scholarly journals Mining the archives: a cross-platform analysis of gene expression profiles in archival formalin-fixed paraffin-embedded (FFPE) tissue

2015 ◽  
Author(s):  
Anna Francina Webster ◽  
Paul Zumbo ◽  
Jennifer Fostel ◽  
Jorge Gandara ◽  
Susan D Hester ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Ffpe Tissue ◽  
Rna Seq ◽  
Preservation Method ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for transcriptomic-based research into the molecular basis of disease. However, use of FFPE samples in gene expression studies has been limited by technical challenges resulting from degradation of nucleic acids. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues using two DNA microarray protocols and two whole transcriptome sequencing (RNA-seq) library preparation methodologies. The ribo-depletion protocol outperformed the other three methods by having the highest correlations of differentially expressed genes (DEGs) and best overlap of pathways between FRO and FFPE groups. We next tested the effect of sample time in formalin (18 hours or 3 weeks) on gene expression profiles. Hierarchical clustering of the datasets indicated that test article treatment, and not preservation method, was the main driver of gene expression profiles. Meta- and pathway analyses indicated that biological responses were generally consistent for 18-hour and 3-week FFPE samples compared to FRO samples. However, clear erosion of signal intensity with time in formalin was evident, and DEG numbers differed by platform and preservation method. Lastly, we investigated the effect of age in FFPE block on genomic profiles. RNA-seq analysis of 8-, 19-, and 26-year-old control blocks using the ribo-depletion protocol resulted in comparable quality metrics, including expected distributions of mapped reads to exonic, UTR, intronic, and ribosomal fractions of the transcriptome. Overall, our results suggest that FFPE samples are appropriate for use in genomic studies in which frozen samples are not available, and that ribo-depletion RNA-seq is the preferred method for this type of analysis in archival and long-aged FFPE samples.

scholarly journals Mining the Archives: A Cross-Platform Analysis of Gene Expression Profiles in Archival Formalin-Fixed Paraffin-Embedded Tissues

2015 ◽  
Vol 148 (2) ◽  
pp. 460-472 ◽  
Author(s):  
A. Francina Webster ◽  
Paul Zumbo ◽  
Jennifer Fostel ◽  
Jorge Gandara ◽  
Susan D. Hester ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Cross Platform ◽  
Formalin Fixed

scholarly journals FFPE samples from cavitational ultrasonic surgical aspirates are suitable for RNA profiling of gliomas

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0255168
Author(s):  
Cristina Alenda ◽  
Estefanía Rojas ◽  
Luis M. Valor
Keyword(s):  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Solid Material ◽  
Rna Seq ◽  
Rna Profiling ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
First Time

During surgical procedures for gliomas, tissue material obtained from cavitational ultrasonic surgical aspirators (CUSAs) is generally discarded but can actually exceed the amount and quality of certain tumour core resections (TCRs). Despite reports indicating the suitability of CUSA-derived material for diagnosis and research, its use is still marginal. We extended these conclusions to formalin-fixed, paraffin-embedded (FFPE) samples, the most common format for archival tumour tissue in anatomical pathology departments, by conducting for the first time RNA-seq analysis in CUSA aspirates. We compared the molecular diagnosis of somatic mutations used in the clinical routine and the gene expression profiles of fixed solid material from CUSA aspirates and TCRs from the same patients in selected gliomas encompassing grades II to IV. Despite the characteristic heterogeneity of gliomas, we found substantial similarities between the corresponding aspirates and TCRs that included transcriptional signatures associated with glioma subtypes. Based on these results, we confirmed that CUSA-fixed biomaterials from glioma surgeries are appropriate for downstream applications and biomarkers screening.

scholarly journals Gene expression profiles of gliomas in formalin-fixed paraffin-embedded material

2011 ◽  
Vol 106 (3) ◽  
pp. 538-545 ◽  
Author(s):  
L A M Gravendeel ◽  
J J de Rooi ◽  
P H C Eilers ◽  
M J van den Bent ◽  
P A E Sillevis Smitt ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals Gene Expression Profiles in Formalin-Fixed, Paraffin-Embedded Tissues Obtained with a Novel Assay for Microarray Analysis

2004 ◽  
Vol 50 (12) ◽  
pp. 2384-2386 ◽  
Author(s):  
Marina Bibikova ◽  
Joanne M Yeakley ◽  
Eugene Chudin ◽  
Jing Chen ◽  
Eliza Wickham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Microarray Analysis ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

scholarly journals Impact of RNA Extraction and Target Capture Methods on RNA Sequencing Using Formalin-Fixed, Paraffin Embedded Tissues

2019 ◽  
Author(s):  
Christopher A. Hilker ◽  
Aditya V. Bhagwate ◽  
Jin Sung Jang ◽  
Jeffrey G Meyer ◽  
Asha A. Nair ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Gene ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Seq ◽  
Gene Detection ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Whole Transcriptome ◽  
Formalin Fixed

