ArticleBMC genomics2014
Influence of RNA extraction methods and library selection schemes on RNA-seq data.
Article in BMC genomics, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 95 papers.
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95 citing papers in PubMed, 188 citations in OpenAlex.
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- The Translatome Map: RNC-Seq vs. Ribo-Seq for Profiling of HBE, A549, and MCF-7 Cell Lines.International journal of molecular sciences · 2024Article
- A scalable and cost-efficient rRNA depletion approach to enrich RNAs for molecular biology investigations.RNA (New York, N.Y.) · 2024Article
- Dual RNA-seq in filarial nematodes andFrontiers in microbiology · 2024Article
- Associations between DNA methylation and gene regulation depend on chromatin accessibility during transgenerational plasticity.BMC biology · 2023Article
- Transcriptomics for Clinical and Experimental Biology Research: Hang on a Seq.Advanced genetics (Hoboken, N.J.) · 2023Review
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- In Vitro and Silico Studies on the N-Doped Carbon Dots Potential in ACE2 Expression Modulation.ACS omega · 2023Article
35 more citing papers are in PubMed but not listed here.
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Authors and funding
9 authors at 2 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundGene expression analysis by RNA sequencing is now widely used in a number of applications surveying the whole transcriptomes of cells and tissues. The recent introduction of ribosomal RNA depletion protocols, such as RiboZero, has extended the view of the polyadenylated transcriptome to the poly(A)- fraction of the RNA. However, substantial amounts of intronic transcriptional activity has been reported in RiboZero protocols, raising issues regarding their potential nuclear origin and the impact on the actual sequence depth in exonic regions.
resultsUsing HEK293 human cells as source material, we assessed here the impact of the two commonly used RNA extraction methods and of the library construction protocols (rRNA depletion versus mRNA) on 1) the relative abundance of intronic reads and 2) on the estimation of gene expression values. We benchmarked the rRNA depletion-based sequencing with a specific analysis of the cytoplasmic and nuclear transcriptome fractions, suggesting that the large majority of the intronic reads correspond to unprocessed nuclear transcripts rather than to independent transcriptional units. We show that Qiagen or TRIzol extraction methods retain differentially nuclear RNA species, and that consequently, rRNA depletion-based RNA sequencing protocols are particularly sensitive to the extraction methods.
conclusionsWe could show that the combination of Trizol-based RNA extraction with rRNA depletion sequencing protocols led to the largest fraction of intronic reads, after the sequencing of the nuclear transcriptome. We discuss here the impact of the various strategies on gene expression and alternative splicing estimation measures. Further, we propose guidelines and a double selection strategy for minimizing the expression biases, without loss of information.
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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 OpenQuestion graph.