Nucleic Acids Research, Vol 27, Issue 22 4436-4443, Copyright © 1999 by Oxford University Press
N Masuda, T Ohnishi, S Kawamoto, M Monden and K Okubo
Formalin-fixed archival samples are known to be poor materials for
molecular biological applications. We conducted a series of experiments to
understand the alterations in RNA in fixed tissue. We found that
formalin-fixed tissue was resistant to solubilization by chaotropic agents.
However, proteinase K completely solubilized the fixed tissue and enabled
the extraction of almost the same amount of RNA as from a fresh sample. The
extracted RNA did not show apparent degradation. However, as reported,
successful PCR amplification was limited to short targets. The nature of
such 'fixed' RNA was analyzed using synthetic homo-oligo RNAs. The
heterogeneous increase in molecular weight of the RNAs, measured by
MALDI-TOF mass spectrometry, showed that all four bases showed addition of
mono-methylol (-CH(2)OH) groups at various rates. The modification rate
varied from 40% for adenine to 4% for uracil. In addition, some adenines
underwent dimerization through methylene bridging. The majority of the
methylol groups, however, could be removed from bases by simply elevating
the temperature in formalin- free buffer. This demodification proved
effective in restoring the template activity of RNA from fixed tissue. The
improvement in PCR results suggested that more than half of the
modification was removed by this demodification.
ARTICLES
Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples
Institute for Molecular and Cellular Biology, Osaka University, 1-3, Yamada-oka, Suita, Osaka 565-0871, Japan.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Z. Ma, W.-O. Lui, A. Fire, and S. S. Dadras Profiling and Discovery of Novel miRNAs from Formalin-Fixed, Paraffin-Embedded Melanoma and Nodal Specimens J. Mol. Diagn., September 1, 2009; 11(5): 420 - 429. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Bohmann, G. Hennig, U. Rogel, C. Poremba, B. M. Mueller, P. Fritz, S. Stoerkel, and K.-L. Schaefer RNA Extraction from Archival Formalin-Fixed Paraffin-Embedded Tissue: A Comparison of Manual, Semiautomated, and Fully Automated Purification Methods Clin. Chem., September 1, 2009; 55(9): 1719 - 1727. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Bandi, S. Zbinden, M. Gugger, M. Arnold, V. Kocher, L. Hasan, A. Kappeler, T. Brunner, and E. Vassella miR-15a and miR-16 Are Implicated in Cell Cycle Regulation in a Rb-Dependent Manner and Are Frequently Deleted or Down-regulated in Non-Small Cell Lung Cancer Cancer Res., July 1, 2009; 69(13): 5553 - 5559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Gazziero, V Guzzardo, E Aldighieri, and A Fassina Morphological quality and nucleic acid preservation in cytopathology J. Clin. Pathol., May 1, 2009; 62(5): 429 - 434. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U Siebolts, H Varnholt, U Drebber, H-P Dienes, C Wickenhauser, and M Odenthal Tissues from routine pathology archives are suitable for microRNA analyses by quantitative PCR J. Clin. Pathol., January 1, 2009; 62(1): 84 - 88. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. W. Gardiner, J. P. Teifke, B. K. Podell, and D. A. Kamstock Fibropapilloma of the glans penis in a horse J Vet Diagn Invest, November 1, 2008; 20(6): 816 - 819. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-Y. Chung, T. Braunschweig, R. Williams, N. Guerrero, K. M. Hoffmann, M. Kwon, Y. K. Song, S. K. Libutti, and S. M. Hewitt Factors in Tissue Handling and Processing That Impact RNA Obtained From Formalin-fixed, Paraffin-embedded Tissue J. Histochem. Cytochem., November 1, 2008; 56(11): 1033 - 1042. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhang, J. Chen, T. Radcliffe, D. P. LeBrun, V. A. Tron, and H. Feilotter An Array-Based Analysis of MicroRNA Expression Comparing Matched Frozen and Formalin-Fixed Paraffin-Embedded Human Tissue Samples J. Mol. Diagn., November 1, 2008; 10(6): 513 - 519. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Hartmann, V. Fernandez, V. Moreno, J. Valls, L. Hernandez, F. Bosch, P. Abrisqueta, W. Klapper, M. Dreyling, E. Hoster, et al. Five-Gene Model to Predict Survival in Mantle-Cell Lymphoma Using Frozen or Formalin-Fixed, Paraffin-Embedded Tissue J. Clin. Oncol., October 20, 2008; 26(30): 4966 - 4972. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Szafranska, T. S. Davison, J. Shingara, M. Doleshal, J. A. Riggenbach, C. D. Morrison, S. Jewell, and E. Labourier Accurate Molecular Characterization of Formalin-Fixed, Paraffin-Embedded Tissues by microRNA Expression Profiling J. Mol. Diagn., September 1, 2008; 10(5): 415 - 423. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Malumbres, J. Chen, R. Tibshirani, N. A. Johnson, L. H. Sehn, Y. Natkunam, J. Briones, R. Advani, J. M. Connors, G. E. Byrne, et al. Paraffin-based 6-gene model predicts outcome in diffuse large B-cell lymphoma patients treated with R-CHOP Blood, June 15, 2008; 111(12): 5509 - 5514. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Doleshal, A. A. Magotra, B. Choudhury, B. D. Cannon, E. Labourier, and A. E. Szafranska Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues J. Mol. Diagn., May 1, 2008; 10(3): 203 - 211. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Sgroi Real-Time PCR Biomarker Analysis Using Routine Pathology Specimens Am. Assoc. Cancer Res. Educ. Book, April 12, 2008; 2008(1): 529 - 530. [Full Text] [PDF]
|
|
|
|
|
|
B. S. Knudsen, A. N. Allen, D. F. McLerran, R. L. Vessella, J. Karademos, J. E. Davies, B. Maqsodi, G. K. McMaster, and A. R. Kristal Evaluation of the Branched-Chain DNA Assay for Measurement of RNA in Formalin-Fixed Tissues J. Mol. Diagn., March 1, 2008; 10(2): 169 - 176. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Haque, D. Faury, S. Albrecht, E. Lopez-Aguilar, P. Hauser, M. Garami, Z. Hanzely, L. Bognar, R. F. Del Maestro, J. Atkinson, et al. Gene Expression Profiling from Formalin-Fixed Paraffin-Embedded Tumors of Pediatric Glioblastoma Clin. Cancer Res., November 1, 2007; 13(21): 6284 - 6292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Loudig, E. Milova, M. Brandwein-Gensler, A. Massimi, T. J. Belbin, G. Childs, R. H. Singer, T. Rohan, and M. B. Prystowsky Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT) Nucleic Acids Res., August 1, 2007; 35(15): e94 - e94. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Paik Development and Clinical Utility of a 21-Gene Recurrence Score Prognostic Assay in Patients with Early Breast Cancer Treated with Tamoxifen Oncologist, June 1, 2007; 12(6): 631 - 635. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Coudry, S. I. Meireles, R. Stoyanova, H. S. Cooper, A. Carpino, X. Wang, P. F. Engstrom, and M. L. Clapper Successful Application of Microarray Technology to Microdissected Formalin-Fixed, Paraffin-Embedded Tissue J. Mol. Diagn., February 1, 2007; 9(1): 70 - 79. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Scicchitano, D. A. Dalmas, M. A. Bertiaux, S. M. Anderson, L. R. Turner, R. A. Thomas, R. Mirable, and R. W. Boyce Preliminary Comparison of Quantity, Quality, and Microarray Performance of RNA Extracted From Formalin-fixed, Paraffin-embedded, and Unfixed Frozen Tissue Samples J. Histochem. Cytochem., November 1, 2006; 54(11): 1229 - 1237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Talantov, J. Baden, T. Jatkoe, K. Hahn, J. Yu, Y. Rajpurohit, Y. Jiang, C. Choi, J. S. Ross, D. Atkins, et al. A Quantitative Reverse Transcriptase-Polymerase Chain Reaction Assay to Identify Metastatic Carcinoma Tissue of Origin J. Mol. Diagn., July 1, 2006; 8(3): 320 - 329. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hamatani, H. Eguchi, K. Takahashi, K. Koyama, M. Mukai, R. Ito, M. Taga, W. Yasui, and K. Nakachi Improved RT-PCR Amplification for Molecular Analyses with Long-term Preserved Formalin-fixed, Paraffin-embedded Tissue Specimens J. Histochem. Cytochem., July 1, 2006; 54(7): 773 - 780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Delfour, P. Roger, C. Bret, M.