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Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction
Published on: August 21, 2016
Optimization of gene expression microarray protocol for formalin-fixed paraffin-embedded tissues
Nevin Belder1, Öznur Coşkun2, Beyza Doğanay Erdoğan3
1Ankara University, Biotechnology Institute, Ankara, Turkey.
Abstract:
Formalin-fixed paraffin-embedded (FFPE) tissue is a widely available clinical specimen for retrospective studies. The possibility of long-term clinical follow-up of FFPE samples makes them a valuable source to evaluate links between molecular and clinical information. Working with FFPE samples in the molecular research area, especially using high-throughput molecular techniques such as microarray gene expression profiling, has come into prominence. Because of the harmful effects of formalin fixation process such as degradation of nucleic acids, cross-linking with proteins, and chemical modifications on DNA and RNA, there are some limitations in gene expression profiling studies using FFPE samples. To date many studies have been conducted to evaluate gene expression profiling using microarrays (Thomas et al., Thomas et al. (2013) [1]; Scicchitano et al., Scicchitano et al. (2006) [2]; Frank et al., Frank et al. (2007) [3]; Fedorowicz et al., Fedorowicz et al. (2009) [4]). However, there is still no generally accepted, efficient and standardized procedure for microarray analysis of FFPE samples. This paper describes the microarray data presented in our recently accepted to be published article showing a standard protocol from deparaffinization of FFPE tissue sections and RNA extraction to microarray gene expression analysis. Here we represent our data in detail, deposited in the gene expression omnibus (GEO) database with the accession number GSE73883. Four combinations of two different cRNA/cDNA preparation and labeling protocols with two different array platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P) were evaluated to determine which combination gives the best percentage of present call. The study presents a dataset for comparative analysis which has a potential in terms of providing a robust protocol for gene expression profiling with FFPE tissue samples.
Insights
This study evaluates standard protocols for microarray gene expression profiling using formalin-fixed paraffin-embedded (FFPE) tissues. It provides a detailed dataset to establish a robust method for analyzing FFPE samples, crucial for linking molecular and clinical data.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Formalin-fixed paraffin-embedded (FFPE) tissues are valuable for retrospective studies due to long-term clinical follow-up.
- Challenges exist in gene expression profiling of FFPE samples due to nucleic acid degradation and chemical modifications from formalin fixation.
- Existing microarray analysis protocols for FFPE samples lack standardization.
Purpose of the Study:
- To describe and present detailed microarray data for a standard protocol for gene expression profiling using FFPE tissue samples.
- To evaluate different combinations of RNA preparation, labeling protocols, and array platforms for optimal FFPE microarray analysis.
- To provide a dataset for comparative analysis to establish a robust protocol.
Main Methods:
- Deparaffinization of FFPE tissue sections and RNA extraction.
- Evaluation of four combinations of two different cRNA/cDNA preparation and labeling protocols.
- Analysis using two different array platforms: Affymetrix Human Genome U133 Plus 2.0 and U133_X3P.
- Data deposited in the Gene Expression Omnibus (GEO) database under accession number GSE73883.
Main Results:
- Comparative analysis of four protocol combinations across two array platforms was performed.
- The percentage of present calls was used as a metric to determine the best performing combination.
- Detailed data is available for further analysis and protocol development.
Conclusions:
- The study presents a comprehensive dataset and evaluation of protocols for gene expression profiling using FFPE samples.
- The findings aim to contribute to the development of a standardized and efficient protocol for FFPE microarray analysis.
- This work supports the use of FFPE tissues for linking molecular information with clinical outcomes.
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