AbstractFormalin fixed paraffin embedded (FFPE) tissues are commonly used biospecimen for clinical diagnosis. However, RNA degradation is extensive when isolated from FFPE blocks making it challenging for whole transcriptome profiling (RNA-seq). Here, we examined RNA isolation methods, quality metrics, and the performance of RNA-seq using different approaches with RNA isolated from FFPE and fresh frozen (FF) tissues. We evaluated FFPE RNA extraction methods using six different tissues and five different methods. The reproducibility and quality of the prepared libraries from these RNAs were assessed by RNA-seq. We next examined the performance and reproducibility of RNA-seq for gene expression profiling with FFPE and FF samples using targeted (Kinome capture) and whole transcriptome capture based sequencing. Finally, we assessed Agilent SureSelect All-Exon V6+UTR capture and the Illumina TruSeq RNA Access protocols for their ability to detect known gene fusions in FFPE RNA samples. Although the overall yield of RNA varied among extraction methods, gene expression profiles generated by RNA-seq were highly correlated (>90%) when the input RNA was of sufficient quality (≥DV200 30%) and quantity (≥ 100 ng). Using gene capture, we observed a linear relationship between gene expression levels for shared genes that were captured using either All-Exon or Kinome kits. Gene expression correlations between the two capture-based approaches were similar using RNA from FFPE and FF samples. However, TruSeq RNA Access protocol provided significantly higher exon and junction reads when compared to the SureSelect All-Exon capture kit and was more sensitive for fusion gene detection. Our study established pre and post library construction QC parameters that are essential to reproducible RNA-seq profiling using FFPE samples. We show that gene capture based NGS sequencing is an efficient and highly reproducible strategy for gene expression measurements as well as fusion gene detection.

scholarly journals Expression Concordance of 325 Novel RNA Biomarkers between Data Generated by NanoString nCounter and Affymetrix GeneChip

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Lucas Delmonico ◽  
Said Attiya ◽  
Joan W. Chen ◽  
John C. Obenauer ◽  
Edward C. Goodwin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Pearson Correlation ◽  
Gene Expression Profiles ◽  
Standard Operating Procedure ◽  
Driver Genes ◽  
Technology Platforms ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Rna Biomarkers

Background. With the development of new drug combinations and targeted treatments for multiple types of cancer, the ability to stratify categories of patient populations and to develop companion diagnostics has become increasingly important. A panel of 325 RNA biomarkers was selected based on cancer-related biological processes of healthy cells and gene expression changes over time during nonmalignant epithelial cell organization. This “cancer in reverse” approach resulted in a panel of biomarkers relevant for at least 7 cancer types, providing gene expression profiles representing key cellular signaling pathways beyond mutations in “driver genes.” Objective. To further investigate this biomarker panel, the objective of the current study is to (1) validate the assay reproducibility for the 325 RNA biomarkers and (2) compare gene expression profiles side by side using two technology platforms. Methods and Results. We have mapped the 325 RNA transcripts and in a custom NanoString nCounter expression panel to be compared to all potential probe sets in the Affymetrix Human Genome U133 Plus 2.0. The experiments were conducted with 10 unique biological formalin-fixed paraffin-embedded (FFPE) breast tumor samples. Each site extracted RNA from four sections of 10-micron thick FFPE tissue over three different days by two different operators using an optimized standard operating procedure and quality control criteria. Samples were analyzed using mas5 in BioConductor and NanoStringNorm in R. Pearson correlation showed reproducibility between sites for all 60 samples with r=0.995 for Affymetrix and r=0.999 for NanoString. Correlation in multiple days and multiple users was for Affymetrix r=0.962−0.999 and for NanoString r=0.982−0.991. Conclusion. The 325 RNA biomarkers showed reproducibility in two technology platforms with moderate to high concordance. Future directions include performing clinical validation studies and generating rationale for patient selection in clinical trials using the technically validated assay.

scholarly journals HYPERsol: flash-frozen results from archival FFPE tissue for clinical proteomics

2019 ◽  
Author(s):  
Dylan M. Marchione ◽  
Ilyana Ilieva ◽  
Benjamin A. Garcia ◽  
Darryl J. Pappin ◽  
John P. Wilson ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Ffpe Tissue ◽  
Clinical Proteomics ◽  
High Yield ◽  
Proteome Coverage ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Proteomics Research ◽  
Formalin Fixed

Massive formalin-fixed, paraffin-embedded (FFPE) tissue archives exist worldwide, representing a potential gold mine for clinical proteomics research. However, current protocols for FFPE proteomics lack standardization, efficiency, reproducibility, and scalability. Here we present High-Yield Protein Extraction and Recovery by direct SOLubilization (HYPERsol), an optimized workflow using adaptive-focused acoustics (AFA) ultrasonication and S-Trap sample processing that enables proteome coverage and quantification from FFPE samples comparable to that achieved from flash-frozen tissue (average R = 0.936).

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