-L. Berthe, P. Rochaix, N. Kalfa, P. Raynaud, F. Bibeau, T. Maudelonde, and N. Boulle RCL2, a New Fixative, Preserves Morphology and Nucleic Acid Integrity in Paraffin-Embedded Breast Carcinoma and Microdissected Breast Tumor Cells J. Mol. Diagn., May 1, 2006; 8(2): 157 - 169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-S. Chu, Q. Liang, Y. Tang, R. King, K. Wong, M. Gong, M. Wei, J. Liu, S.-H. Feng, S.-C. Lo, et al. Ultrasound-accelerated Tissue Fixation/Processing Achieves Superior Morphology and Macromolecule Integrity with Storage Stability J. Histochem. Cytochem., May 1, 2006; 54(5): 503 - 513. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S C C Wong, A T C Chan, J K C Chan, and Y M D Lo Nuclear {beta}-catenin and Ki-67 expression in choriocarcinoma and its pre-malignant form J. Clin. Pathol., April 1, 2006; 59(4): 387 - 392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Paik Methods for Gene Expression Profiling in Clinical Trials of Adjuvant Breast Cancer Therapy Clin. Cancer Res., February 1, 2006; 12(3): 1019s - 1023s. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Craig, H. Al-Timimi, and N. J. Bernier Differential Increase in Forebrain and Caudal Neurosecretory System Corticotropin-Releasing Factor and Urotensin I Gene Expression Associated with Seawater Transfer in Rainbow Trout Endocrinology, September 1, 2005; 146(9): 3851 - 3860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yamashita and Y. Okada Mechanisms of Heat-induced Antigen Retrieval: Analyses In Vitro Employing SDS-PAGE and Immunohistochemistry J. Histochem. Cytochem., January 1, 2005; 53(1): 13 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. W. Olsen and X. Feng The Minnesota Grading System of Eye Bank Eyes for Age-Related Macular Degeneration Invest. Ophthalmol. Vis. Sci., December 1, 2004; 45(12): 4484 - 4490. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Gloghini, B. Canal, U. Klein, L. Dal Maso, T. Perin, R. Dalla-Favera, and A. Carbone RT-PCR Analysis of RNA Extracted from Bouin-Fixed and Paraffin-Embedded Lymphoid Tissues J. Mol. Diagn., November 1, 2004; 6(4): 290 - 296. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Zhou, F. Lu, C. Latham, D. S. Zander, and G. A. Visner Heme Oxygenase-1 Expression in Human Lungs with Cystic Fibrosis and Cytoprotective Effects against Pseudomonas Aeruginosa In Vitro Am. J. Respir. Crit. Care Med., September 15, 2004; 170(6): 633 - 640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. T. Beggs, A. R. Holmes, R. D. Cannon, and A. M. Rich Detection of Candida albicans mRNA in Archival Histopathology Samples by Reverse Transcription-PCR J. Clin. Microbiol., May 1, 2004; 42(5): 2275 - 2278. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Struckmann, P. Schraml, R. Simon, K. Elmenhorst, M. Mirlacher, J. Kononen, and H. Moch Impaired Expression of the Cell Cycle Regulator BTG2 Is Common in Clear Cell Renal Cell Carcinoma Cancer Res., March 1, 2004; 64(5): 1632 - 1638. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Gjerdrum, B. S. Sorensen, E. Kjeldsen, F. B. Sorensen, E. Nexo, and S. Hamilton-Dutoit Real-Time Quantitative PCR of Microdissected Paraffin-Embedded Breast Carcinoma: An Alternative Method for HER-2/neu Analysis J. Mol. Diagn., February 1, 2004; 6(1): 42 - 51. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. B. Stanton, C. C. Brown, S. Poet, T. P. Lipscomb, J. Saliki, and S. Frasca Jr. RETROSPECTIVE DIFFERENTIATION OF CANINE DISTEMPER VIRUS AND PHOCINE DISTEMPER VIRUS IN PHOCIDS J. Wildl. Dis., January 1, 2004; 40(1): 53 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Kornmann, W. Schwabe, S. Sander, M. Kron, J. Strater, S. Polat, E. Kettner, H. F. Weiser, W. Baumann, H. Schramm, et al. Thymidylate Synthase and Dihydropyrimidine Dehydrogenase mRNA Expression Levels: Predictors for Survival in Colorectal Cancer Patients Receiving Adjuvant 5-Fluorouracil Clin. Cancer Res., September 15, 2003; 9(11): 4116 - 4124. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Schofield, C. Westwater, E. E. Paulling, P. J. Nicholas, and E. Balish Detection of Candida albicans mRNA from Formalin-Fixed, Paraffin-Embedded Mouse Tissues by Nested Reverse Transcription-PCR J. Clin. Microbiol., February 1, 2003; 41(2): 831 - 834. