RIN score as measured by the Agilent Bioanalyzer, expression levels of the genes FOS, IL1B, IL8, and GAPDH, and detection of qPCR inhibition. In addition, the expression levels of two new biomarkers, FOSB and TNFRSF10c, developed and validated within the SPIDIA project, as indicators of ex vivo gene expression changes in stored EDTA blood, were also included in order to improve the evaluation of highly labile RNA targets. The results of these two SPIDIA RNA EQA studies have been compiled and will be used by the European Committee for Standardization to propose an evidence-based Technical Specification for pre-analytical handling of blood for RNA-based in vitro diagnostics. Because blood from one donor was not of sufficient volume to provide specimens for all of the participating laboratories, we determined the effect of blood pooling on gene expression. The results Axitinib demonstrated that differential gene expression was observed between pooled and non-pooled blood for the IL1B transcript. Consequently, specimen pooling was abandoned as a proficiency specimen strategy in the second EQA. Blood from two donors were collected and aliquoted into proficiency specimens, and the participating laboratories were randomized into two groups, each group receiving blood specimens from only one donor. Relative to the first SPIDIA-RNA EQA, other modifications were introduced including controlled shipping conditions and defined time and temperature storage conditions of proficiency specimens prior to RNA extraction. One hundred twenty-two applications were received from 21 different European countries, 109 laboratories returned the extracted RNA to the SPIDIA facility by the established deadline. During the first SPIDIA-RNA EQA, there were 124 applications, and 93 laboratories returned RNA samples to the SPIDIA facility. The high response rate from the laboratories for both EQAs indicated a high level of interest and participation both in terms of the number of laboratories enrolled as well as the number of returned RNA samples. The survey queried current laboratory policies and practices specific to specimen handling. Respondents were asked to provide information on blood collection and extraction protocols. The analysis of the survey from the second SPIDIA RNAEQA confirmed the results obtained during the first EQA, which was the preference to use commercially available extraction kits. The majority of the laboratories collected blood in K2EDTA tubes, whereas others used PAXgene tubes. The quality of the extracted RNA samples was evaluated for yield and purity by UV analysis. Purified RNA was most often stored at 280uC, and the predominant downstream analytical methods were PCR technologies. Other aspects of sample handling and analysis protocols were more variable and included the volume of blood used and time and temperature of specimen storage post-phlebotomy. Using the same approach adopted in the first SPIDIA EQA, we evaluated the quality of RNA returned to the SPIDIA facility by participating laboratories.