Abstract
Induced sputum is a non-invasive method, which allows collecting cells from airways. Gene expression analysis from sputum cells has been used to understand the underlying mechanisms of airway diseases such as asthma or Chronic Obstructive Pulmonary Disease (COPD). Suitable reference genes for normalisation of target mRNA levels between sputum samples have not been defined so far.
The current study assessed the expression stability of 9 common reference genes in sputum samples from 14 healthy volunteers, 12 asthmatics and 12 COPD patients. Using three different algorithms (geNorm, NormFinder and BestKeeper), we identified HPRT1, GNB2L1 as the most optimal reference genes to use for normalisation of RT-qPCR data from sputum cells. The higher expression stability of HPRT1 and GNB2L1 were confirmed in a validation set of patients including 9 healthy controls, 5 COPD and 5 asthmatic patients. In this group, the RNA extraction and RT-PCR methods differed which attested that these genes remained the most reliable whatever the method used to extract the RNA, generate complementary DNA or amplify it. Finally, an example of relative quantification of gene expression linked to eosinophils or neutrophils provided more accurate results after normalisation with the reference genes identified as the most stable compared to the least stable and confirmed our findings.
Footnotes
This manuscript has recently been accepted for publication in the European Respiratory Journal. It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article.
Conflict of interest: Dr. Moermans has nothing to disclose.
Conflict of interest: Dr. Deliège has nothing to disclose.
Conflict of interest: Dr. Pirottin has nothing to disclose.
Conflict of interest: Dr. Poulet has nothing to disclose.
Conflict of interest: Dr. GUIOT has nothing to disclose.
Conflict of interest: Dr. Henket has nothing to disclose.
Conflict of interest: I have no conflicts of interest
Conflict of interest: Dr. Louis reports grants from GSK, grants and personal fees from Novartis, grants from Chiesi, personal fees from Astra Zeneca, outside the submitted work.
This is a PDF-only article. Please click on the PDF link above to read it.
- Copyright ©ERS 2019