Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions

AbstractGlycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

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Optimizations for identifying reference genes in bone and cartilage bioengineering

BMC Biotechnology ◽

10.1186/s12896-021-00685-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Fei Xiong ◽

Xiangyun Cheng ◽

Chao Zhang ◽

Roland Manfred Klar ◽

Tao He

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Adipose Tissue ◽

Real Time ◽

Reference Gene ◽

Muscle Tissue ◽

Reference Genes ◽

Target Genes ◽

The Stability ◽

And Cartilage

Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) remains one of the best-established techniques to assess gene expression patterns. However, appropriate reference gene(s) selection remains a critical and challenging subject in which inappropriate reference gene selction can distort results leading to false interpretations. To date, mixed opinions still exist in how to choose the most optimal reference gene sets in accodrance to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes were the most feasible for the identification of reference genes in a bone and cartilage bioengineering experimental setting. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme that would subsequently ensure stable and accurate interpretation of gene expression in bone and cartilage bioengineering. Results The stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed that different schemes do indeed have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values with eventual normalization of target genes showed that the different selection schemes also have a significant effect on the eventual normalization of target genes. Conclusions Based on these results, the proposed cut-off value of Vn/n + 1 under 0.15, according to the geNorm algorithm, should be considered with caution. For cell only experiments, at least rBMSCs, a Vn/n + 1 under 0.15 is sufficient in RT-qPCR studies. However, when using certain tissue types such as skeletal muscle and adipose tissue the minimum Vn/n + 1 should be used instead as this provides a far superior mode of generating accurate gene expression results. We thus recommended that when the stability and variation of a candidate reference genes in a specific study is unclear the minimum Vn/n + 1 should always be used as this ensures the best and most accurate gene expression value is achieved during RT-qPCR assays.

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The carbon source-dependent pattern of antimicrobial activity and gene expression in Pseudomonas donghuensis P482

Scientific Reports ◽

10.1038/s41598-021-90488-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marta Matuszewska ◽

Tomasz Maciąg ◽

Magdalena Rajewska ◽

Aldona Wierzbicka ◽

Sylwia Jafra

Keyword(s):

Gene Expression ◽

Antimicrobial Activity ◽

Carbon Source ◽

Reference Genes ◽

Plant Pathogens ◽

Plant Protection ◽

Carbon Sources ◽

Unknown Compound ◽

Fungal Plant Pathogens ◽

The Impact

AbstractPseudomonas donghuensis P482 is a tomato rhizosphere isolate with the ability to inhibit growth of bacterial and fungal plant pathogens. Herein, we analysed the impact of the carbon source on the antibacterial activity of P482 and expression of the selected genes of three genomic regions in the P482 genome. These regions are involved in the synthesis of pyoverdine, 7-hydroxytropolone (7-HT) and an unknown compound (“cluster 17”) and are responsible for the antimicrobial activity of P482. We showed that the P482 mutants, defective in these regions, show variations and contrasting patterns of growth inhibition of the target pathogen under given nutritional conditions (with glucose or glycerol as a carbon source). We also selected and validated the reference genes for gene expression studies in P. donghuensis P482. Amongst ten candidate genes, we found gyrB, rpoD and mrdA the most stably expressed. Using selected reference genes in RT-qPCR, we assessed the expression of the genes of interest under minimal medium conditions with glucose or glycerol as carbon sources. Glycerol was shown to negatively affect the expression of genes necessary for 7-HT synthesis. The significance of this finding in the light of the role of nutrient (carbon) availability in biological plant protection is discussed.

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Selection of reference genes for gene expression analysis in Liriodendron hybrids’ somatic embryogenesis and germinative tissues

Scientific Reports ◽

10.1038/s41598-021-84518-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Tingting Li ◽

Weigao Yuan ◽

Shuai Qiu ◽

Jisen Shi

Keyword(s):

Gene Expression ◽

Somatic Embryogenesis ◽

Reference Genes ◽

Gene Selection ◽

Elongation Factor ◽

Reverse Transcription Pcr ◽

Expression Of Genes ◽

Suitable Reference ◽

Functional Analyses ◽

Elongation Factor 1

AbstractThe differential expression of genes is crucial for plant somatic embryogenesis (SE), and the accurate quantification of gene expression levels relies on choosing appropriate reference genes. To select the most suitable reference genes for SE studies, 10 commonly used reference genes were examined in synchronized somatic embryogenic and subsequent germinative cultures of Liriodendron hybrids by using quantitative real-time reverse transcription PCR. Four popular normalization algorithms: geNorm, NormFinder, Bestkeeper and Delta-Ct were used to select and validate the suitable reference genes. The results showed that elongation factor 1-gamma, histone H1 linker protein, glyceraldehyde-3-phosphate dehydrogenase and α-tubulin were suitable for SE tissues, while elongation factor 1-gamma and actin were best for the germinative organ tissues. Our work will benefit future studies of gene expression and functional analyses of SE in Liriodendron hybrids. It is also serves as a guide of reference gene selection in early embryonic gene expression analyses for other woody plant species.

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Temporal changes in DNA methylation and RNA expression in a small song bird: within- and between-tissue comparisons

BMC Genomics ◽

10.1186/s12864-020-07329-9 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Melanie Lindner ◽

Irene Verhagen ◽

Heidi M. Viitaniemi ◽

Veronika N. Laine ◽

Marcel E. Visser ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Environmental Conditions ◽

Brain Regions ◽

Temporal Changes ◽

Egg Laying ◽

Gene Body ◽

Cpg Site ◽

Repeated Sampling ◽

Over Time

Abstract Background DNA methylation is likely a key mechanism regulating changes in gene transcription in traits that show temporal fluctuations in response to environmental conditions. To understand the transcriptional role of DNA methylation we need simultaneous within-individual assessment of methylation changes and gene expression changes over time. Within-individual repeated sampling of tissues, which are essential for trait expression is, however, unfeasible (e.g. specific brain regions, liver and ovary for reproductive timing). Here, we explore to what extend between-individual changes in DNA methylation in a tissue accessible for repeated sampling (red blood cells (RBCs)) reflect such patterns in a tissue unavailable for repeated sampling (liver) and how these DNA methylation patterns are associated with gene expression in such inaccessible tissues (hypothalamus, ovary and liver). For this, 18 great tit (Parus major) females were sacrificed at three time points (n = 6 per time point) throughout the pre-laying and egg-laying period and their blood, hypothalamus, ovary and liver were sampled. Results We simultaneously assessed DNA methylation changes (via reduced representation bisulfite sequencing) and changes in gene expression (via RNA-seq and qPCR) over time. In general, we found a positive correlation between changes in CpG site methylation in RBCs and liver across timepoints. For CpG sites in close proximity to the transcription start site, an increase in RBC methylation over time was associated with a decrease in the expression of the associated gene in the ovary. In contrast, no such association with gene expression was found for CpG site methylation within the gene body or the 10 kb up- and downstream regions adjacent to the gene body. Conclusion Temporal changes in DNA methylation are largely tissue-general, indicating that changes in RBC methylation can reflect changes in DNA methylation in other, often less accessible, tissues such as the liver in our case. However, associations between temporal changes in DNA methylation with changes in gene expression are mostly tissue- and genomic location-dependent. The observation that temporal changes in DNA methylation within RBCs can relate to changes in gene expression in less accessible tissues is important for a better understanding of how environmental conditions shape traits that temporally change in expression in wild populations.

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