ArticleBMC medical genomics2022
Quality control recommendations for RNASeq using FFPE samples based on pre-sequencing lab metrics and post-sequencing bioinformatics metrics.
Article in BMC medical genomics, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 19 citations in OpenAlex.
- Integration of bulk RNA-seq pipeline metrics for assessing low-quality samples.BMC bioinformatics · 2026Article
- Development of an End-to-End Total RNA Sequencing Quality Control Framework for Blood-Based Biomarker Discovery.The Journal of molecular diagnostics : JMD · 2025Article
- Transcriptomics and epigenomics datasets of primary brain cancers in formalin-fixed paraffin embedded format.Scientific data · 2025Article
- Comparing RNA extraction protocols from formalin-fixed paraffin-embedded microcore samples.PloS one · 2025Article
- A cost-effective and scalable approach for DNA extraction from FFPE tissues.Biology methods & protocols · 2025Article
- Evaluating cell type deconvolution in FFPE breast tissue: application to benign breast disease.NAR genomics and bioinformatics · 2024Review
- Comparison of RNA-Sequencing Methods for Degraded RNA.International journal of molecular sciences · 2024Article
- Differential expression analysis identifies a prognostically significant extracellular matrix-enriched gene signature in hyaluronan-positive clear cell renal cell carcinoma.Scientific reports · 2024Article
- Angio-Long Noncoding RNA MALAT1 (rs3200401) and MIAT (rs1061540) Gene Variants in Ovarian Cancer.Epigenomes · 2024Article
- A comprehensive assessment of exome capture methods for RNA sequencing of formalin-fixed and paraffin-embedded samples.BMC genomics · 2023Review
- Proteomic analysis to identification of hypoxia related markers in spinal tuberculosis: a study based on weighted gene co-expression network analysis and machine learning.BMC medical genomics · 2023Article
- A practical spatial analysis method for elucidating the biological mechanisms of cancers with abdominal dissemination in vivo.Scientific reports · 2022Article
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Authors and funding
18 authors at 4 institutions in 1 country.
Funding
Abstract
backgroundFormalin-fixed, paraffin-embedded (FFPE) tissues have many advantages for identification of risk biomarkers, including wide availability and potential for extended follow-up endpoints. However, RNA derived from archival FFPE samples has limited quality. Here we identified parameters that determine which FFPE samples have the potential for successful RNA extraction, library preparation, and generation of usable RNAseq data.
methodsWe optimized library preparation protocols designed for use with FFPE samples using seven FFPE and Fresh Frozen replicate pairs, and tested optimized protocols using a study set of 130 FFPE biopsies from women with benign breast disease. Metrics from RNA extraction and preparation procedures were collected and compared with bioinformatics sequencing summary statistics. Finally, a decision tree model was built to learn the relationship between pre-sequencing lab metrics and qc pass/fail status as determined by bioinformatics metrics.
resultsSamples that failed bioinformatics qc tended to have low median sample-wise correlation within the cohort (Spearman correlation < 0.75), low number of reads mapped to gene regions (< 25 million), or low number of detectable genes (11,400 # of detected genes with TPM > 4). The median RNA concentration and pre-capture library Qubit values for qc failed samples were 18.9 ng/ul and 2.08 ng/ul respectively, which were significantly lower than those of qc pass samples (40.8 ng/ul and 5.82 ng/ul). We built a decision tree model based on input RNA concentration, input library qubit values, and achieved an F score of 0.848 in predicting QC status (pass/fail) of FFPE samples.
conclusionsWe provide a bioinformatics quality control recommendation for FFPE samples from breast tissue by evaluating bioinformatic and sample metrics. Our results suggest a minimum concentration of 25 ng/ul FFPE-extracted RNA for library preparation and 1.7 ng/ul pre-capture library output to achieve adequate RNA-seq data for downstream bioinformatics analysis.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the Socratic graph.