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Updated: May 27, 2026
Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
Published on: May 5, 2018
Expression analysis on archival material: comparison of 5 commercially available RNA isolation kits for FFPE material
Carolien Boeckx1, An Wouters, Bea Pauwels
1Center for Oncological Research Antwerp, Laboratory of Cancer Research and Clinical Oncology, University of Antwerp, Wilrijk, Belgium.
Background:
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.
Methods:
Five commercially available RNA isolation kits were tested and the concentration, purity, integrity, and raw cycle threshold values were determined.
Results:
The mean total RNA concentrations were as follows: Qiagen 25957±19417 ng, Ambion 8249±2898 ng, SA Biosciences 8070±3700 ng, and Macherey-Nagel 622±394 ng. The mean A260/A280 ratios were as follows: Qiagen: 1.81, SA Biosciences: 0.66, Ambion: 1.03, and Macherey-Nagel: 1.04. The mean A260/A230 ratios were as follows: Qiagen: 1.88, SA Biosciences: 1.61, Ambion: 1.54, and Macherey-Nagel: 1.88. The RNA extractions from Epicentre could not be measured by the Nanodrop and, therefore, were excluded from further analysis. The mean RNA integrity number (range, 2.09 to 2.47) and the mean raw cycle threshold values (range, 33.43 to 35.37) were more or less the same for all the tested RNA isolation kits.
Conclusions:
Altogether, on the basis of the number of adequate isolations, the kit from Qiagen seems to be the most appropriate kit to be used in our further studies that require RNA isolation from FFPE material.
