Mamessier E, Boniface S, Dupuy P, Reynaud-Gaubert M, Vervloet D, Magnan A
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.
J Immunol Methods. 2003 Sep;280(1-2):37-47. doi: 10.1016/s0022-1759(03)00230-8.
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.
痰液检查正越来越多地被用作研究气道炎症的一种非侵入性方法。然而，由于回收的细胞数量较少，痰液的技术应用仍然有限。为了扩展痰液检查的应用，我们开发并标准化了逆转录聚合酶链反应（RT-PCR），这是一种在诱导痰液样本中检测mRNA的灵敏且特异的技术。使用酚-氯仿提取法从含有低至50至80000个细胞的样本中提取总RNA。RT-PCR已成功应用于β-肌动蛋白、干扰素-γ（IFN-γ）、白细胞介素-4（IL-4）和肿瘤坏死因子-β（TGF-β）基因的检测。该方案提供了一种从少量诱导痰液细胞中提取总RNA的简单技术。它允许用简单的方法对气道中的细胞因子基因表达进行半定量研究。
