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A n Australian study is the first to question routine use in asthma research of two genes widely used for standardising quantitative mRNA assays. The expression of β actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) needs careful validation first, say the researchers.
They used a reverse transcriptase (RT)-PCR method that does not rely on internal standards to measure β actin and GAPDH mRNA in bronchiolar lavage (BAL) fluid cells and bronchial biopsy samples from 92 asthmatic and 26 normal control volunteers.
Correlation coefficients for amounts of β actin and GAPDH mRNA indicated that expression was not comparable at the resolution of the method. GAPDH mRNA varied among biopsy samples and was about five times lower in BAL fluid cells than β actin mRNA. Furthermore, asthmatic subjects showed lower β actin and GAPDH expression overall, but not using inhaled corticosteroids resulted in tenfold lower GAPDH than in the controls and subjects who did. This was also true for β actin mRNA in BAL fluid cells. β actin and GAPDH mRNA were expressed similarly in biopsy material, and regardless of whether inhaled corticosteroids were used or not.
Interleukin (IL)-2 mRNA expression was similar in BAL fluid cells from asthmatic and control groups, but it showed significant differences between asthmatic subjects who did not use inhaled corticosteroids and controls and subjects who did when expressed relative to β actin, owing to that gene’s non-uniform expression.
Use of β actin and GAPDH genes as internal standards assumes uniform and constant expression, but they are probably regulated differently.