Paraffin Embedding Contributes to RNA Aggregation, Reduced RNA Yield, and Low RNA Quality  David L. Evers, Junkun He, Yeon Ho Kim, Jeffrey T. Mason, Timothy J. O'Leary  The Journal of Molecular Diagnostics  Volume 13, Issue 6, Pages 687-694 (November 2011) DOI: 10.1016/j.jmoldx.2011.06.007 Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Effect of histologic processing steps on the size of recoverable PCR amplicons. RNA bound to paramagnetic beads was formalin-fixed, passed through graded alcohols and xylene, and embedded in paraffin. After reversing these steps, samples were heated at 70°C for 30 minutes in 1× TAE buffer (pH 9). Equal amounts of RNA were reverse-transcribed into cDNA that was used as a template for the PCR amplifications of sequences corresponding to the Ku80 mRNA from HeLa cells. The left lanes are ϕX174 DNA/HaeIII ladders. Images are inverted from agarose gels stained using ethidium bromide. The Journal of Molecular Diagnostics 2011 13, 687-694DOI: (10.1016/j.jmoldx.2011.06.007) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Effect of histologic processing steps on the electrophoretic mobility of total cellular RNA. RNA isolated from HeLa cells was formalin-fixed or not (native/untreated RNA), passed through graded alcohols and xylene, and paraffin-embedded. After reversing these steps, samples were either untreated or heated at 70°C for 30 minutes in 1× TAE buffer (pH 9). Equal amounts of RNA were heated in loading buffer at 65°C for 10 minutes and loaded onto denaturing agarose gels. 28S and 18S indicate migration of ribosomal subunits. Images stained using ethidium bromide are inverted. Lanes labeled with an asterisk indicate samples that contained visibly insoluble material before and after all heating steps. The Journal of Molecular Diagnostics 2011 13, 687-694DOI: (10.1016/j.jmoldx.2011.06.007) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Heating of formaldehyde-treated RNA in nonpolar solvent was sufficient to produce high-molecular-weight species. Samples were processed and reverse processed as indicated, and equal amounts of RNA were heated in loading buffer at 65°C for 10 minutes and loaded onto denaturing agarose gels. Images stained using ethidium bromide are inverted. Lanes labeled with an asterisk indicate samples that contained visible insoluble material before and after all heating steps. Left panel: Heating fixed RNA in nonpolar solvents produces aggregates. The first lane contained a 1-kb DNA ladder. Middle panel: Effect of time on conversion to high-molecular-weight RNA in xylene heated at 60°C. Right panel: Effect of temperature on conversion to high-molecular-weight RNA in xylene heated for 60 minutes. The Journal of Molecular Diagnostics 2011 13, 687-694DOI: (10.1016/j.jmoldx.2011.06.007) Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions