Study of cytokine gene expression in small cell samples: use in induced sputum

Sputum examination is being increasingly used as a non-invasive method for the study of airway inflammation. However, the technical applications of sputum are still limited because of the small number of cells recovered. In attempt to extend applications of sputum examinations, we developed and standardised, the reverse transcriptase-polymerase chain reaction (RT-PCR), a sensitive and specific technique of detection of mRNA, in induced sputum samples. Total RNA were extracted from samples containing as few as 50 to 80,000 cells, using a phenol-chloroform extraction method. RT-PCR was successfully tested on beta-actin, interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and tumour necrosis factor-beta (TGF-beta) genes. This protocol provides a simple technique to extract total RNA from a few number of induced sputum cells. It permits the semi-quantitatively study of cytokine gene expression in airways with simple means.