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From RNA isolation to microarray analysis: Comparison of methods in FFPE tissues.
Belder, Nevin; Coskun, Öznur; Doganay Erdogan, Beyza; Ilk, Ozlem; Savas, Berna; Ensari, Arzu; Özdag, Hilal.
Afiliación
  • Belder N; Ankara University, Biotechnology Institute, Ankara, Turkey.
  • Coskun Ö; Ankara University, School of Medicine, Department of Pathology, Ankara, Turkey.
  • Doganay Erdogan B; Ankara University, School of Medicine, Department of Biostatistics, Ankara, Turkey.
  • Ilk O; Middle East Technical University, Department of Statistics, Ankara, Turkey.
  • Savas B; Ankara University, School of Medicine, Department of Pathology, Ankara, Turkey.
  • Ensari A; Ankara University, School of Medicine, Department of Pathology, Ankara, Turkey.
  • Özdag H; Ankara University, Biotechnology Institute, Ankara, Turkey. Electronic address: hilalozdag@gmail.com.
Pathol Res Pract ; 212(8): 678-85, 2016 Aug.
Article en En | MEDLINE | ID: mdl-27161306
BACKGROUND: Genome-wide gene expression profiling analysis of FFPE tissue samples is indispensable for cancer research and provides the opportunity to evaluate links between molecular and clinical information, however, working with FFPE samples is challenging due to extensive cross-linking, fragmentation and limited quantities of nucleic acid. Thus, processing of FFPE tissue samples from RNA extraction to microarray analysis still needs optimization. MATERIALS AND METHODS: In this study, a modified deparaffinization protocol was conducted prior to RNA isolation. Trizol, Qiagen RNeasy FFPE and Arcturus PicoPure RNA Isolation kits were used in parallel to compare their impact on RNA isolation. We also evaluated the effect of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) on the percentage of present calls. RESULTS: Our optimization study shows that the Qiagen RNeasy FFPE kit with modified deparaffinization step gives better results (RNA quantity and quality) than the other two isolation kits. The Ribo-SPIA protocol gave a significantly higher percentage of present calls than the 3' IVT cDNA amplification and labeling system. However, no significant differences were found between the two array platforms. CONCLUSION: Our study paves the way for future high-throughput transcriptional analysis by optimizing FFPE tissue sample processing from RNA isolation to microarray analysis.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN / Perfilación de la Expresión Génica / Análisis de Matrices Tisulares / Secuenciación de Nucleótidos de Alto Rendimiento Tipo de estudio: Guideline Límite: Humans Idioma: En Revista: Pathol Res Pract Año: 2016 Tipo del documento: Article País de afiliación: Turquía Pais de publicación: Alemania
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: ARN / Perfilación de la Expresión Génica / Análisis de Matrices Tisulares / Secuenciación de Nucleótidos de Alto Rendimiento Tipo de estudio: Guideline Límite: Humans Idioma: En Revista: Pathol Res Pract Año: 2016 Tipo del documento: Article País de afiliación: Turquía Pais de publicación: Alemania