Abstract 280: RNA isolation from formalin-fixed paraffin-embedded material: A comparison of five commercially available RNA isolation kits

Abstract

Abstract Formalin-fixed paraffin-embedded (FFPE) tissue is the most common tissue specimen widely available. Moreover, long clinical follow-up is on hand. Therefore, FFPE material is a precious source of material for identifying predictive and/or prognostic biomarkers in cancer research on the basis of gene expression. However, the main drawback of FFPE tissue is the significant reduction in quantity and quality of the extracted RNA. The aim of this study is the comparison of different commercially available kits for the RNA isolation in FFPE tissue material. Five commercially available kits were compared and evaluated to identify the most optimal RNA isolation procedure for FFPE material. The FFPE sections used in this study derived from cervical cancer patients treated by surgery. Three paraffin sections (10 μm) per patient were used to isolate RNA, according to the manufacturer's protocol. The following RNA isolation kits were used: Rneasy® FFPE isolation kit (Qiagen), RecoverAllTM Total Nucleic Acid isolation kit (Ambion), ArrayGradeTM FFPE RNA isolation kit (SA Biosciences), NucleoSpin® FFPE RNA isolation kit (Macherey-Nagel) and QuickExtractTM FFPE RNA Extraction Kit (Epicentre® Biotechnologies). RNA extraction was carried out in RNAse free environment. The concentration and purity (A260/A280 ratio and A260/A230 ratio) were measured using a Nanodrop ND-1000 spectrophotometer. The integrity of the isolated RNA was assessed by capillary electrophoresis with an Agilent 2100 Bioanalyzer using Agilent RNA 6000 Series Nano kits, expressed in RNA integrity numbers (RIN). The mean total RNA eluated by the different kits were as follows: Qiagen 25957 ± 19417ng, Ambion 8249 ± 2898ng, SA Biosciences 8070 ± 3700ng and Machery-Nagel 622 ± 394ng. The A260/A280 and A260/A230 ratio was used to evaluate the purity of the samples. The mean A260/A280 ratios were as follows: Qiagen: 1.81 ± 0.23, SA Biosciences: 0.66 ± 0.36, Ambion: 1.03 ± 0.37 and Machery-Nagel: 1.04 ± 0.61 and for the mean A260/A230 ratios: Qiagen: 1.88 ± 0.09, SA Biosciences: 1.61 ± 0.32, Ambion: 1.54 ± 0.30 and Machery-Nagel: 1.88 ± 0.61. The RNA extractions from Epicentre® could not be measured by Nanodrop and, therefore, were excluded from further analysis. The mean RIN values were as follows: Qiagen: 2.19 ± 0,37, SA Biosciences: 2.09 ± 0.38, Ambion: 2.44 ± 0.12 and Macherey-Nagel: 2.30 ± 0.44. Based on the Nanodropdata, the most consistent results were obtained with the Rneasy® FFPE Isolation Kit (Qiagen). However, RNA isolated with RecoverAllTM Total Nucleic Acid isolation kit (Ambion) gave slightly higher RIN values. Taken these results together, the kit from Qiagen appears the most appropriate kit to be used in our further studies that require RNA isolation from FFPE material. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 280. doi:10.1158/1538-7445.AM2011-280