Xiang X, Qiu D, Hegele R D, Tan W C
Respiratory Division, Department of Medicine, National University of Singapore, 119260, Singapore, Singapore.
J Virol Methods. 2001 May;94(1-2):129-35. doi: 10.1016/s0166-0934(01)00284-1.
Examination of sputum specimens can be used for monitoring airway inflammation by means of immunological and molecular techniques. RNA extraction is essential for measurement of cytokine gene expression and for detection of many viral pathogens in sputum. In this study, three RNA extraction methods used commonly (acid guanidinium thiocyanate-phenol-chloroform extraction, Trizol and RNeasy Mini kit) were compared on the sputum of 14 patients who had chronic obstructive pulmonary disease. The effect of dithiothreitol pre-treatment on sputum RNA extraction was also investigated. The yield and purity of total RNA were determined by spectrophotometry. Reverse transcription-polymerase chain reaction (RT-PCR) results of the house keeping gene (glyceraldehyde-3-phosphate dehydrogenase) and RNA sequences specific to common respiratory viruses were compared. The results showed that (1) total RNA extracted with Trizol had highest yield and purity among the three RNA extraction methods; (2) there was no significant difference among the three RNA extraction methods on the house keeping gene and viral detection by RT-PCR; (3) dithiothreitol pre-treatment did not improve either the purity of total RNA, or RT-PCR signal. Moreover, dithiothreitol treatment reduced significantly the yield of total RNA. The results of the study indicate that the Trizol method appears to be superior for total RNA extraction from sputum, and dithiothreitol pre-treatment does not increase the efficiency of RNA extraction and RNA detection in sputum specimens.
痰液标本检查可通过免疫和分子技术用于监测气道炎症。RNA提取对于测量细胞因子基因表达以及检测痰液中的多种病毒病原体至关重要。在本研究中，对14例慢性阻塞性肺疾病患者的痰液，比较了三种常用的RNA提取方法（酸性硫氰酸胍-苯酚-氯仿提取法、Trizol法和RNeasy Mini试剂盒法）。还研究了二硫苏糖醇预处理对痰液RNA提取的影响。通过分光光度法测定总RNA的产量和纯度。比较了管家基因（甘油醛-3-磷酸脱氢酶）的逆转录-聚合酶链反应（RT-PCR）结果以及常见呼吸道病毒特异性的RNA序列。结果表明：（1）在三种RNA提取方法中，用Trizol提取的总RNA产量和纯度最高；（2）三种RNA提取方法在管家基因和通过RT-PCR检测病毒方面无显著差异；（3）二硫苏糖醇预处理既未提高总RNA的纯度，也未改善RT-PCR信号。此外，二硫苏糖醇处理显著降低了总RNA的产量。该研究结果表明，Trizol法似乎在从痰液中提取总RNA方面更具优势，并且二硫苏糖醇预处理不会提高痰液标本中RNA提取和RNA检测的效率。
