Dataset Information

Impact of RNA integrity and blood sample storage conditions on the gene expression analysis.

ABSTRACT: Background:The reliability of RNA sequencing (RNA-seq) output is affected by the quality of RNAs, which is in turn dependent on the quality of samples. Therefore, the purposes of this study were to reconsider the threshold of the RNA integrity number (RIN) and propose a simple and efficient storage scheme of blood samples for RNA-seq. Patients and methods:The RNAs were extracted from blood samples that were stored at different conditions and used for sequencing. The bioinformatic analyses were performed to evaluate the impact of RNA integrity and blood sample storage conditions on the gene expression analysis. Results:Our outcomes showed that the samples with RIN values more than 5.3 scarcely affected the quantitative results of RNA-seq, and the influence of inherent cellular physiological processes on RNA-seq output could be negligible. Conclusion:The blood samples stored at 4°C within 7 days with RIN values more than 5.3 were available for RNA-seq.

SUBMITTER: Shen Y

PROVIDER: S-EPMC6016255 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Impact of RNA integrity and blood sample storage conditions on the gene expression analysis.

Shen Yanting Y Li Rui R Tian Fei F Chen Zhenzhu Z Lu Na N Bai Yunfei Y Ge Qinyu Q Lu Zuhong Z

OncoTargets and therapy 20180620

<h4>Background</h4>The reliability of RNA sequencing (RNA-seq) output is affected by the quality of RNAs, which is in turn dependent on the quality of samples. Therefore, the purposes of this study were to reconsider the threshold of the RNA integrity number (RIN) and propose a simple and efficient storage scheme of blood samples for RNA-seq.<h4>Patients and methods</h4>The RNAs were extracted from blood samples that were stored at different conditions and used for sequencing. The bioinformatic ...[more]

PMID: 29950862

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Project description:Storage of biological specimens is crucial in the life and medical sciences. Storage conditions for samples can be different for a number of reasons, and it is unclear what effect this can have on the inferred microbiome composition in metagenomics analyses. Here, we assess the effect of common storage temperatures (deep freezer, -80°C; freezer, -20°C; refrigerator, 5°C; room temperature, 22°C) and storage times (immediate sample processing, 0 h; next day, 16 h; over weekend, 64 h; longer term, 4, 8, and 12 months) as well as repeated sample freezing and thawing (2 to 4 freeze-thaw cycles). We examined two different pig feces and sewage samples, unspiked and spiked with a mock community, in triplicate, respectively, amounting to a total of 438 samples (777 Gbp; 5.1 billion reads). Storage conditions had a significant and systematic effect on the taxonomic and functional composition of microbiomes. Distinct microbial taxa and antimicrobial resistance classes were, in some situations, similarly affected across samples, while others were not, suggesting an impact of individual inherent sample characteristics. With an increasing number of freeze-thaw cycles, an increasing abundance of Firmicutes, Actinobacteria, and eukaryotic microorganisms was observed. We provide recommendations for sample storage and strongly suggest including more detailed information in the metadata together with the DNA sequencing data in public repositories to better facilitate meta-analyses and reproducibility of findings. IMPORTANCE Previous research has reported effects of DNA isolation, library preparation, and sequencing technology on metagenomics-based microbiome composition; however, the effect of biospecimen storage conditions has not been thoroughly assessed. We examined the effect of common sample storage conditions on metagenomics-based microbiome composition and found significant and, in part, systematic effects. Repeated freeze-thaw cycles could be used to improve the detection of microorganisms with more rigid cell walls, including parasites. We provide a data set that could also be used for benchmarking algorithms to identify and correct for unwanted batch effects. Overall, the findings suggest that all samples of a microbiome study should be stored in the same way. Furthermore, there is a need to mandate more detailed information about sample storage and processing be published together with DNA sequencing data at the International Nucleotide Sequence Database Collaboration (ENA/EBI, NCBI, DDBJ) or other repositories.

Dataset Information

Impact of RNA integrity and blood sample storage conditions on the gene expression analysis.

Publications

Impact of RNA integrity and blood sample storage conditions on the gene expression analysis.

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