Optimization of the isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissue: The armed forces institute of pathology experience and literature review*, **

Krafft, Amy E.; Duncan, Beverly; Bijwaard, Karen E.; Taubenberger, Jeffery K.; Lichy, Jack H.

doi:10.1016/s1084-8592(97)80032-x

Cited by 142 publications

(103 citation statements)

References 57 publications

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“…In the past, it was thought that RNA extracted from paraffin tissue sections was too degraded to permit analysis (30). Many early attempts to extract RNA from formalin-fixed, paraffin-embedded tissues yielded low quantity and poor-quality material.…”

Section: Discussionmentioning

confidence: 99%

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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%

“…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). Although these methods can yield RNA suitable for PCR amplification and analysis, the effect of prolonged formalin fixation has not been well studied.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2002

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“…RNA extraction and reverse transcription were performed as described previously. 19 Real-time PCR cycles were 2 min at 501C, followed by 10 min at 951C and 40 cycles of 15 s at 951C, and 1 min at 601C. Synthetic DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) with three known fusion-transcript sequences were used as positive controls.…”

Section: Gene Rearrangement Studiesmentioning

confidence: 99%

t(11;18)(q21;q21) in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue in stomach: a study of 48 cases

et al. 2009

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Gastric extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MZL-MALT) is speculated to be immune mediated and is notable for responding to treatment by Helicobacter pylori eradication. However, the gastric MZL-MALT with t(11;18)(q21;q21) has been shown to be resistant to treatment by H. pylori eradication. We studied the molecular, immunohistochemical, and histological aspects of 48 cases of gastric MZL-MALT and used a reverse transcription real-time PCR assay to assess the presence of a t(11;18)(q21;q21) in formalin-fixed, paraffin-embedded tissue. Florescence in situ hybridization for t(11:18)(q21;q21) was used to confirm the real-time PCR results. Three distinct morphological subtypes were recognized: monocytoid, small lymphocytic, and plasmacytoid. Morphology, immunophenotype, and immunoglobulin heavy chain (IgH) gene rearrangement were correlated with the results of the t(11:18)(q21;q21) assay. Of the 48 analyzed cases, 15 (31%) were positive for t(11;18)(q21;q21) and 33 (69%) were monoclonal for IgH gene rearrangement. Of the 15, 13 (87%) cases with t(11;18)(q21;q21) translocation showed IgH gene rearrangement by PCR. Of the 33 t(11;18)(q21;q21)-negative cases tested, 20 cases (61%) showed IgH gene rearrangement. The 15 t(11;18)(q21;q21) translocation-positive cases had either monocytoid (12 of 15) or small lymphocytic morphology (3 of 15). Aberrant expression of CD43 was observed in 8 of 15 (53%) t(11;18)(q21;q21)-positive cases and 21 of 31 (68%) t(11;18)(q21;q21)-negative cases. Our data show that t(11;18)(q21;q21)-positive MZL-MALTs frequently show monocytoid morphology, less often small lymphocytic morphology, and not purely plasmacytoid morphology. Identification of a t(11;18)(q21;q21) by reverse transcription real-time PCR is highly specific for MZL-MALT and helps in the diagnosis of MZL-MALT. Studying the correlation between this translocation and morphological features may increase our understanding of the role of this translocation in the pathogenesis and the clinical behavior of gastric MZL-MALT.

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“…As we found no viral DNA, it was not surprising that all our cases were negative for latent nuclear antigen, corroborating the immunohistochemical findings on inflammatory myofibroblastic tumor from varying sites reported by Yamamoto et al 22 and pediatric cases reported by Mergan et al 23 Molecular identification of HHV-8 is known to produce both false-positive and false-negative results. [24][25][26] The latter is thought, in part, to be related to HHV-8 sequence variation, which can range up to 35% in certain regions of the viral genome, as in ORFK1. 26 The use of a single primer set based on a sequence with a high level of sequence variation can lower the PCR sensitivity and, therefore, the rate of HHV-8 detection.…”

Section: Discussionmentioning

confidence: 99%

“…Based on our experience and data in the literature, 250 base pairs are often beyond the upper size limit of what can readily be amplified by PCR from formalin-fixed, paraffin-embedded tissue. 24 False-positive results are thought to be due to utilization of high cycle protocols and/or complimentary primer sets, as in nested PCR. Through a set of validation tests, Pan et al, 25 demonstrated that nested PCR is readily contaminated, even in strictly controlled environments, and use of standard PCR with more than one set of primers is recommended.…”

Section: Discussionmentioning

confidence: 99%

Absence of human herpesvirus-8 in pulmonary inflammatory myofibroblastic tumor: immunohistochemical and molecular analysis of 20 cases

et al. 2007

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Inflammatory myofibroblastic tumors are uncommon lesions composed of spindled myofibroblasts within a variable background of collagen and inflammatory cells. Although the true nature of these lesions is not fully elucidated, identification of consistent cytogenetic alterations in the anaplastic lymphoma kinase (ALK) gene suggests that they may be neoplastic. A small number of inflammatory myofibroblastic tumors have been reported to harbor human herpesvirus-8 (HHV-8), implicating the virus in its pathogenesis. In this study, 20 cases of pulmonary inflammatory myofibroblastic tumor were analyzed for the presence of HHV-8 with immunohistochemical and molecular methods. In all cases, antibodies to the latent nuclear antigen of the virus were applied. Four open reading frames (ORFs), including ORFs K2, 16, 26, and 72, were targeted utilizing realtime polymerase chain reaction (PCR). The cohort included 9 men and 11 women with a mean age of 37 years

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Optimization of the isolation and amplification of RNA from formalin-fixed, paraffin-embedded tissue: The armed forces institute of pathology experience and literature review*, **

Cited by 142 publications

References 57 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

t(11;18)(q21;q21) in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue in stomach: a study of 48 cases

Absence of human herpesvirus-8 in pulmonary inflammatory myofibroblastic tumor: immunohistochemical and molecular analysis of 20 cases

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