Abstract B32: High-throughput solution for microRNA (miRNA) isolation from stabilized whole blood in a GLP setting: Development and comparison to established procedures.

Abstract

Abstract Introduction: Beside the analysis of messenger RNA (mRNA) expression changes, profiling of miRNAs in human whole blood holds much promise for the development of genetic markers of disease. Many scientific groups in academia and in life science companies investigate the use of miRNAs as diagnostic tools at the moment. As these small RNAs become more and more important, effective and reliable isolation procedures are urgently needed. Especially standardization and exclusion of human errors require new automated solutions which are able to replace established manual processes without losing quality and quantity of the needed analytes. In this study we compare a newly developed and optimized procedure on QIAGEN's BioRobot platform to previously published automated (Kruhøffer et al., 2007, JMD 9: 452–458) as well as manual procedures. Material & Methods: Whole blood from consented healthy adults was collected into PAXgene Blood RNA Tubes and subjected to RNA isolation. The RNA quality and quantity were determined by using a Beckman photometer and Agilent Bioanalyzer. miRNA yields were determined by quantitative RT-PCR using commercially available probe based (Life Technologies) assays on a BioMark (Fluidigm) instrument. Due to the small reaction volume of 9 nanoliters on the BioMark arrays a pre-amplification of the cDNA was performed. Therefore 14 amplification cycles were run with a 1:100 diluted primer set which was the same that was used in the following detection assays. Results: Compared to the originally published partly automated method the workflow could be further streamlined resulting in a fully automated procedure. With this automation the time needed for manual steps could be reduced by more than 50%. In parallel, the quality and quantity of the isolated RNA (large and small RNA species) was not affected. This is demonstrated by high RIN values (>7) and low DNA contamination levels <0.1%. With respect to miRNA enrichment, the performance of the optimized protocol was at least comparable to the current manual or partly automated methods. Conclusion: This comparison demonstrates the high efficiency of this optimized, fully automated isolation procedure for miRNAs from stabilized whole blood. The new procedure for purification of miRNAs provides the standardization required in a GLP setting. All miRNA enrichment protocols, the PAXgene Blood miRNA Kit and the PAXgene Blood RNA MDx Kit are for research use only. Not for use in diagnostic procedures. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B32.