The Effect of Dithiothreitol on the Transcriptome of Induced Sputum Cells

61.6 ± 8.9% pred. and no respiratory tract infection/exacerbation or anti-inflammatory treatment within the 4 weeks prior to sputum induction). The samples were processed – with or without DTT – to transcriptome analysis, using an Agilent (4 × 44K) platform [4] . In order to obtain an overview of the general effects of DTT, pooled samples (600 ng each) of four RNA preparations from these patients – processed either with or without DTT – were analyzed. Processing of the sputum samples without DTT was performed by adding 8 volumes of PBS with subsequent mechanical homogenization of the sputum sample according to the method of Hector et al. [5] . Processing of the sputum samples with DTT followed a standard protocol adding 4 volumes of DTT to the sputum cell pellet. The results are displayed in figure 1 . The comprehensive dataset is given in the online repository. We observed a strong effect of DTT on the transcriptomes with 2,906 genes differentially regulated on a logarithm base 2 value of the expression ratio (log-2 fold change) of higher than 2.0 (1,755 genes upregulated and 1,151 genes downregulated by DTT). On a log-2 fold change higher than 2.5, a total of 1,384 genes were still affected (917 genes upregulated and 467 downregulated by DTT). Among these, some play a role in COPD, such as interleukin 8 which was clearly upregulated by DTT (log-2 fold change = 5.2). For a complete listing of all genes, see online supplementary material (www.karger.com/doi/10.1159/000348392). We conclude that the effect of DTT needs to be taken into account when transcriptome analysis is applied to induced sputum samples.