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

Macabeo-Ong, Maricris; Ginzinger, David G.; Dekker, Nusi P.; McMillan, Alex; Regezi, Joseph A.; Wong, David T.; Jordan, Richard C.

doi:10.1097/01.mp.0000026054.62220.fc

Cited by 125 publications

(80 citation statements)

References 31 publications

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“…All oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA, USA). Primers for EGFR (Macabeo-Ong et al, 2002), and GAPDH and HMBS primers (Tomlins et al, 2005) were as described previously. The forward primer for ERK1 was 5 0 -CAA CAT GAA GGC CCG AAA CTA CC-3 0 and the reverse primer for ERK1 was 5 0 -TAA CAT CCG GTC CAG CAG GTC A AG-3 0 .…”

Section: Rna Isolation and Quantitative Pcrmentioning

confidence: 99%

Role of epidermal growth factor receptor degradation in gemcitabine-mediated cytotoxicity

et al. 2006

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We have recently reported that treatment with gemcitabine, a potent chemotherapeutic agent and radiation sensitizer, stimulates phosphorylation of the epidermal growth factor receptor (EGFR). Because phosphorylation of EGFR is known to precede receptor degradation, we hypothesized that gemcitabine treatment may also result in EGFR degradation. In two human head and neck cancer cell lines, UMSCC-1 and UMSCC-6, we demonstrated an approximately 80% decrease in total EGFR levels at 72 h after a 2-h treatment with 1 lM gemcitabine. Neither cisplatin nor 5-fluorouracil, which are used to treat head and neck cancer, caused EGFR degradation. EGFR downregulation did not occur at the level of transcription, as assessed by reverse transcription-polymerase chain reaction (RT-PCR), but instead occurred via phosphorylation and ubiquitination of the receptor along a proteosome/lysosome-mediated pathway. Inhibition of EGFR degradation, by either pretreatment with the EGFR tyrosine kinase inhibitor gefitinib or by exposure to the proteosome/lysosome inhibitor MG132, significantly reduced gemcitabine-induced cell death. These results suggest that EGFR degradation may be a novel mechanism for gemcitabine-mediated cell death. These findings also indicate that caution should be exercised when combining gemcitabine with agents that may prevent EGFR degradation, such as EGFR tyrosine kinase inhibitors administered in a suboptimal sequence or proteosome inhibitors.

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Section: Rna Isolation and Quantitative Pcrmentioning

confidence: 99%

Role of epidermal growth factor receptor degradation in gemcitabine-mediated cytotoxicity

et al. 2006

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“…[7][8][9][10][11][12][13][14][15][16][17][18] Moreover, several studies have provided new methods to optimize DNA and RNA extraction from archival formalin-fixed tissues, 18 -25 whereas other studies have investigated the effect of duration of fixation on quantitative RT-PCR analyses. 10,12,13,26 There is a paucity of data in the use of other fixatives that incorporate picric acid (Bouin's solution), or mercuric chloride (B5, Zenker's, Helly's, and Ridley's solutions), or tannic acid as nucleic acid preserving agents. 9,27,28 Only one group of researchers reported that neither DNA nor RNA could successfully be extracted from highly cross-linking fixa-tives such as glutaraldehyde, modified formalins containing mercuric chloride, and Bouin's fixative.…”

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confidence: 99%

RT-PCR Analysis of RNA Extracted from Bouin-Fixed and Paraffin-Embedded Lymphoid Tissues

Gloghini

Canal

Klein

et al. 2004

The Journal of Molecular Diagnostics

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In the present study, we have investigated whether RNA can be efficiently isolated from Bouin-fixed or formalin-fixed, paraffin-embedded lymphoid tissue specimens. To this aim, we applied a new and simple method that includes the combination of proteinase K digestion and column purification. By this method, we demonstrated that the amplification of long fragments could be accomplished after a pre-heating step before cDNA synthesis associated with the use of enzymes that work at high temperature. By means of PCR using different primers for two examined genes (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]-and CD40), we amplified segments of cDNA obtained by reverse transcription of the isolated RNA extracted from Bouin-fixed or formalin-fixed paraffin-embedded tissues. Amplified fragments of the expected sizes were obtained for both genes tested indicating that this method is suitable for the isolation of high-quality RNA.

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“…Gene expression analyses in FFPE tissues have been largely limited to in situ hybridization or PCR analyses of RNA (Ben-Ezra et al 1991;Nouri Aria et al 1993;Goldsworthy et al 1999;Hayden et al 2001;Lewis et al 2001;Macabeo-Ong et al 2001;Qian et al 2001). Although informative, conventional PCR and in situ hybridization typically evaluate gene expression of only a few genes simultaneously.…”

mentioning

confidence: 99%

Preliminary Comparison of Quantity, Quality, and Microarray Performance of RNA Extracted From Formalin-fixed, Paraffin-embedded, and Unfixed Frozen Tissue Samples

Scicchitano

Dalmas

Bertiaux

et al. 2006

J Histochem Cytochem.

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S U M M A R Y Microarrays have been used to simultaneously monitor the expression of thousands of genes from biological samples, an approach that can potentially uncover previously unrecognized functions of genes. Microarray analyses can rarely be conducted retrospectively because of the requirement for RNA to be obtained from fresh or unfixed frozen tissues. Archived pathology specimens would need to be used for retrospective analyses, and these are typically preserved as formalin-fixed, paraffin-embedded (FFPE) tissue. Formalin-fixed tissues have been shown to yield compromised RNA compared with that obtained from frozen tissue. To begin to assess the performance of RNA extracted from FFPE samples on a microarray format, we compared RNA from a model system of pelleted lipopolysaccharidestimulated human bone marrow stromal cells that were snap frozen with RNA from FFPE cells. RNA integrity and Affymetrix quality control parameters were assessed, and differentially regulated genes were analyzed with Ingenuity Pathway Analysis software. Results demonstrate that both snap-frozen and FFPE samples yielded intact RNA suitable for amplification prior to Affymetrix GeneChip analysis. Although some transcriptional information was lost with RNA extracted from the FFPE samples, Ingenuity Pathway Analysis revealed that the major pathways identified as affected by drug treatment were similar. Results show that FFPE samples are amenable to Affymetrix GeneChip analysis, expanding the possibility for expression profiling on archived tissue blocks in pathology laboratories.

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

Cited by 125 publications

References 31 publications

Role of epidermal growth factor receptor degradation in gemcitabine-mediated cytotoxicity

Role of epidermal growth factor receptor degradation in gemcitabine-mediated cytotoxicity

RT-PCR Analysis of RNA Extracted from Bouin-Fixed and Paraffin-Embedded Lymphoid Tissues

Preliminary Comparison of Quantity, Quality, and Microarray Performance of RNA Extracted From Formalin-fixed, Paraffin-embedded, and Unfixed Frozen Tissue Samples

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