Short communication Gene expression measurements in the context of epidemiological studies High-throughput genomic technologies allow to measure large-scale gene expression profiles in a time-efficient way. These methodological advances have spread the range of potential applications and allow gene expres- sion analyses to be used in the context of epidemiolog- ical studies (1, 2). However, in epidemiological study settings, other sources of measurement error have to be considered than in clinical or laboratory research: distance between location of blood collection and further processing, different fieldworkers involved in the studies, and, particularly in international studies, different laboratories and laboratory staff are likely to affect quality of the obtained gene expression data. In the present study, we had the possibility to analyse gene expression data of two epidemiological studies [PARSIFAL (3) and PASTURE (4)]. We compared centre-associated variations in RNA quanti- ties obtained using two sampling methods. Further- more, we tested the comparability of low-density arrays, a high-throughput method, with the conven- tional single tube assay to assess gene expression. Finally, the PARSIFAL study allowed us to follow up RNA quality, as multiple measures of the same samples at different time points were available. The aim was to evaluate sources of measurement error in gene expres- sion data in order to avoid these factors in future investigations. Background: Gene expression measurements became an attractive tool to assess biological responses in epidemiological studies. However, collection of blood samples poses various technical problems. We used gene expression data from two epidemiological studies to evaluate differences between sampling methods, comparability of two methods for measuring RNA levels and stability of RNA samples over time. Methods: For the PARSIFAL study, PBLC of 1155 children were collected using EDTA tubes in two countries. In the PASTURE study, tubes containing RNA- stabilizing solutions (PAXgene Ò Blood RNA Tubes; PreAnalytiX) were used to collect cord blood leucocytes of 982 children in five countries. Real-time PCR (conventional single tube assay and high-throughput low density arrays) was used to quantify expression of various innate immunity genes. In 77 PARSIFAL samples, gene expression was measured repeatedly during prolonged storage. Results: In PARSIFAL (EDTA tubes) the median RNA yield after extrac- tion significantly differed between the two centres (70 and 34 ng/ll). Collecting blood into an RNA-stabilizing solution markedly reduced differences in RNA yield in PASTURE (range of medians 91–107 ng/ll). The agreement [Spearman rank correlation (r)] between repeated measurements of gene expression de- creased with increasing storage time [e.g., for CD14: r (first/second measure- ment) = 0.35; r (first/third measurement) = 0.03]. RNA levels measured with either the conventional method or low-density arrays were comparable (r > 0.9). Conclusion: Collecting blood samples into tubes containing an RNA-stabilizing solution increases RNA yield and reduces its variability. Long-term storage of samples may lead to RNA degradation, requiring special attention in longitu- dinal studies. C. Bieli 1 , R. Frei 2 , V. Schickinger 2 , J. Steinle 2 , C. Bommer 2 , S. Loeliger 2 , C. Braun-Fahrländer 1 , E. von Mutius 3 , G. Pershagen 4 , R. Lauener 2 1 Institute of Social and Preventive Medicine, University of Basel, Basel, Switzerland; 2 University of Zurich, ChildrenÕs Hospital, Zurich, Switzerland; 3 University ChildrenÕs Hospital, Munich, Germany; 4 Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden Key words: blood sampling; epidemiology; gene expression; innate immunity; PAXgene; RNA degradation Christian Bieli, MD Institute of Social and Preventive Medicine University of Basel Steinengraben 49 4051 Basel Switzerland C. Bieli and R. Frei contributed equally Accepted for publication 10 March 2008 Abbreviations: CBLC, cord blood leucocytes; EDTA, ethylene diamine tetraacetic acid; LDA, low density array; PARSIFAL, prevention of allergy risk factors for sensitization in children related to farming and anthroposophic lifestyle; PASTURE, protection against allergy: study in rural environments; PBLC, peripheral blood leucocytes; rtPCR, real-time polymerase chain reaction; STA, (conventional) single tube assay. Allergy 2008: 63: 1633–1636 Ó 2008 The Authors Journal compilation Ó 2008 Blackwell Munksgaard DOI: 10.1111/j.1398-9995.2008.01744.x 1633