RNA From Decades-Old Archival Tissue Blocks for Retrospective Studies

Mizuno, Takuya; Nagamura, Hiroko; Iwamoto, Keisuke S.; Ito, Takashi; Fukuhara, Tatsuro; Tokunaga, Masayoshi; Tokuoka, Shoji; Mabuchi, Kiyóhiko; Seyama, Toshio

doi:10.1097/00019606-199808000-00004

Cited by 90 publications

(63 citation statements)

References 27 publications

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“…Although these methods can yield RNA suitable for PCR amplification and analysis, the effect of prolonged formalin fixation has not been well studied. Previous studies that have examined this issue in a controlled manner have used fixation times of Յ24 hours (3,5,24,34,35). Although this duration may be suitable for small tissues obtained in some hospital settings, it is likely shorter than those typically used for larger specimens or those obtained in outpatient settings or in mail-in biopsy services.…”

Section: Discussionmentioning

confidence: 99%

Effect of Duration of Fixation on Quantitative Reverse Transcription Polymerase Chain Reaction Analyses

et al. 2002

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Increasingly, there is the need to analyze gene expression in tumor tissues and correlate these findings with clinical outcome. Because there are few tissue banks containing enough frozen material suitable for large-scale genetic analyses, methods to isolate and quantify messenger RNA (mRNA) from formalin-fixed, paraffin-embedded tissue sections are needed. Recovery of RNA from routinely processed biopsies and quantification by the polymerase chain reaction (PCR) has been reported; however, the effects of formalin fixation have not been well studied. We used a proteinase K-salt precipitation RNA isolation protocol followed by TaqMan quantitative PCR to compare the effect of formalin fixation for 24, 48, and 72 hours and for 1 week in normal (2), oral epithelial dysplasia (3), and oral squamous cell carcinoma (4) specimens yielding 9 fresh and 36 formalin-fixed samples. We also compared mRNA and protein expression levels using immunohistochemistry for epidermal growth factor receptor (EGFR), matrix metalloproteinase (MMP)-1, p21, and vascular endothelial growth factor (VEGF) in 15 randomly selected and routinely processed oral carcinomas. We were able to extract RNA suitable for quantitative reverse transcription (RT) from all fresh (9/9) and formalinfixed (36/36) specimens fixed for differing lengths of time and from all (15/15) randomly selected oral squamous cell carcinoma. We found that prolonged formalin fixation (>48 h) had a detrimental effect on quantitative RT polymerase chain reaction results that was most marked for MMP-1 and VEGF but less evident for p21 and EGFR. Comparisons of quantitative RT polymerase chain reaction and immunohistochemistry showed that for all markers, except p21, there was good correlation between mRNA and protein levels. p21 mRNA was overexpressed in only one case, but protein levels were elevated in all but one tumor, consistent with the established translational regulation of p21. These results show that RNA can be reliably isolated from formalin-fixed, paraffinembedded tissue sections and can produce reliable quantitative RT-PCR data. However, results for some markers are adversely affected by prolonged formalin fixation times.

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Section: Discussionmentioning

confidence: 99%

Effect of Duration of Fixation on Quantitative Reverse Transcription Polymerase Chain Reaction Analyses

et al. 2002

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“…Although RNA can be extracted from such tissue (Rupp & Locker 1988), extensive degradation can occur before (Mizuno et al 1998) or during (Klimecki et al 1994) the formalin fixation process. Furthermore, formalin fixation generates cross-links between nucleic acids and proteins and covalently modifies RNA, making subsequent RNA extraction, RT and quantification analysis problematic (Masuda et al 1999).…”

Section: Template Preparationmentioning

confidence: 99%

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Bustin

2002

Journal of Molecular Endocrinology

2,232

1,674

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The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

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“…A short time interval between sampling and fixation also seems to be important for efficient amplification Mizuno et al, 1998). The needle liver biopsies analyzed in the present study were immediately fixed in formalin after being taken, and the fixation time was no longer than 24 hours.…”

