TY - JOUR T1 - Pitfalls of RNA isolation from sputum in COPD JF - European Respiratory Journal JO - Eur Respir J VL - 44 IS - Suppl 58 SP - P995 AU - Csilla Páska AU - Imre Barta AU - Krisztina Kelemen AU - Balázs Antus Y1 - 2014/09/01 UR - http://erj.ersjournals.com/content/44/Suppl_58/P995.abstract N2 - Aims: Sputum could be a valuable source for molecular analysis of respiratory diseases; however, various methodological problems have to be overcome. We attempted to maximize the quality and the quantity of RNA by optimizing sputum processing, RNA isolation and transcription methods.Methods: Sputum samples (n=20) were obtained from COPD patients at our Institute, and processed using dithiothreitol (DTT), n-acetyl cysteine or RNAlater solutions. Three different RNA isolation methods were tested. DNA contamination was tested by reverse transcription negative controls with GAPDH and universal bacterial primers. RNA quality was assessed by real-time PCR yielding GAPDH amplicons of different length. Data are presented as mean±SD.Results: Sputum cell counts varied between 0.95-7´106 cells/g sputum. Isolated RNA content was 332.9±322.2 ng/g sputum. All three processing methods yielded similar amount of RNA, with a slight advantage for DTT (p=NS). Cell integrity was only preserved in DTT. DNase treatment diminished the yield by 30-50%. Surprisingly, in most samples several rounds of DNase treatment was needed to clean the samples from the contaminating DNA. The choice of RNA isolation kit, the speed and the temperature of centrifugation significantly influenced the amount of RNA. The choice of reverse transcriptase had a lesser effect on the PCR products.Conclusion: The quality and the quantity of sputum RNA depends on several factors during the isolation. However, the biggest challenge is the elimination of bacterial DNA, which is of high importance, since contaminating bacterial background might mask the human RNA in gene expression studies. ER -