2009
DOI: 10.1097/pdm.0b013e3181a06f42
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Validation of Putative Reference Genes for Normalization of Q-RT-PCR Data From Paraffin-embedded Lymphoid Tissue

Abstract: Normalization of quantitative reverse transcription-PCR (Q-RT-PCR) data to appropriate tissue-specific reference genes is an essential part of interpreting the results. This study aimed to determine the most appropriate reference genes for normalizing gene expressions in lymphatic tissue, represented by non-neoplastic lymph nodes and diffuse large B-cell lymphomas, by using 2 statistical software applications, geNorm and NormFinder. In addition, we wanted to validate the usefulness of paraffin-embedded samples… Show more

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Cited by 13 publications
(16 citation statements)
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“…This approach has demonstrated clinical utility (Clark-Langone et al, 2010). Our study utilized several additional measures to validate the technique, including primers designed to span introns, primers that are non-homologous with other human cDNA sequences, and inclusion of negative and positive controls (cDNA vectors with verified DNA sequence of each gene) for each reaction (April et al, 2009; Green et al, 2009; Sanchez-Navarro et al, 2010). Furthermore, the generated PCR product was confirmed with examination of melting peaks and by agarose gel electrophoresis (Christophi et al, 2012b).…”
Section: Resultsmentioning
confidence: 99%
“…This approach has demonstrated clinical utility (Clark-Langone et al, 2010). Our study utilized several additional measures to validate the technique, including primers designed to span introns, primers that are non-homologous with other human cDNA sequences, and inclusion of negative and positive controls (cDNA vectors with verified DNA sequence of each gene) for each reaction (April et al, 2009; Green et al, 2009; Sanchez-Navarro et al, 2010). Furthermore, the generated PCR product was confirmed with examination of melting peaks and by agarose gel electrophoresis (Christophi et al, 2012b).…”
Section: Resultsmentioning
confidence: 99%
“…Other studies report equivalent performance among differentially preserved biospecimens (1,28,29). Of the studies that reported decreased success in FFPE biospecimens, differences were attributed to RNA degradation, leading to alterations in transcript abundance and length (2,3,23,24). Importantly, such alterations are not global, as transcript-specific effects have been noted (30).…”
Section: Methodsmentioning
confidence: 99%
“…Analytical parameters such as amplicon size and priming method can be manipulated to improve assay success. While RT-PCR success with FFPE biospecimens is influenced by amplicon size (2,23,24,28), reliable qRT-PCR data has been obtained with both FFPE and frozen biospecimens for amplicons between 54 and 105 bp in length (2). One study reported that low percent present rates for FFPE biospecimens increased when samples were primed with both oligo dT and random hexamer primers and analyzed by Exon arrays (25), while another study reported an increase following transcript repair (24).…”
Section: Methodsmentioning
confidence: 99%
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