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 Biological Validation of RNA Sequencing Data From Formalin-Fixed Paraffin-Embedded Primary Melanomas

2018 ◽  
pp. 1-19 ◽  
Author(s):  
Lawrence N. Kwong ◽  
Mariana Petaccia De Macedo ◽  
Lauren Haydu ◽  
Aron Y. Joon ◽  
Michael T. Tetzlaff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Cancer Genome ◽  
The Cancer Genome Atlas ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Cancer Genome Atlas ◽  
Formalin Fixed ◽  
Genome Atlas

Purpose Initiatives such as The Cancer Genome Atlas and International Cancer Genome Consortium have generated high-quality, multiplatform molecular data from thousands of frozen tumor samples. Although these initiatives have provided invaluable insight into cancer biology, a tremendous potential resource remains largely untapped in formalin-fixed, paraffin-embedded (FFPE) samples that are more readily available but which can present technical challenges because of crosslinking of fragile molecules such as RNA. Materials and Methods We extracted RNA from FFPE primary melanomas and assessed two gene expression platforms—genome-wide RNA sequencing and targeted NanoString—for their ability to generate coherent biologic signals. To do so, we generated an improved approach to quantifying gene expression pathways. We refined pathway scores through correlation-guided gene subsetting. We also make comparisons to The Cancer Genome Atlas and other publicly available melanoma datasets. Results The comparison of the gene expression patterns to each other, to established biologic modules, and to clinical and immunohistochemical data confirmed the fidelity of biologic signals from both platforms using FFPE samples to known biology. Moreover, correlations with patient outcome data were consistent with previous frozen-tissue–based studies. Conclusion FFPE samples from previously difficult-to-access cancer types, such as small primary melanomas, represent a valuable and previously unexploited source of analyte for RNA sequencing and NanoString platforms. This work provides an important step toward the use of such platforms to unlock novel molecular underpinnings and inform future biologically driven clinical decisions.

scholarly journals Spatial transcriptomics of tumor microenvironment in formalin-fixed paraffin-embedded breast cancer

2020 ◽  
Author(s):  
Lou Romanens ◽  
Prasad Chaskar ◽  
Jean-Christophe Tille ◽  
Stephan Ryser ◽  
Nicolas Liaudet ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Exome Capture ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Laser Capture ◽  
Formalin Fixed

AbstractTumor samples are conserved in clinical practice in formalin-fixed paraffin-embedded (FFPE) blocks. Formalin fixation chemically alters nucleic acids, rendering transcriptomic analysis challenging. RNA-sequencing is usually performed on tumor bulk, without distinction of cell subtypes or location. Here we describe the development of a robust method for RNA extraction and exome-capture RNA-sequencing of laser-capture microdissected tumor cells (TC) and stromal immune cells (TIL) based on their morphology. We applied this method on 7 tumor samples (surgical or core needle biopsy) of triple-negative breast cancer (TNBC) stored in FFPE blocks over 3-10 years. Unsupervised clustering and principal component analysis showed a clear separation between gene-expression profile of TIL and TC. TIL were enriched in markers of B cells (CD79B, PAX5 and BLNK) and T cells (CD2, CD3D and CD8B) whereas tumor cells expressed epithelial markers (EPCAM, MUC1 and KRT8). Microenvironment cell populations-counter (MCP)-counter deconvolution showed an enrichment in adaptive immune cell signatures in microdissected TIL. Transcripts of immune checkpoints were differentially expressed in TIL and TC. We further validated our results by qRT-PCR and multispectral immunohistochemistry. In conclusion, we showed that combining laser-capture microdissection and RNA-sequencing on archived FFPE blocks is feasible and allows spatial transcriptional characterization of tumor microenvironment.

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.

Clinical relevance of Ki67 gene expression analysis using formalin-fixed paraffin-embedded breast cancer specimens

Breast Cancer ◽  
2012 ◽  
Vol 20 (3) ◽  
pp. 262-270 ◽  
Author(s):  
Satoko Yamamoto ◽  
Mutsuko Ibusuki ◽  
Yutaka Yamamoto ◽  
Peifen Fu ◽  
Saori Fujiwara ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Expression Analysis ◽  
Clinical Relevance ◽  
Gene Expression Analysis ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals Use of Formalin-Fixed Paraffin-Embedded Samples for Gene Expression Studies in Breast Cancer Patients

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0123194 ◽  
Author(s):  
Valeria Musella ◽  
Maurizio Callari ◽  
Eleonora Di Buduo ◽  
Manuela Scuro ◽  
Matteo Dugo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Cancer Patients ◽  
Breast Cancer Patients ◽  
Expression Studies ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Gene Expression Studies ◽  
Formalin Fixed

scholarly journals Comparison of gene expression profiling by reverse transcription quantitative PCR between fresh frozen and formalin-fixed, paraffin-embedded breast cancer tissues

BioTechniques ◽  
2010 ◽  
Vol 48 (5) ◽  
pp. 389-397 ◽  
Author(s):  
Iker Sánchez-Navarro* ◽  
Angelo Gámez-Pozo* ◽  
Manuel González-Barón ◽  
Álvaro Pinto-Marín ◽  
David Hardisson ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Reverse Transcription ◽  
Expression Profiling ◽  
Formalin Fixed Paraffin ◽  
Reverse Transcription Quantitative Pcr ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Cancer Tissues ◽  
Formalin Fixed

scholarly journals Analyses and interpretation of whole-genome gene expression from formalin-fixed paraffin-embedded tissue: an illustration with breast cancer tissues

BMC Genomics ◽  
2010 ◽  
Vol 11 (1) ◽  
Author(s):  
Muhammad G Kibriya ◽  
Farzana Jasmine ◽  
Shantanu Roy ◽  
Rachelle M Paul-Brutus ◽  
Maria Argos ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Whole Genome ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Cancer Tissues ◽  
Formalin Fixed
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