ArticleBMC genomics2015
Experimental validation of methods for differential gene expression analysis and sample pooling in RNA-seq.
Article in BMC genomics, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 91 papers.
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Who cites it
91 citing papers in PubMed.
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- Transcriptome Analysis of Trigeminal Ganglion and Medullary Dorsal Horn in Mice to Identify Potential Targets for Pulpitis-Induced Pain.ACS omega · 2025Article
- Identification of genetic factors influencing flavonoid biosynthesis through pooled transcriptome analysis in mungbean sprouts.Frontiers in plant science · 2025Article
- Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of nuclear-encoded noncoding RNAs.FASEB journal : official publication of the Federation of American Societies for Experimental Biology · 2024Article
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- Identification of Biomarkers Associated With Paget's Disease of Bone and Bone Metastasis From Breast Cancer Patients.Cancer reports (Hoboken, N.J.) · 2024Article
- Transcriptome Analysis of Left Versus Right Intrinsic Laryngeal Muscles Associated with Innervation.The Laryngoscope · 2024Article
- Challenges and best practices in omics benchmarking.Nature reviews. Genetics · 2024Review
- Class III Peroxidases in the Peach (Plants (Basel, Switzerland) · 2024Article
- Next-Generation Sequencing Application: A Systematic Approach for High-Quality RNA Isolation from Skeletal Muscles.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Differentially Expressed Genes in Response to a Squalene-Supplemented Diet Are Accurate Discriminants of Porcine Non-Alcoholic Steatohepatitis.International journal of molecular sciences · 2023Article
31 more citing papers are in PubMed but not listed here.
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Authors and funding
10 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundMassively parallel cDNA sequencing (RNA-seq) experiments are gradually superseding microarrays in quantitative gene expression profiling. However, many biologists are uncertain about the choice of differentially expressed gene (DEG) analysis methods and the validity of cost-saving sample pooling strategies for their RNA-seq experiments. Hence, we performed experimental validation of DEGs identified by Cuffdiff2, edgeR, DESeq2 and Two-stage Poisson Model (TSPM) in a RNA-seq experiment involving mice amygdalae micro-punches, using high-throughput qPCR on independent biological replicate samples. Moreover, we sequenced RNA-pools and compared their results with sequencing corresponding individual RNA samples.
resultsFalse-positivity rate of Cuffdiff2 and false-negativity rates of DESeq2 and TSPM were high. Among the four investigated DEG analysis methods, sensitivity and specificity of edgeR was relatively high. We documented the pooling bias and that the DEGs identified in pooled samples suffered low positive predictive values.
conclusionsOur results highlighted the need for combined use of more sensitive DEG analysis methods and high-throughput validation of identified DEGs in future RNA-seq experiments. They indicated limited utility of sample pooling strategies for RNA-seq in similar setups and supported increasing the number of biological replicate samples.
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