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. N. Abrahamsen, T. Steiniche, E. Nexo, S. J. Hamilton-Dutoit, and B. S. Sorensen Towards Quantitative mRNA Analysis in Paraffin-Embedded Tissues Using Real-Time Reverse Transcriptase-Polymerase Chain Reaction: A Methodological Study on Lymph Nodes from Melanoma Patients J. Mol. Diagn., February 1, 2003; 5(1): 34 - 41. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Srinivasan, D. Sedmak, and S. Jewell Effect of Fixatives and Tissue Processing on the Content and Integrity of Nucleic Acids Am. J. Pathol., December 1, 2002; 161(6): 1961 - 1971. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Noura, H. Yamamoto, T. Ohnishi, N. Masuda, T. Matsumoto, O. Takayama, H. Fukunaga, Y. Miyake, M. Ikenaga, M. Ikeda, et al. Comparative Detection of Lymph Node Micrometastases of Stage II Colorectal Cancer by Reverse Transcriptase Polymerase Chain Reaction and Immunohistochemistry J. Clin. Oncol., October 15, 2002; 20(20): 4232 - 4241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-R. Shi, R. J. Cote, L. Wu, C. Liu, R. Datar, Y. Shi, D. Liu, H. Lim, and C. R. Taylor DNA Extraction from Archival Formalin-fixed, Paraffin-embedded Tissue Sections Based on the Antigen Retrieval Principle: Heating Under the Influence of pH J. Histochem. Cytochem., August 1, 2002; 50(8): 1005 - 1011. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Farrand, L. Jovanovic, B. Delahunt, B. McIver, I. D. Hay, N. L. Eberhardt, and S. K. G. Grebe Loss of Heterozygosity Studies Revisited: Prior Quantification of the Amplifiable DNA Content of Archival Samples Improves Efficiency and Reliability J. Mol. Diagn., August 1, 2002; 4(3): 150 - 158. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Thomas, T. J. Giordano, D. Sanders, J. S. Biermann, and L. Baker Absence of HER2/neu Gene Expression in Osteosarcoma and Skeletal Ewing's Sarcoma Clin. Cancer Res., March 1, 2002; 8(3): 788 - 793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Gillespie, C. J.M. Best, V. E. Bichsel, K. A. Cole, S. F. Greenhut, S. M. Hewitt, M. Ahram, Y. B. Gathright, M. J. Merino, R. L. Strausberg, et al. Evaluation of Non-Formalin Tissue Fixation for Molecular Profiling Studies Am. J. Pathol., February 1, 2002; 160(2): 449 - 457. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Karsten, V. M. D. Van Deerlin, C. Sabatti, L. H. Gill, and D. H. Geschwind An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis Nucleic Acids Res., January 15, 2002; 30(2): e4 - e4. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Schoenberg Fejzo and D. J. Slamon Frozen Tumor Tissue Microarray Technology for Analysis of Tumor RNA, DNA, and Proteins Am. J. Pathol., November 1, 2001; 159(5): 1645 - 1650. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Raida, W. Schwabe, P. Hausler, A. B. P. Van Kuilenburg, A. H. Van Gennip, D. Behnke, and K. Hoffken Prevalence of a Common Point Mutation in the Dihydropyrimidine Dehydrogenase (DPD) Gene within the 5'-Splice Donor Site of Intron 14 in Patients with Severe 5-Fluorouracil (5-FU)- related Toxicity Compared with Controls Clin. Cancer Res., September 1, 2001; 7(9): 2832 - 2839. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-R. Shi, R. J. Cote, and C. R. Taylor Antigen Retrieval Techniques: Current Perspectives J. Histochem. Cytochem., August 1, 2001; 49(8): 931 - 938. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Specht, T. Richter, U. Muller, A. Walch, M. Werner, and H. Hofler Quantitative Gene Expression Analysis in Microdissected Archival Formalin-Fixed and Paraffin-Embedded Tumor Tissue Am. J. Pathol., February 1, 2001; 158(2): 419 - 429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Masuda, Y. Tamaki, I. Sakita, M. Ooka, T. Ohnishi, M. Kadota, N. Aritake, K. Okubo, and M. Monden Clinical Significance of Micrometastases in Axillary Lymph Nodes Assessed by Reverse Transcription-Polymerase Chain Reaction in Breast Cancer Patients Clin. Cancer Res., November 1, 2000; 6(11): 4176 - 4185. [Abstract] [Full Text] [PDF]
|
|