BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tumor material represents a valuable resource for the analysis of RNA-based biomarkers, both in research laboratories and in routine clinical testing. A robust and automated RNA-extraction method with a high sample throughput is required. METHODS: We evaluated extraction performance for 4 silica-based RNA-extraction protocols: (a) a fully automated, bead-based RNA-isolation procedure; (b) its manual counterpart; (c) a semiautomated bead-based extraction system; and (d) a manual column-based extraction kit. RNA from 360 sections (90 sections per extraction method) of 30 FFPE tumor blocks up to 20 years of age was purified and analyzed by quantitative reverse-transcription PCR for ESR1 (estrogen receptor 1), PGR (progesterone receptor), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)], and RPL37A (ribosomal protein L37a). RESULTS: The semiautomated protocol gave the best yield. The 3 bead-based methods showed good across-method correlations in both yield and relative mRNA amounts (r = 0.86-0.95 and 0.98, respectively). In contrast, correlations between any of the bead-based methods and the manual column-based method were worse (r = 0.77-0.95 and 0.96, respectively). The fully automated method showed the lowest variation from section to section (root mean square error, 0.32-0.35 Cq, where Cq is the quantification cycle) and required the least hands-on time (1 h). CONCLUSIONS: The fully automated RNA-purification method showed the best reproducibility in gene expression analyses of neighboring sections of tissue blocks between 3 and 20 years of age and required the least overall and hands-on times. This method appears well suited for high-throughput RNA analyses in both routine clinical testing and translational research studies with archived FFPE material.
BACKGROUND:Formalin-fixed paraffin-embedded (FFPE) tumor material represents a valuable resource for the analysis of RNA-based biomarkers, both in research laboratories and in routine clinical testing. A robust and automated RNA-extraction method with a high sample throughput is required. METHODS: We evaluated extraction performance for 4 silica-based RNA-extraction protocols: (a) a fully automated, bead-based RNA-isolation procedure; (b) its manual counterpart; (c) a semiautomated bead-based extraction system; and (d) a manual column-based extraction kit. RNA from 360 sections (90 sections per extraction method) of 30 FFPE tumor blocks up to 20 years of age was purified and analyzed by quantitative reverse-transcription PCR for ESR1 (estrogen receptor 1), PGR (progesterone receptor), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)], and RPL37A (ribosomal protein L37a). RESULTS: The semiautomated protocol gave the best yield. The 3 bead-based methods showed good across-method correlations in both yield and relative mRNA amounts (r = 0.86-0.95 and 0.98, respectively). In contrast, correlations between any of the bead-based methods and the manual column-based method were worse (r = 0.77-0.95 and 0.96, respectively). The fully automated method showed the lowest variation from section to section (root mean square error, 0.32-0.35 Cq, where Cq is the quantification cycle) and required the least hands-on time (1 h). CONCLUSIONS: The fully automated RNA-purification method showed the best reproducibility in gene expression analyses of neighboring sections of tissue blocks between 3 and 20 years of age and required the least overall and hands-on times. This method appears well suited for high-throughput RNA analyses in both routine clinical testing and translational research studies with archived FFPE material.
Authors: C Brüggemann; M C Kirchberger; S M Goldinger; B Weide; A Konrad; M Erdmann; D Schadendorf; R S Croner; L Krähenbühl; K C Kähler; C Hafner; W Leisgang; F Kiesewetter; R Dummer; G Schuler; M Stürzl; L Heinzerling Journal: J Cancer Res Clin Oncol Date: 2017-06-14 Impact factor: 4.553
Authors: Berit M Pfitzner; Bianca Lederer; Judith Lindner; Christine Solbach; Knut Engels; Mahdi Rezai; Karel Dohnal; Hans Tesch; Martin L Hansmann; Christoph Salat; Michaela Beer; Andreas Schneeweiss; Peter Sinn; Agnes Bankfalvi; Silvia Darb-Esfahani; Gunter von Minckwitz; Bruno V Sinn; Ralf Kronenwett; Karsten Weber; Carsten Denkert; Sibylle Loibl Journal: Mod Pathol Date: 2017-12-22 Impact factor: 7.842
Authors: José Luis García-Giménez; Marta Seco-Cervera; Trygve O Tollefsbol; Carlos Romá-Mateo; Lorena Peiró-Chova; Pablo Lapunzina; Federico V Pallardó Journal: Crit Rev Clin Lab Sci Date: 2017-12-11 Impact factor: 6.250
Authors: Carsten Denkert; Bruno Valentin Sinn; Yasmin Issa; Berit Maria Müller; Andrea Maisch; Michael Untch; Gunter von Minckwitz; Sibylle Loibl Journal: Breast Care (Basel) Date: 2011-08-29 Impact factor: 2.860
Authors: Ute Schaal; Sandra Grenz; Susanne Merkel; Tilman T Rau; Michel V Hadjihannas; Elisabeth Kremmer; Priya Chudasama; Roland S Croner; Jürgen Behrens; Michael Stürzl; Elisabeth Naschberger Journal: Int J Colorectal Dis Date: 2013-05-24 Impact factor: 2.571