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.