Abstract 1706: Comparison of automated and manual RNA isolation from stabilized peripheral blood samples (PBL) for expression analysis of colorectal cancer patients

Abstract

Abstract Background and aims: PBL based molecular diagnostics requires automated sample processing and evaluation of the isolated RNA for RT-PCR analysis. Our aim was to compare the automated PBL RNA isolation on MagNA Pure 96 with conventional manual RNA isolation by the PAXgene Blood RNA kit and to determine the purity, recovery, RNA integrity (RIN) and sensitivity of downstream RT-PCR applications. Materials and Methods: PBL samples were taken from 25 CRC and 19 healthy normal patients into PAXgene Blood RNA Tubes (PreAnalytix). Total RNA was manually isolated using the PAXgene Blood RNA Kit (Qiagen) for 2 blood samples of each patient. In parallel, the MagNA Pure 96 Cellular RNA Kit LV and MagNA Pure 96 instrument (MP96) (Roche) were applied for total RNA extraction from the other 2 blood samples of each patient. Total RNA concentration was measured using a NanoDrop spectrophotometer. RIN was determined on a Bioanalyzer 2100 (Agilent) using the RNA6000 Nano Kit. Reverse transcription was carried out from 1ug total RNA using Transcriptor First Strand cDNA Synthesis Kit (Roche). The expression of 6 CRC markers from previous microarray analysis (ASGR2, CLIP1, ARHGAP18, 1569263_at, C19orf43, MALAT1) was determined by quantitative real-time PCR on LightCycler 480 System (Roche). Results: The MP96 isolation resulted in higher RNA yields (MP96: 5.99±2.04 ug/tube; manual: 5.65±2.75 ug/tube). The purity OD260/280 of manually isolated samples (2.09 ± 0.03) was equal to that of MP96 isolated ones (2.01 ± 0.05), all samples fulfilled the requirement. The OD230/260 was significantly better in case of MP96 isolated samples (1.85 ± 0.23) compared to manually isolated ones (1.52 ± 0.57) (p<0.0001). The RNA integrity was acceptable in both isolation method, however the manual protocol provided more intact RNA with slightly higher RNA integrity numbers (RIN manual: 9,19 ± 0,26; RIN MP96: 8,25 ± 0,35; p<0.0001). No significant RIN differences were detected between prospective and archived samples. Comparison of both extraction methods and evaluation by RT-PCR showed a linear regression (r = 0.98). In real-time PCR experiments, ARHGAP18 (spec.:80%, sens.:75%) and ASGR2 (spec.:70%, sens.:75%) were proved to be the best differentiating individual markers. The CTs of the RNA extracted by MP96 were lower than those of the manual extraction (means higher expression) reflecting the higher mRNA yield/ratio of the MP96 method. Conclusion: Automated RNA isolation from stabilized PBL samples on MP96 device provides a high quality, purity and yield of total RNA. The CTs of the RNA extracted by MP96 were better than those of the manual extraction. The MP96 extraction is a fully automated, fast and reliable method with 96 RNA extractions within 85 min, compared to the equally precise, but time consuming manual extraction by the PAXgene Blood RNA kit. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1706. doi:1538-7445.AM2012-1706