Quantitative RT-PCR from Fixed Paraffin-Embedded Tissues by Capillary Electrophoresis

Stanta, Giorgio; Bonin, Serena; Lugli, Maurizio

doi:10.1385/1-59259-116-7:253

Cited by 3 publications

(1 citation statement)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…More recent studies have shown that RNA can be extracted successfully from formalin-fixed paraffinembedded tissue section and used for cDNA template generation and subsequent PCR (4,5,32,33). The most effective isolation methods involve the use of a concentrated proteinase K digestion step to solubilize tissue proteins and reverse monomethyl nucleotide modification to RNA (24,30).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2002

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

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

et al. 2002

View full text Add to dashboard Cite

show abstract

Models of Biobanking and Tissue Preservation: RNA Quality in Archival Samples in Pathology Laboratories and “In VivoBiobanking” by Tumor Xenografts in Nude Mice—Two Models of Quality Assurance in Pathology

Stanta

Bonin

Machado

et al. 2011

Biopreservation and Biobanking

View full text Add to dashboard Cite

Tissue banks represent essential resources and platforms for biomedical research serving basic, translational, and clinical research projects. In this article, we describe 2 models of biobanking and tissue preservation with different approaches and aims. Archive tissue biobanking is described here as a resource of residual pathology tissues for translational research, which represents the huge clinical heterogeneity. In this context, managing of tissues and RNA quality in archive tissue are discussed. The other model of tissue biobanking is referred to as xenograft tissue banking, which represents an alternative method for obtaining large amounts of tissue, over an indefinite period, in so far as the tumor can be transferred in vivo over generations, maintaining the histological and genetic particularities. A description of the method and examples of the application are given with particular emphasis on sarcomas (Ewing's sarcoma/primitive neuroectodermal sarcoma, synovial sarcomas, and rhabdomyosarcomas) and early stages of tumor angiogenesis in sarcomas.

show abstract

Overview: The Application of Capillary Electrophoresis for DNA Polymorphism Analysis

Mitchelson

Capillary Electrophoresis of Nucleic Acids

View full text Add to dashboard Cite

Quantitative RT-PCR from Fixed Paraffin-Embedded Tissues by Capillary Electrophoresis

Cited by 3 publications

References 6 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

Models of Biobanking and Tissue Preservation: RNA Quality in Archival Samples in Pathology Laboratories and “In VivoBiobanking” by Tumor Xenografts in Nude Mice—Two Models of Quality Assurance in Pathology

Overview: The Application of Capillary Electrophoresis for DNA Polymorphism Analysis

Contact Info

Product

Resources

About