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 Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yulia Newton ◽  
Andrew J. Sedgewick ◽  
Luis Cisneros ◽  
Justin Golovato ◽  
Mark Johnson ◽  
...  
Keyword(s):  
Large Scale ◽  
Clinical Decision Making ◽  
Rna Extraction ◽  
Transcriptome Profiling ◽  
Clinical Decision ◽  
Extraction Methods ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Whole Transcriptome ◽  
Formalin Fixed

Abstract Transcriptome profiling can provide information of great value in clinical decision-making, yet RNA from readily available formalin-fixed paraffin-embedded (FFPE) tissue is often too degraded for quality sequencing. To assess the clinical utility of FFPE-derived RNA, we performed ribo-deplete RNA extractions on > 3200 FFPE slide samples; 25 of these had direct FFPE vs. fresh frozen (FF) replicates, 57 were sequenced in 2 different labs, 87 underwent multiple library analyses, and 16 had direct microdissected vs. macrodissected replicates. Poly-A versus ribo-depletion RNA extraction methods were compared using transcriptomes of TCGA cohort and 3116 FFPE samples. Compared to FF, FFPE transcripts coding for nuclear/cytoplasmic proteins involved in DNA packaging, replication, and protein synthesis were detected at lower rates and zinc finger family transcripts were of poorer quality. The greatest difference in extraction methods was in histone transcripts which typically lack poly-A tails. Encouragingly, the overall sequencing success rate was 81%. Exome coverage was highly concordant in direct FFPE and FF replicates, with 98% agreement in coding exon coverage and a median correlation of whole transcriptome profiles of 0.95. We provide strong rationale for clinical use of FFPE-derived RNA based on the robustness, reproducibility, and consistency of whole transcriptome profiling.

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 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 EPND-03. RNA-SEQ GENE EXPRESSION ANALYSIS OF 106 FORMALIN-FIXED PARAFFIN-EMBEDDED PAEDIATRIC EPENDYMOMAS

2017 ◽  
Vol 19 (suppl_4) ◽  
pp. iv15-iv15
Author(s):  
Timothy Ritzmann ◽  
Hazel Rogers ◽  
Andrew M Donson ◽  
Rebecca Chapman ◽  
Lisa Storer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Rna Seq ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals Functional Comparison of Different Exome Capture-based Methods for Transcriptomic Profiling of Formalin-Fixed Paraffin-Embedded Tumor Samples

2021 ◽  
Author(s):  
Kyrillus S. Shohdy ◽  
Rohan Bareja ◽  
Michael Sigouros ◽  
David C. Wilkes ◽  
Princesca Dorsaint ◽  
...  
Keyword(s):  
Gene Expression ◽  
Exome Capture ◽  
Rna Seq ◽  
Transcriptomic Profiling ◽  
Immune Genes ◽  
Formalin Fixed Paraffin ◽  
Ffpe Tumor ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Formalin Fixed

AbstractBackgroundThe need for fresh frozen (FF) tissue limits implementing RNA sequencing (RNA-seq) in the clinic. The majority of clinical samples are processed in clinical laboratories and stored as formalin-fixed, paraffin-embedded (FFPE) tissues. Exome capture has recently emerged as a promising approach for RNA-seq from FFPE samples. Multiple exome capture platforms are now available. However, their performances have not been systematically compared.MethodsTranscriptomic analysis of 32 FFPE tumor samples from 11 patients was performed using three exome capture-based methods: Agilent SureSelect V6, TWIST NGS Exome, and IDT XGen Exome Research Panel. We compared these methods to TruSeq RNA-seq of fresh frozen (FF-TruSeq) tumor samples from the same patients. We assessed the recovery of clinically relevant biological features, including the expression of key immune genes, expression outliers often associated with actionable genes, gene expression-based subtypes, and fusions using each of these capture methods.ResultsThe Spearman’s correlation coefficients between global expression profiles of the three capture-based methods and matched FF tumor samples, analyzed using TruSeq RNA-seq, were high (rho = 0.72-0.9, p < 0.05). There was a significant correlation between the expression of key immune genes between individual capture-based methods and FF-TruSeq (rho = 0.76-0.88, p < 0.05). All three exome capture-based methods reliably detected the outlier expression of actionable genes, including ERBB2, MET, NTRK1, and PPARG, initially detected in FF-TruSeq. In urothelial cancer samples, the Agilent assay was associated with the highest molecular subtyping agreement with FF-TruSeq (Cohen’s k = 0.7, p < 0.01). Both Agilent and IDT detected all the clinically relevant fusions which were initially identified in FF-TruSeq.ConclusionAll exome capture-based methods had comparable performance and concordance with FF-TruSeq. These findings provide a path for the transcriptomic profiling of vast numbers of FFPE currently stored in biobanks. For specific applications such as fusion detection and gene expression-based subtyping, some methods performed better. By enabling the interrogation of FFPE tumor samples, our findings open the door for implementing RNA-seq in the clinic to guide precision oncology approaches.

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