Detection of viral RNA from paraffin-embedded tissues after prolonged formalin fixation

doi:10.1016/j.jcv.2008.09.003

Journal of Clinical Virology

Volume 44, Issue 1, January 2009, Pages 39-42

https://doi.org/10.1016/j.jcv.2008.09.003 Get rights and content

Abstract

Background

Isolating amplifiable RNA from formalin-fixed, paraffin-embedded (FFPE) tissues is more difficult than isolating DNA because of RNases, chemical modification of the RNA, and cross-linking of nucleic acids and proteins. Tissues containing infectious disease agents that require biosafety level (BSL)-3 and -4 necessitate fixation times of 21 and 30 days, respectively.

Objective

To improve procedures for extracting RNA from these FFPE tissues and detect the RNA with the more sensitive TaqMan^®-based reverse transcriptase (RT)-PCR.

Study design

Through a single modification of a commercially available kit, we were able to extract amplifiable RNA and detect West Nile virus (WNV), Marburg virus (MARV), and Ebola virus (EBOV)-infected tissues using TaqMan^® assays.

Results

Formalin fixation results in an approximately 2 log₁₀ reduction in detection limit when compared to fresh tissues. Increasing proteinase K digestion (24 h) improved extraction of amplifiable RNA from FFPE tissues. The TaqMan^® results were comparable to more traditional detection results such as virus isolation.

Conclusion

This improved extraction procedure for obtaining RNA combined with the TaqMan^® RT-PCR assays permit retrospective and prospective studies on FFPE tissues infected with BSL-3 and -4 pathogens.

Introduction

PCR analysis of tissues is used frequently for detecting and identifying infectious disease agents because it provides improved sensitivity compared to other techniques, such as immunohistochemistry (IHC) and in situ hybridization. Traditionally, fresh or frozen tissue is used as a source of RNA or DNA, which limits PCR analysis to prospective studies. Recently, DNA extraction methods of formalin-fixed, paraffin-embedded (FFPE) tissues and PCR analysis have become possible.1, 2 However, similar analyses with RNA are problematic due to degradation by autologous RNases and the fixation and paraffin-embedding procedures. Extracting RNA from FFPE tissues is more difficult, requiring several days and the use of complex extraction methods. After extraction, the RNA is severely degraded to fragments averaging 100–300 nucleotides long.3, 4, 5 In addition, formalin fixation modifies RNA by adding methylol groups (–CH₂OH) to nucleotides⁶ which can interfere with reverse transcription of RNA that reduces PCR efficiency.

TaqMan^® assays are real-time quantitative RT-PCR assays that precisely and linearly measure amplicon accumulation during the exponential phase of the reaction by detecting the increase in fluorescent emission. Coupled with 75–150 bp amplicon sizes, TaqMan^® RT-PCR is ideal for analyzing RNA from FFPE tissues.

Clinical tissue samples are routinely formalin-fixed for less than 24 h followed by paraffin embedding. At the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) standard operating procedures require tissues infected with BSL-3 and -4 bioagents to be fixed in 10% neutral-buffered formalin (NBF) for 21 (BSL-3) or 30 days (BSL-4) prior to molecular pathology studies. However, prolonged fixation is thought to result in irreversible modifications to the RNA.⁶ Our objective was to improve procedures for extracting RNA from these extensively formalin-fixed tissues and subsequently detect the RNA with TaqMan^® RT-PCR assays.

Section snippets

FFPE tissue samples

Brain tissue from suckling mice experimentally infected intracranially with WNV strain NY99, along with additional WNV strain NY99-infected FFPE brain, liver, spleen, and kidney tissues from American crows (Corvus brachyrhynchos) were taken during necropsy and fixed in 10% NBF for 21 days, processed routinely, and paraffin-embedded. Following proteinase K pretreatment, WNV antigen detection was done by incubation with a rabbit hyperimmune polyclonal serum against WNV and a peroxidase-conjugated

RNA extraction optimization

Attempts to extract amplifiable WNV RNA from FFPE tissues using the unmodified commercial kits were unsuccessful (data not shown). Only increasing the proteinase K digestion from 2 to 24 h resulted in the recovery of RNA that could be detected in the TaqMan^® assay.

