Introduction: Gene expression analysis in peripheral blood is an important tool in molecular diagnostics, monitoring diseases at the molecular level, clinical research, and clinical trials of new drugs. Since the introduction of the PAXgene Blood RNA System, researchers have expressed a need for a low-throughput, automated (LTA) solution for RNA preparation from blood collected in PAXgene Blood RNA Tubes, with purification based on the proven silica-membrane spin-column technology in the PAXgene Blood RNA Kit. The aim of this research study was to develop and optimize a protocol for LTA RNA purification using PAXgene Blood RNA Tubes and the (manual) PAXgene Blood RNA Kit on a robotic platform. (Note: The combination of the PAXgene Blood RNA Kit on the robotic platform is for research use only and not for use in diagnostic procedures. It has not received clearance or approval for clinical use in the US, Canada or Europe.) Methods: Replicate blood samples were collected in PAXgene Blood RNA Tubes, and starting from the resuspended nucleic acid pellet, RNA was purified using either the manual procedure according to the PAXgene Blood RNA Kit handbook (reference protocol) or the new automated purification procedure (test protocol). To enable comparison of the test and reference protocol performance, purified RNA from both protocols was analyzed spectrophotometrically, by capillary gel electrophoresis, in real-time quantitative RT-PCR (RQ-RT-PCR), and in PCR assays to measure RNA yield, purity, integrity, RQ-RT-PCR inhibition, and traces of genomic DNA (gDNA). Results: The new automated PAXgene Blood RNA protocol was highly reliable in that there were no failures in sample processing. Furthermore, the quantity and quality of purified RNA was comparable between both protocols: Greater than 95% of samples processed on the instrument yielded RNA of >3micrograms per tube, all samples contained <1% (w/w) residual gDNA, no RQ-RT-PCR inhibition, and A260/A280 ratios of the purified RNA were between 1.8 and 2.2. Conclusion: This research study demonstrates that the new automated protocol using proven silica-membrane spin-column technology and existing chemistry with the new robotic platform provides an efficient and reliable alternative LTA solution to the manual protocol. Automated processing reduces user interaction and hands-on time, resulting in a more convenient workflow compared to the manual PAXgene Blood RNA procedure.
First AACR Centennial Conference on Translational Cancer Medicine-- Nov 4-8, 2007; Singapore