BACKGROUND: It has recently being emphasized that gene-expression profiles are important clinical decision-making tools, and as such must be standardized across hospital laboratories in the same way as pathological investigations. In this study our objective was to independently establish a standardized gene-expression assay system using routinely processed, formalin-fixed, paraffin-embedded (FFPE) tissues. METHODS: To verify gene expression by quantitative real-time polymerase chain reaction, the most stably expressed reference genes were explored using 30 matched FFPE and fresh frozen (FF) tissues. FFPE specimens from 290 female breast cancer patients were used for further RNA extraction; ESR1 and PGR were measured using 203 matched FFPE and FF specimens and normalized to these reference genes. RESULTS: RNA extracted from FFPE specimens was highly degraded, but almost the same selection of genes was identified-TAF, PUM1, and ACTB, and, for FFPE specimens only, FKBP15. Eventually 88.6% of all the FFPE samples were identified as quantitatively and qualitatively adequate for downstream analysis. The results revealed good correlation and excellent concordance with ERα and PgR protein expression evaluated by immunohistochemistry. Moreover, the distribution of ESR1 and PGR gene expression values was quite reasonable, reflecting differences between the transcriptional mechanisms of the respective genes. CONCLUSIONS: We successfully confirmed that our gene-expression analysis system provides good quality control for larger scale assays; it may therefore be suitable for development, in the near future, of a multiple gene assay as a routine clinical judgment tool.
BACKGROUND: It has recently being emphasized that gene-expression profiles are important clinical decision-making tools, and as such must be standardized across hospital laboratories in the same way as pathological investigations. In this study our objective was to independently establish a standardized gene-expression assay system using routinely processed, formalin-fixed, paraffin-embedded (FFPE) tissues. METHODS: To verify gene expression by quantitative real-time polymerase chain reaction, the most stably expressed reference genes were explored using 30 matched FFPE and fresh frozen (FF) tissues. FFPE specimens from 290 female breast cancerpatients were used for further RNA extraction; ESR1 and PGR were measured using 203 matched FFPE and FF specimens and normalized to these reference genes. RESULTS: RNA extracted from FFPE specimens was highly degraded, but almost the same selection of genes was identified-TAF, PUM1, and ACTB, and, for FFPE specimens only, FKBP15. Eventually 88.6% of all the FFPE samples were identified as quantitatively and qualitatively adequate for downstream analysis. The results revealed good correlation and excellent concordance with ERα and PgR protein expression evaluated by immunohistochemistry. Moreover, the distribution of ESR1 and PGR gene expression values was quite reasonable, reflecting differences between the transcriptional mechanisms of the respective genes. CONCLUSIONS: We successfully confirmed that our gene-expression analysis system provides good quality control for larger scale assays; it may therefore be suitable for development, in the near future, of a multiple gene assay as a routine clinical judgment tool.
Authors: Michael J Gerdes; Anup Sood; Christopher Sevinsky; Andrew D Pris; Maria I Zavodszky; Fiona Ginty Journal: Front Oncol Date: 2014-12-18 Impact factor: 6.244
Authors: Beata Ostasiewicz; Paweł Ostasiewicz; Kamila Duś-Szachniewicz; Katarzyna Ostasiewicz; Piotr Ziółkowski Journal: Mol Med Rep Date: 2016-04-27 Impact factor: 2.952