RNA extraction efficiency

Detection of GAPDH RNA extracted from paired frozen and fixed tissues by real-time PCR indicated FFPE tissues had a shift of 6–8 cycles in C_T value (later) than the corresponding frozen tissues (Table 1), indicating a significant

Discussion

Formalin is the most commonly used fixative in pathology laboratories. Extracting DNA from FFPE tissues has become routine¹³ but RNA extraction remains difficult and inefficient, due to degradation or modification of the RNA. Formalin fixation-induced cross-linking between proteins and DNA or RNA is probably the most significant result of formalin fixation13, 14, 15 and is most likely the greatest obstacle for obtaining RT-PCR-amplifiable RNA. Cross-linking preserves cellular structure, but

Acknowledgements

The authors acknowledge Robert M.C. Worthing and Jeff F. Brubaker for their technical contributions to this work. We thank Lou Sileo (National Wildlife Disease Center, USGS) for providing the WNV-infected crow tissues, Lisa Hensley and Thomas W. Geisbert (Pathology Division, USAMRIID) for providing the EBOV-infected NHP tissues, and Catherine L. Wilhelmsen (Pathology Division, USAMRIID) and Michael Hevey (Virology Division, USAMRIID) for providing the MARV-infected NHP tissues. We thank David

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However, there is a high cost associated with storage and preservation of large numbers of frozen samples for a prolonged period (Joon-Yong et al., 2006) resulting in limited availability of such samples for retrospective studies. With the exception of cases where the virus needs to be isolated, most laboratories receiving clinical or post-mortem samples routinely have such samples fixed in 10% neutral buffered formalin (McKinney et al., 2009). Although formalin-fixed and paraffin-embedded (FFPE) material still remains the optimal sample material for microscopic tissue-based techniques such as immunohistochemistry and histopathology, archived FFPE samples are potentially an alternative source of nucleic acids for molecular analysis and pathogen identification (Adam, 2012).
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In this study, we used a multi-species RT-qPCR method originally developed to detect mRNA in tissue homogenate samples (Wang et al., 2011) and applied it to effectively detect a specific mRNA in formalin-fixed tissues with or without paraffin-embedding by targeting mRNA sequences as short as 24 nucleotides.
Target sizes ranging from 24 to 91 nucleotides were evaluated using this multi-species RT-qPCR assay. Data generated with FFPE tissues demonstrated that use of short target sequences relieved the dependence on RNA quality and could reliably quantify mRNA. This method was highly sensitive, reproducible, and had a dynamic range of five orders of magnitude. Importantly, this method could quantify mRNA in prolonged formalin-fixed and FFPE tissue, where conventional RT-qPCR assays failed. Moreover, a similar result for small interfering RNA (siRNA)-mediated Apob mRNA knockdown was obtained from tissues fixed in formalin solution for 3 months to 4 years, and was found to be comparable to results obtained with frozen liver tissues.
Therefore, the method presented here allows for preclinical and clinical retrospective and prospective studies on mRNA derived from archived FFPE and prolonged formalin-fixed tissue.
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Published by Elsevier B.V.

Detection of viral RNA from paraffin-embedded tissues after prolonged formalin fixation

Abstract

Background

Objective

Study design

Results

Conclusion

Introduction

Section snippets

FFPE tissue samples

RNA extraction optimization

RNA extraction efficiency

Discussion

Acknowledgements

Int Hepatol Commun

Am J Pathol

Mol Cell Probes

Mol Cell Probes

J Virol Methods

J Virol Methods

J Mol Diagn

Am J Pathol

Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction

J Clin Pathol

Detection of human papilloma virus DNA sequences by polymerase chain reaction

BioTechniques

Detection of hepatitis C virus RNA in more than 20-year-old paraffin-embedded liver tissue

Lab Invest

RNA extracted from paraffin-embedded human tissues is amenable to analysis by PCR amplification

BioTechniques

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

Nucleic Acids Res