Project description:Tissue sample acquisition is a limiting step in many studies. There are many thousands of formalin fixed paraffin embedded archival blocks collected around the world, but in contrast relatively few fresh frozen samples in tumor banks. Once samples are fixed in formalin the RNA is degraded and traditional methods for gene expression profiling are not suitable. In this study we have evaluated the whole genome DASL assay from Illumina to perform transcriptomic analysis from archived breast tumor tissue fixed in formalin paraffin embedded blocks. We profiled 76 familial breast tumors from cases carrying a BRCA1, BRCA2 or ATM mutation, or from non-BRCA1/2 families. We found that replicate samples correlated well with each other (r2=0.9-0.98). In 12/15 cases, the matched formalin-fixed and frozen samples predicted the same tumor molecular subtypes with confidence. These results demonstrate that the whole genome DASL assay is a valuable tool to profile degraded RNA from archival FFPE material. This assay will enable transcriptomic analysis of a large number of archival samples that are stored in pathology archives around the globe and consequently will have the potential to improve our understanding and characterisation of many diseases.
Project description:Tissue sample acquisition is a limiting step in many studies. There are many thousands of formalin fixed paraffin embedded archival blocks collected around the world, but in contrast relatively few fresh frozen samples in tumor banks. Once samples are fixed in formalin the RNA is degraded and traditional methods for gene expression profiling are not suitable. In this study we have evaluated the whole genome DASL assay from Illumina to perform transcriptomic analysis from archived breast tumor tissue fixed in formalin paraffin embedded blocks. We profiled 76 familial breast tumors from cases carrying a BRCA1, BRCA2 or ATM mutation, or from non-BRCA1/2 families. We found that replicate samples correlated well with each other (r2=0.9-0.98). In 12/15 cases, the matched formalin-fixed and frozen samples predicted the same tumor molecular subtypes with confidence. These results demonstrate that the whole genome DASL assay is a valuable tool to profile degraded RNA from archival FFPE material. This assay will enable transcriptomic analysis of a large number of archival samples that are stored in pathology archives around the globe and consequently will have the potential to improve our understanding and characterisation of many diseases. RNA was extracted from FFPE Familial breast tumours and analysed using the WG-DASL assay for Illumina.
Project description:This SuperSeries is composed of the following subset Series: GSE23384: Gene profiling using archival formalin-fixed paraffin-embedded breast cancer specimens can generate informative microarray data: A comparison with matched fresh fine needle aspiration biopsy samples (FFPE samples) GSE23385: Gene profiling using archival formalin-fixed paraffin-embedded breast cancer specimens can generate informative microarray data: A comparison with matched fresh fine needle aspiration biopsy samples (FNA samples) Refer to individual Series
Project description:This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3’ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE.
Project description:<p>The overarching goal of the study was to better understand the evolution of melanoma. To do this, we collected archival tissue of melanomas adjacent to their intact, remnant precursor lesions. We also collected archival tissue of primary melanomas and their matching metastases. From the formalin-fixed paraffin-embedded (FFPE) blocks, we microdissected non-neoplastic (normal), precursor, and descendent portions of tissue. Both RNA and DNA were extracted from the microdissected tissues and subjected to next generation sequencing. In summary, we performed matched DNA/RNA sequencing of melanoma/precursor/normal trios, allowing us to trace the genetic and transcriptomic evolution of melanoma.</p>
Project description:Archival formalin-fixed paraffin-embedded (FFPE) tissue samples hold a wealth of transcriptomic information; however, little is known about potential artifacts. Previously, we identified a consistent shift in global RNA-sequencing profiles between matching frozen and FFPE samples. We hypothesized that this shift was from fixing fresh tissue in formalin. To test this idea, RNA-sequencing was performed on liver samples collected from male mice treated with 600 ppm of a reference chemical (phenobarbital, 600 ppm phenobarbital) or vehicle control for 7 days. Samples were divided into: (1) fresh-frozen (FR); (2) directly fixed in 10% buffered formalin for 18 hours followed by paraffin embedding (FFPE); (3) frozen then fixed as FFPE (FR>FFPE); or (4) frozen then fixed in 70% ethanol followed by paraffin embedding (FR>OH) (n=6/group/condition). Direct fixation resulted in 2946 differentially expressed genes (DEGs), 98% of which were down-regulated. Freezing prior to fixation resulted in ≥95% fewer DEGs vs. FR, indicating that most formalin-derived transcriptional effects occurred with fixation. This was supported by follow-up studies, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways with formalin fixation. Notably, formalin fixation in the parent study did not significantly impact chemical response profiles, which were consistent with CAR/PXR activation and 600 ppm phenobarbital exposure. Our results demonstrate distinct transcriptional effects of formalin fixation that could impact gene expression studies using FFPE samples.
Project description:Abstract: Sufficient quantity of genomic DNA can be a bottleneck in genome-wide analysis of clinical tissue samples. DNA polymerase Phi29 can be used for the random-primed amplification of whole genomes, although the amplification may introduce bias in gene dosage. We have performed a detailed investigation of this technique in archival fresh-frozen and formalin-fixed/paraffin-embedded tumor DNA by using cDNA microarray-based comparative genomic hybridization. Phi29 amplified DNA from matched pairs of fresh-frozen and formalin-fixed/paraffin-embedded tumor samples with similar efficiency. The distortion in gene dosage representation in the amplified DNA was nonrandom and reproducibly involved distinct genomic loci. Regional amplification efficiency was significantly linked to regional GC content of the template genome. The biased gene representation in amplified tumor DNA could be effectively normalized by using amplified reference DNA. Our data suggest that genome-wide gene dosage alterations in clinical tumor samples can be reliably assessed from a few hundred tumor cells. Therefore, this amplification method should lend itself to high-throughput genetic analyses of limited sources of tumor, such as fine-needle biopsies, laser-microdissected tissue, and small paraffin-embedded specimens. This SuperSeries is composed of the SubSeries listed below.
Project description:Renal epithelial neoplasms have characteristic chromosomal imbalances that can be used for classification. We have previously shown that virtual karyotypes (v-karyotype) derived from SNP microarrays can be performed on formalin-fixed paraffin embedded (FFPE) tissue samples but a direct comparison with karyotypes obtained by conventional cytogenetics has not been done. 20 archival FFPE tumor samples were analyzed with Affymetrix 10K 2.0 or 250K Nsp SNP microarrays.
Project description:Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients, to enable the decision of whether or not it is feasible to apply RNAseq technology to archival tissue. Core biopsies were obtained with a 16g needle from 16 patients undergoing partial or full nephrectomy. The RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol.