RT-PCR Analysis of RNA Extracted from Bouin-Fixed and Paraffin-Embedded Lymphoid Tissues Annunziata Gloghini, Barbara Canal, Ulf Klein, Luigino Dal Maso,

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RT-PCR Analysis of RNA Extracted from Bouin-Fixed and Paraffin-Embedded Lymphoid Tissues Annunziata Gloghini, Barbara Canal, Ulf Klein, Luigino Dal Maso, Tiziana Perin, Riccardo Dalla-Favera, Antonino Carbone The Journal of Molecular Diagnostics Volume 6, Issue 4, Pages 290-296 (November 2004) DOI: 10.1016/S1525-1578(10)60524-7 Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Representative analysis of RNA isolated from frozen tissue and Bouin-fixed tissue using the Agilent Bioanalyzer (see Material and Methods). Left: Picture of the gel; the fragment sizes of the RNA ladder are indicated. Right: The corresponding histograms of the samples; fluorescence and time are indicated. For the size of the peaks of the RNA ladder (bottom), see left. The 28S and 18S rRNA peaks are indicated in the top histogram. The Journal of Molecular Diagnostics 2004 6, 290-296DOI: (10.1016/S1525-1578(10)60524-7) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Products of RT-PCR performed on RNA isolated from a Bouin-fixed tissue samples reverse transcripted at 42°C (lanes 1 and 2) or at 60°C (lanes 3 and 4) using primers for CD40. M, DNA marker. The Journal of Molecular Diagnostics 2004 6, 290-296DOI: (10.1016/S1525-1578(10)60524-7) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 RT-PCR for the detection of a 921-bp GAPDH fragment. A: RNA isolated from a Bouin-fixed (lanes 1 to 4, C−) reactive lymph node and reverse transcripted at 60°C by using random hexamer or oligo (dT)20. C−, negative control; M, DNA marker. B: RNA isolated from Bouin-fixed (lanes 1, C1−) or formalin-fixed (lanes 2, C2−) reactive lymph node and reverse transcripted at 60°C. C−, negative control; M, DNA marker. The Journal of Molecular Diagnostics 2004 6, 290-296DOI: (10.1016/S1525-1578(10)60524-7) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Real time RT-PCR: standard curves for β 2 microglobulin in cDNA obtained from matched frozen, Bouin-fixed, and formalin-fixed, paraffin-embedded tissues. A strong linear correlation between the CT values and the log of the input cDNA amount (correlation coefficients ranging from 0.97 to 1.0) was obtained. Amplification efficiency in matching frozen, Bouin-fixed, and formalin-fixed tissue samples is comparable, as indicated by similar slopes of the regression lines. The Journal of Molecular Diagnostics 2004 6, 290-296DOI: (10.1016/S1525-1578(10)60524-7) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions