Using RNA-Seq Data to Evaluate Reference Genes Suitable for Gene Expression Studies in Soybean

PloS One
Aldrin Kay-Yuen YimHon-Ming Lam

Abstract

Differential gene expression profiles often provide important clues for gene functions. While reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an important tool, the validity of the results depends heavily on the choice of proper reference genes. In this study, we employed new and published RNA-sequencing (RNA-Seq) datasets (26 sequencing libraries in total) to evaluate reference genes reported in previous soybean studies. In silico PCR showed that 13 out of 37 previously reported primer sets have multiple targets, and 4 of them have amplicons with different sizes. Using a probabilistic approach, we identified new and improved candidate reference genes. We further performed 2 validation tests (with 26 RNA samples) on 8 commonly used reference genes and 7 newly identified candidates, using RT-qPCR. In general, the new candidate reference genes exhibited more stable expression levels under the tested experimental conditions. The three newly identified candidate reference genes Bic-C2, F-box protein2, and VPS-like gave the best overall performance, together with the commonly used ELF1b. It is expected that the proposed probabilistic model could serve as an important tool to identify stable refere...Continue Reading

References

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Citations

Apr 19, 2017·International Journal of Molecular Sciences·Qi JiaHon-Ming Lam
Feb 3, 2019·Aging Cell·Alejandra Hernandez-SeguraMarco Demaria
Feb 16, 2020·Journal of Experimental Botany·Yee-Shan KuHon-Ming Lam
May 24, 2020·The Plant Journal : for Cell and Molecular Biology·Fabricio B MachadoThiago M Venancio
Feb 26, 2019·Physiology and Molecular Biology of Plants : an International Journal of Functional Plant Biology·Ragini SinhaAnil Kumar Singh
Feb 26, 2021·Genome Biology·Mohieddin JafariNaser Ansari-Pour
Jul 11, 2021·The Plant Journal : for Cell and Molecular Biology·Xin WangHon-Ming Lam

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Datasets Mentioned

BETA
SRR1185323
SRR1737747

Methods Mentioned

BETA
RNA-Seq
PCR
environmental stress
RNA assay

Software Mentioned

Trimmomatic
Primer
NormFinder
BestKeeper
geomean
GeNorm
RefFinder
Ref
Phytozome
Finder

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