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Feasibility and Interpretation of the Genome-wide Infinium Methylation Data Derived from Ligated Formalin Fixed Paraffin Embedded Paired Tumor and Normal Tissue.

ABSTRACT: Formalin-fixed paraffin-embedded (FFPE) samples are a highly desirable resource for epigenetic studies, but there is no suitable platform to assay genome-wide methylation in these widely available resources. Recently, Thirwell et al. (2010) have reported a modified ligation-based DNA repair protocol to prepare FFPE DNA for the Infinium methylation assay. In this study, we have tested the accuracy of methylation data obtained with this modification by comparing paired fresh-frozen (FF) and FFPE colon tissue from colorectal cancer patients. We report locus-specific correlation and concordance of tumor-specific differentially-methylated loci (DML), both of which were not previously assessed.

ORGANISM(S): Homo sapiens

PROVIDER: GSE33181 | GEO | 2014/10/23

SECONDARY ACCESSION(S): PRJNA149199

REPOSITORIES: GEO

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Project description:Formalin-fixed, paraffin-embedded (FFPE) samples are a highly desirable resource for epigenetic studies, but there is no suitable platform to assay genome-wide methylation in these widely available resources. Recently, Thirlwell et al. (2010) have reported a modified ligation-based DNA repair protocol to prepare FFPE DNA for the Infinium methylation assay. In this study, we have tested the accuracy of methylation data obtained with this modification by comparing paired fresh-frozen (FF) and FFPE colon tissue (normal and tumor) from colorectal cancer patients. We report locus-specific correlation and concordance of tumor-specific differentially methylated loci (DML), both of which were not previously assessed.We used Illumina's Infinium Methylation 27K chip for 12 pairs of FF and 12 pairs of FFPE tissue from tumor and surrounding healthy tissue from the resected colon of the same individual, after repairing the FFPE DNA using Thirlwell's modified protocol.For both tumor and normal tissue, overall correlation of ? values between all loci in paired FF and FFPE was comparable to previous studies. Tissue storage type (FF or FFPE) was found to be the most significant source of variation rather than tissue type (normal or tumor). We found a large number of DML between FF and FFPE DNA. Using ANOVA, we also identified DML in tumor compared to normal tissue in both FF and FFPE samples, and out of the top 50 loci in both groups only 7 were common, indicating poor concordance. Likewise, while looking at the correlation of individual loci between FFPE and FF across the patients, less than 10% of loci showed strong correlation (r ? 0.6). Finally, we checked the effect of the ligation-based modification on the Infinium chemistry for SNP genotyping on an independent set of samples, which also showed poor performance.Ligation of FFPE DNA prior to the Infinium genome-wide methylation assay may detect a reasonable number of loci, but the numbers of detected loci are much fewer than in FF samples. More importantly, the concordance of DML detected between FF and FFPE DNA is suboptimal, and DML from FFPE tissues should be interpreted with great caution.

Project description:This study was performed to evaluated RNA extraction and gene expression analysis of FFPE specimen stored for more than 20 years. Using long time stored FFPE material; large retrospective studies correlating molecular features with therapeutic response and clinical outcome, can be performed. Quantitative PCR (qPCR) was used to evaluate RNA extraction methods and to compare gene expression profiles of FFPE and fresh frozen (FF) tissue. Extracted RNA was subsequently subjected to microarray analysis and compared to qPCR data. The Ambion RecoverAll kit appears to be particularly suited for RNA extraction of long time stored FFPE tissues. Gene expression analysis using Affymetrix platform displayed a high degree of correlation for endogenous control genes comparing FF and FFPE tissues. We conclude that high quality gene expression signatures can be recognized using Affymetrix gene expression platform on FFPE tissue stored for more than 20 years. However, a general interpretation must be done with caution as different FFPE procedures have varying effects on RNA quality. Three RNA extraction methods from Roche (Basel, Switzerland), Ambion (Austin, TX, USA) and Qiagen (Hilden, Germany), designed for FFPE material was used. The methods were compared using qPCR. For the qPCR analysis, two different concentrations of input cDNA were used (20ng and 100ng) and 32 human endogenous control genes were examined. RNA from the Ambion FFPE kit was further analyzed by using microarray. Amplification of RNA prior to microarray analysis was performed using Nugen technologies (San Carlos, CA, USA). Nugen has developed amplification kits both for FFPE and FF materials: WT- ovation FFPE RNA amplification System V2 (Nugen FFPE), Ovation FF RNA Amplification System V2 (Nugen V2 FF) and Ovation FF Pico RNA Amplification System (Nugen PICO FF). The Nugen FFPE kit, designed for FFPE material was only used for FFPE tissue. The Nugen Pico FF kit, designed to target small amounts of FF RNA (>500 pg) and the Nugen V2 FF kit designed to target total FF RNA, were only used for FF tissue. Affymetrix standard amplification protocol (Affy FF) designed for FF RNA was also included as the standard method for amplification.

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Feasibility and Interpretation of the Genome-wide Infinium Methylation Data Derived from Ligated Formalin Fixed Paraffin Embedded Paired Tumor and Normal Tissue.

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