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.
Mamessier
E
E
UPRES EA 3287, Univ-Mediterranée, Pathologie respiratoire liée à l'environnement, Hôpital Ste Marguerite, Service de Pneumo-allergologie, 270 Bd de Ste Marguerite, 13009 Marseilles, France.
Boniface
S
S
Dupuy
P
P
Reynaud-Gaubert
M
M
Vervloet
D
D
Magnan
A
A
D000199:Actins / Q000235:genetics; D002725:Chloroform; D016207:Cytokines / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics; D015847:Interleukin-4 / Q000235:genetics; D019800:Phenol; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D013183:Sputum / Q000166:cytology / Q000276:immunology*; D016212:Transforming Growth Factor beta / Q000235:genetics
D000199:Actins; D016207:Cytokines; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D015847:Interleukin-4; D019800:Phenol; D002725:Chloroform; D007371:Interferon-gamma