Detection of viral RNA from paraffin-embedded tissues after prolonged formalin fixation
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 CT 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
References (26)
- et al.
Detection of hepatitis C virus genome in paraffin-embedded tissues by nested reverse transcription polymerase chain reaction
Int Hepatol Commun
(1994) - et al.
Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection
Am J Pathol
(2003) - et al.
Development and evaluation of a fluorogenic 5′-nuclease assay to identify Marburg virus
Mol Cell Probes
(2001) - et al.
Development of a novel internal positive control for Taqman based assays
Mol Cell Probes
(2005) - et al.
The duration of fixation influences the yield of HCV cDNA-PCR products from formalin-fixed, paraffin-embedded liver tissue
J Virol Methods
(1994) - et al.
Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives
J Virol Methods
(1993) - et al.
Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction
J Mol Diagn
(2000) - et al.
Quantitative gene expression analysis in microdissected archival formalin-fixed and paraffin-embedded tumor tissue
Am J Pathol
(2001) - et al.
Tissue extraction of DNA and RNA and analysis by the polymerase chain reaction
J Clin Pathol
(1990) - et al.
Detection of human papilloma virus DNA sequences by polymerase chain reaction
BioTechniques
(1990)
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
Cited by (42)
A Novel Tissue Preservation and Transport Solution as a Substitute for Formalin
2023, Laboratory InvestigationEvaluation of positive Rift Valley fever virus formalin-fixed paraffin embedded samples as a source of sequence data for retrospective phylogenetic analysis
2017, Journal of Virological MethodsCitation Excerpt :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).
Reliable quantification of mRNA in archived formalin-fixed tissue with or without paraffin embedding
2015, Journal of Pharmacological and Toxicological MethodsCitation Excerpt :In other words, short target sequences are much more accessible for quantitation than long target sequences in mRNA derived from fixed tissues. Previous studies indicate that tissue fixation in formalin results in a Ct shift of up to six cycles using conventional RT-qPCR when compared to fresh tissues, and the extent of the increase in Ct value is dependent on the quality of the RNA isolated from fixed tissues and amplicon size (Koch et al., 2006; McKinney, Moon, Kulesh, Larsen, & Schoepp, 2009). This increase would be much greater when severely damaged RNA was used as template together with a longer amplicon.
Laboratory diagnosis of rabies
2013, Rabies: Scientific Basis of the Disease and Its ManagementLaboratory Diagnosis of Rabies
2013, RabiesSingle-Nucleus RNA-Seq: Open the Era of Great Navigation for FFPE Tissue
2023, International Journal of Molecular Sciences