyeworm (Oxyspirura petrowi).Molecular biology reports · 2024Article
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- Article
138 more citing papers are in PubMed but not listed here.
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundThe use of reverse transcription-polymerase chain reaction (RT-PCR) to measure mRNA levels has led to the common use of beta-actin and GAPDH housekeeping genes as denominators for comparison of samples. Expression of these genes is assumed to remain constant, so normalising for variations in processing and signal quantitation. However, it is well documented that beta-actin and GAPDH expression is upregulated with proliferation, activation, and differentiation. We hypothesised that airway samples which differ in their cellular profiles and activation status have different levels of expression of GAPDH and beta-actin.
methodsThe mRNA for beta-actin, GAPDH, and interleukin (IL)-2 was measured in bronchoalveolar lavage (BAL) fluid cells and endobronchial biopsy tissue by competitive RT-PCR in a cross sectional study of 26 normal controls and 92 asthmatic subjects.
resultsFor both BAL fluid cells and biopsy tissue, asthmatics overall had reduced expression of GAPDH and beta-actin mRNA. In asthmatic subjects not using inhaled corticosteroids (ICS), GAPDH mRNA levels in both BAL fluid and biopsy tissue, and beta-actin mRNA in BAL fluid cells were 10 times lower than samples from both normal controls and from asthmatic subjects using ICS. beta-Actin mRNA in biopsy specimens showed the same pattern of expression, but asthmatic subjects not using ICS were not significantly different from those receiving ICS treatment. IL-2 mRNA levels did not differ between the subject or treatment groups but, when expressed as a ratio with beta-actin, significant differences were seen.
conclusionsbeta-Actin and GAPDH used as denominators of gene expression quantitation in asthma research can cause confounding. Housekeeping genes need careful validation before their use in such quantitative mRNA assays.
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