Evaluation and selection of internal reference genes from two- and six-row U.S. malting barley varieties throughout micromalting for use in RT-qPCR

Abstract In gene expression analysis, sample differences and experimental operation differences are common, but sometimes, these differences will cause serious errors to the results or even make the results meaningless. Finding suitable internal reference genes efficiently to eliminate errors is a challenge. Aside from the need for high efficiency, there is no package for screening endogenous genes available in Python. Here, we introduce ERgene, a Python library for screening endogenous reference genes. It has extremely high computational efficiency and simple operation steps. The principle is based on the inverse process of the internal reference method, and the robust matrix block operation makes the selection of internal reference genes faster than any other method.

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Selection of Suitable Reference Genes Based on Transcriptomic Data in Ginkgo biloba under Different Experimental Conditions

Forests ◽

10.3390/f11111217 ◽

2020 ◽

Vol 11 (11) ◽

pp. 1217

Author(s):

Tingting Zhou ◽

Xiaoming Yang ◽

Fangfang Fu ◽

Guibin Wang ◽

Fuliang Cao

Keyword(s):

Gene Expression ◽

Ginkgo Biloba ◽

Reference Genes ◽

Developmental Stages ◽

Deciduous Tree ◽

Internal Reference ◽

Experimental Conditions ◽

Transcriptomic Data ◽

Suitable Reference ◽

Selection Of

Ginkgo biloba, a deciduous tree species in the Ginkgo family, has a long history of cultivation in China and is widely used in garden landscapes, medicine, food, and health products. However, few reports have focused on the systematic selection of optimal reference genes based on transcriptomic data in G. biloba. The purpose of our research was to select an internal reference gene suitable for different experimental conditions from thirteen candidate reference genes by the delta cycle threshold (ΔCt) method, geNorm, BestKeeper, NormFinder, and RefFinder programs. The reference genes were used for gene expression analyses of Ginkgo biloba. These results showed that elongation factor 1(EF1) and ubiquitin (UBI) were the best choices for samples of different ginkgo genotypes. The expression of UBI and HAS28 presented the most stable at different developmental stages of ginkgo, and EIF3I and RPII were considered as suitable reference genes in different tissues of ginkgo. For methyl jasmonate (MeJA) treatment, ACA and ACT were identified as the optimal reference genes. For cold stress treatment, RPII and EIF4E were chosen for the gene expression normalizations. HAS28 and GAPDH presented the most stable expression for the heat treatment. To validate the above results, a chalcone synthase gene (GbCHS) in ginkgo was amplified by quantitative real-time polymerase chain reaction (qRT-PCR). Our results provide different suitable reference genes for further gene expression studies in ginkgo.

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Selection of Reference Genes for Transcription Studies on Adventitious Root Induction in Olive (Olea Europaea L.) Microshoots Considering Co-expression and Average Transcriptional Stability

10.21203/rs.3.rs-60001/v1 ◽

2020 ◽

Author(s):

Carlos Noceda ◽

Augusto Peixe ◽

Birgit Arnholdt-Schmitt

Keyword(s):

Gene Expression ◽

Gene Expression Data ◽

Reference Genes ◽

Ad Hoc ◽

Root Induction ◽

Expression Data ◽

Internal Reference ◽

Experimental Conditions ◽

Selection Of

Abstract BackgroungSelection of reference genes (RGs) for normalization of PCR-gene expression data includes two crucial steps: determination of the among-sample transcriptionally more stable genes and subsequent choosing of the most suitable genes as internal controls. Both steps can be carried-out through generally accepted strategies each having different strengths and weaknesses. The present study proposes to reinforce normalization of gene expression data by integrating and adding analytical revision at critical steps of those accepted procedures. Especially crucial is to counterbalance a higher representative number of RGs with a correspondent increase in their average transcriptional instability or a generalised co-expression trend among the samples. This methodological study used in vitro olive adventitious rooting as an experimental system, since the underlying morphogenetic process -wich is common to diverse species- is still not completely understood.ResultsFirstly, RG candidates were ranked according to transcriptional stability following a simple statistical method that reduces biasing effects of concomitant, systematic biological variations associated to experimental conditions, such as the variations caused by gene co-regulation. Those types of systematic co-variation are unconsidered by several popular ad hoc informatics programmes. To select the adequate genes among those already ranked, an algorithm of one of the ad hoc informatics programmes (GeNorm) was adapted to allow partial automatization of RG selection for any strategy of transcriptional-gene stability ordering. In order to delve into the resulting possible RG sets suitability for inter-assay comparisons and technical-error compensation, separate statistics were formulated. The achieved results were compared with those obtained by standard stability ranking methods. Finally, a double evaluation was performed to accurately contrast two choice RG sets. The whole strategy was applied to a panel considering several independent factors, but the suitability of the obtained putative RG sets was tested for cases restricted to fewer variables. H2B, OUB and ACT are valid for normalization in transcriptional studies on olive microshoot rooting when comparing treatments, time points and assays.ConclusionsThe set of genes identified as internal reference is now available for wider expression studies on any target gene in similar biological systems. The overall methodology aims to constitute a guide for general application.

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Selection and Validation of Reference Genes Desirable for Gene Expression Analysis by qRT-PCR on Seed Germination of Castanea henryi

10.1101/2021.01.27.428382 ◽

2021 ◽

Author(s):

Bin Liu ◽

Yuting Jiang ◽

Ruqiang Lin ◽

Yuanfang Xiong ◽

Shuzhen Jiang ◽

...

Keyword(s):

Seed Germination ◽

Candidate Genes ◽

Reference Gene ◽

Reference Genes ◽

Stable Gene ◽

Internal Reference ◽

Seed Biology ◽

Unstable Gene ◽

The Stability ◽

Selection Of

AbstractSeed germination is the beginning of the plant’s life cycle, and seed biology is one of the most extensively researched areas in plant physiology, however, Castanea henryi as an important seed plant, the stable internal reference gene during germination is not clear. In this study, seven candidate genes (TUA, TUB, TIF, UBC, RPL21, RPL30, RPL34) were screened out from transcriptome data, we analyzed the expression of seven candidate reference genes in C. henryi at different germination stages with RT–qPCR, and using common algorithms including NormFinder, geNorm and BestKeeper to evaluate the candidate genes stability. The results showed that RPL34 and RPL30 were selected as the most stable genes by NormFinder; TIF was the most stable gene identified by BestKeeper; RPL34 and RPL21 were the most stable genes ranked by geNorm, and TUB was the most unstable gene identified by all of the three software. The RPL34 gene was used as the reference gene, to detected the expression trend of two starch synthetase genes SS1 and SS2 during germination by RT–qPCR, the results of RT–qPCR and transcriptome sequencing were basically consistent, which verified the stability of RPL34 candidate gene. Our result is not only showed functional genes for germination of C. henryi seeds and provide useful guidelines for the selection of reliable reference genes for the normalization of RT– qPCR data for germination of seed plants.

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