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      Determinants of RNA Quality from FFPE Samples

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          Abstract

          The large archives of formalin-fixed paraffin-embedded (FFPE) tissue specimens that exist are a highly valuable source of sample material for molecular biological analysis, including gene expression profiling. However, current data on adverse effects of standard pathological practice on the usefulness of biomolecular analytes obtained from such archived specimens is largely anecdotal. Here, we present a systematic examination of the most relevant parameters for integrity and useability of RNA obtained from FFPE samples, including storage time and conditions, fixation time, and specimen size. The results are particularly relevant for any application relying on cDNA synthesis as an initial step of the procedure, such as RT-PCR, and microarray analysis.

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          Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples.

          T Ohnishi, S Kawamoto, M Monden … (1999)
          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.
          Article 0 comments Cited 153 times – based on 0 reviews     Review now
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            Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces

            Sandrine Imbeaud, Esther Graudens, Virginie Boulanger … (2005)
            While it is universally accepted that intact RNA constitutes the best representation of the steady-state of transcription, there is no gold standard to define RNA quality prior to gene expression analysis. In this report, we evaluated the reliability of conventional methods for RNA quality assessment including UV spectroscopy and 28S:18S area ratios, and demonstrated their inconsistency. We then used two new freely available classifiers, the Degradometer and RIN systems, to produce user-independent RNA quality metrics, based on analysis of microcapillary electrophoresis traces. Both provided highly informative and valuable data and the results were found highly correlated, while the RIN system gave more reliable data. The relevance of the RNA quality metrics for assessment of gene expression differences was tested by Q-PCR, revealing a significant decline of the relative expression of genes in RNA samples of disparate quality, while samples of similar, even poor integrity were found highly comparable. We discuss the consequences of these observations to minimize artifactual detection of false positive and negative differential expression due to RNA integrity differences, and propose a scheme for the development of a standard operational procedure, with optional registration of RNA integrity metrics in public repositories of gene expression data.
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              Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.

              J Stephans, J. Guillermo Esteban, Maureen T Cronin … (2003)
              Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.
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                Author and article information

                Contributors
                : Role: Academic Editor
                Journal
                Journal ID (nlm-ta): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2007
                Publication date (Electronic): 5 December 2007
                Volume: 2
                Issue: 12
                Electronic Location Identifier: e1261
                Affiliations
                [1 ]QIAGEN GmbH, Hilden, Germany
                [2 ]Gemeinschaftspraxis für Pathologie Niendorf und Hamper, Hamburg, Germany
                The Babraham Institute, United Kingdom
                Author notes
                * To whom correspondence should be addressed. E-mail: martin.schlumpberger@ 123456qiagen.com

                Conceived and designed the experiments: MS. Performed the experiments: Sv. Analyzed the data: MS AM KB. Contributed reagents/materials/analysis tools: AM KB. Wrote the paper: MS.

                Article
                Publisher ID: 07-PONE-RA-01859R1
                DOI: 10.1371/journal.pone.0001261
                PMC ID: 2092395
                PubMed ID: 18060057
                SO-VID: deffded6-d24e-4b78-b1c4-1263feac7216
                Copyright © von Ahlfen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                Date received : 26 July 2007
                Date accepted : 31 October 2007
                Page count
                Pages: 7
                Categories
                Subject: Research Article
                Subject: Genetics and Genomics/Gene Expression
                Subject: Pathology/Molecular Pathology

                ScienceOpen disciplines: Uncategorized
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                ScienceOpen disciplines: Uncategorized

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