Section: Soguero Et Almentioning

confidence: 99%

“…Recently, several authors reported that HCV-RNA sequences may also be recovered and amplified by RT-PCR from formalin-fixed, paraffin-embedded (FFPE) liver tissue (Abe et al, 1998;Akyol et al, 1992;Bresters et al, 1992;Edamato et al, 1996;el-Batanony et al, 1994;Guerrero et al, 1997), even when stored for more than 20 years (Mizuno et al, 1998;Soguero et al, 1999). This approach may be valuable for retrospective studies when frozen liver tissue or serum are not available.…”

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confidence: 99%

“…Different strategies, such as prolonged tissue digestion and amplification of only small RNA targets, have been applied to overcome these shortcomings. However, RNA amplification from FFPE tissues more than 10 years old has rarely been achieved, and only the highly conserved 5Ј non-coding region (5ЈNCR) of HCV has so far successfully been amplified from HCV-infected liver tissue (Abe et al, 1998;Bresters et al, 1992;Edamato et al, 1996;Guerrero et al, 1997;Mizuno et al, 1998;Soguero et al, 1999).…”

mentioning

confidence: 99%

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Assessment of Genotype and Molecular Evolution of Hepatitis C Virus in Formalin-Fixed Paraffin-Embedded Liver Tissue from Patients With Chronic Hepatitis C Virus Infection

Soguero

Campo

Ribalta

et al. 2000

Laboratory Investigation

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SUMMARY:Drawbacks of hepatitis C virus (HCV) RNA detection in paraffin-embedded liver tissue have satisfactorily been solved by RT-PCR amplification of the 5Јnon-coding region (5ЈNCR). However, detection of this highly conserved region does not provide information on epidemiological or pathogenetic aspects of HCV infection. This study explores whether other functionally important genetic regions of HCV, such as the hypervariable region 1 (HVR-1) and the interferon sensitivity-determining region (ISDR), can be retrieved from paraffin-embedded liver specimens by RT-PCR, and whether the amplified material is suitable for further molecular analyses. RT-PCR amplification of 5ЈNCR, HVR-1, and ISDR was assessed in RNA extracted from 50 formalin-fixed, paraffin-embedded liver specimens, including 23 needle liver biopsies (11 from patients with non-A, non-B chronic hepatitis diagnosed between 1971 and 1985, 8 from subjects with normal liver histology and 4 from sequential biopsies from a patient with HCV recurrence after liver transplantation), and 27 liver explants from patients undergoing transplantation between 1988 and 1996 (16 with HCV-related cirrhosis and 11 with other disorders). The 5ЈNCR was successfully amplified in 8 of 11 (73%) non-A, non-B chronic hepatitis biopsies and in all of the specimens from patients with serological documentation of HCV infection. There were no false-positive results. HCV genotype was identified by RFLP analysis of the 5ЈNCR in the 13 cases analyzed. HVR-1 and ISDR were amplified in 24 of 28 (86%) samples, which were positive for the 5ЈNCR. Efficient amplification was inversely related to the time of storage. The evolutionary changes of HVR-1 and ISDR were successfully analyzed by direct sequencing of amplificates from the explanted liver and from the sequential liver biopsies in a patient with HCV infection recurrence after transplantation. These observations indicate that paraffin-embedded liver tissue, even when stored for more than 20 years, is appropriate for advanced studies on the molecular biology of HCV. (Lab Invest 2000, 80:851-856).

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RNA From Decades-Old Archival Tissue Blocks for Retrospective Studies

Cited by 90 publications

References 27 publications

Effect of Duration of Fixation on Quantitative Reverse Transcription Polymerase Chain Reaction Analyses

Effect of Duration of Fixation on Quantitative Reverse Transcription Polymerase Chain Reaction Analyses

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Assessment of Genotype and Molecular Evolution of Hepatitis C Virus in Formalin-Fixed Paraffin-Embedded Liver Tissue from Patients With Chronic Hepatitis C Virus Infection

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