Project description:We profiled human DLBCL tumor samples (FF and FFPE matched pairs) to identify the transcripts which are less prone to degradation in FFPE Keywords: DLBCL FF FFPE
Project description:We profiled human DLBCL tumor samples (FF and FFPE matched pairs) to identify the transcripts which are less prone to degradation in FFPE Keywords: DLBCL FF FFPE RNA profiles of human FF and FFPE samples (DLBCL)
Project description:Background: The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging. Results: In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3'-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions. Conclusions: Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2-3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.
Project description:In this study we have performed expression analysis using paired FF-FFPE glioma samples. We show that expression data from FFPE glioma material is concordant with expression data from matched FF tissue, and can be used for molecular profiling in gliomas. In this study we have performed expression analysis using 55 paired FF-FFPE glioma samples (HU133 plus 2.0 arrays (FF) and Human Exon 1.0 ST arrays (FFPE)). The most informative probe sets were selected based on variance This Series contains the FFPE data only. FF data set was previously submitted (GSE16011).
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, 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. We used Illumina’s Infinium Methylation 27 K 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 Thirwell’s modified protocol.
Project description:Background: To date, few studies have systematically characterized microarray gene expression signal performance with degraded RNA from formalin-fixed paraffin-embedded (FFPE) specimens in comparison to intact RNA from unfixed fresh-frozen (FF) specimens. Methodology: RNA was extracted and isolated from paired tumor and normal samples from both FFPE and FF kidney, lung and colon tissue specimens. Microarray signal dynamics on both the raw probe and probeset level were evaluated. A contrast metric was developed to directly compare microarray signal derived from RNA extracted from matched FFPE and FF specimens. Gene-level summaries were then compared to determine the degree of overlap in expression profiles. Results: RNA extracted from FFPE material was more degraded and fragmented than FF, resulting in reduced dynamic range of expression signal. It was found that probe performance is not affected uniformly and declines sharply toward 5¬タル end of genes. The most significant differences in FFPE vs. FF signal were consistent across three tissue types and enriched with ribosomal genes. Significance: Our results show that archived FFPE samples can be used to profile for expression signatures and assess differential expression similar to unfixed tissue sources. This study provides guidelines for application of these methods in the discovery, validation, and clinical application of microarray expression profiling with FFPE material. 53 samples: 16 FF colon, 7 FFPE colon, 6 FF lung, 8 FFPE lung, 8 FF kidney, and 8 FFPE kidney specimens. Samples are paired tumor and normal tissue. 1-4 biological replicates.
Project description:We aimed to identify clinically meaningful biomarkers in pulmonary carcinoid tumors (PCTs), a member of neuroendocrine neoplasms, via profiling miRNAs and mRNAs. Fifty-one individuals who had never smoked tobacco products and developed PCTs between 1997 and 2008 were studied. A group of 47 carcinoids tumors were analyzed; each with paired tumor-adjacent normal tissue samples (at least 5 cm away from the primary tumors). There are 24 pairs of tumor/normal samples in FF tissues, and 23 pairs of tumor/normal samples in FFPE. The expression status of 1145 miRNAs was generated by Illumina miRNA Arrays.
Project description:In this study we have performed expression analysis using paired FF-FFPE glioma samples. We show that expression data from FFPE glioma material is concordant with expression data from matched FF tissue, and can be used for molecular profiling in gliomas.
Project description:DNA copy number changes with or without accompanying copy neutral changes such as unparental disomy (UPD) is a feature of the cancer genome that is linked to cancer development. However, technical problems with archived formalin-fixed, paraffin-embedded (FFPE) tissue samples have limited their general use in genomic profiling studies done using high-density single nucleotide polymorphism (SNP) microarray. To overcome the current problems with the use of this material in the detection of DNA copy number and copy neutral changes, we have devised two new protocols for extracting DNA from FFPE tissue. Genotyping efficiency and accuracy were improved using our novel protocols. After censoring the larger fragments, we obtained call rates for FFPE DNA equivalent to those for FF tissue DNA, with concordance rates between FFPE and FF tumor exceeding 99%. Identical DNA copy number changes were obtained for FFPE and FF; and between two new extraction protocols in tumor samples by using Affymetrix® high-density oligo-based SNP microarray platform. We observed UPD and recurrent gains and losses in tumor samples. Interestingly, we also identified UPD in the 5q and 13q regions in matching normal blood, FF adjacent breast tissue and tumor tissue in two samples. In conclusion, our new two DNA extraction protocols should substantially improve the ability to use archived material to help elucidate the complexity of early-stage breast cancer genomes. Keywords: SNP based array