Validation of reference genes for expression analysis in a murine trauma model combining traumatic brain injury and femoral fracture

Abstract Systemic and local posttraumatic responses are often monitored on mRNA expression level using quantitative real-time PCR (qRT-PCR), which requires normalisation to adjust for confounding sources of variability. Normalisation requests reference (housekeeping) genes stable throughout time and divergent experimental conditions in the tissue of interest, which are crucial for a reliable and reproducible gene expression analysis. Although previous animal studies analysed reference genes following isolated trauma, this multiple-trauma gene expression analysis provides a notable study analysing reference genes in primarily affected (i.e. bone/fracture callus and hypothalamus) and secondarily affected organs (i.e. white adipose tissue, liver, muscle and spleen), following experimental long bone fracture and traumatic brain injury. We considered tissue-specific and commonly used top-ranked reference candidates from different functional groups that were evaluated applying the established expression stability analysis tools NormFinder, GeNorm, BestKeeper and RefFinder. In conclusion, reference gene expression in primary organs is highly time point as well as tissue-specific, and therefore requires careful evaluation for qRT-PCR analysis. Furthermore, the general application of Ppia, particularly in combination with a second reference gene, is strongly recommended for the analysis of systemic effects in the case of indirect trauma affecting secondary organs through local and systemic pathophysiological responses.

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Reference Gene Selection for Normalizing Gene Expression in Ips Sexdentatus (Coleoptera: Curculionidae: Scolytinae) Under Different Experimental Conditions

Frontiers in Physiology ◽

10.3389/fphys.2021.752768 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gothandapani Sellamuthu ◽

Shan Amin ◽

Jan Bílý ◽

Jirí Synek ◽

Roman Modlinger ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Gene Selection ◽

Experimental Conditions ◽

Tissue Specific ◽

Reference Gene Selection ◽

Ips Sexdentatus

Ips sexdentatus (Coleoptera: Curculionidae: Scolytinae) is one of the most destructive and economically important forest pests. A better understanding of molecular mechanisms underlying its adaptation to toxic host compounds may unleash the potential for future management of this pest. Gene expression studies could be considered as one of the key experimental approaches for such purposes. A suitable reference gene selection is fundamental for quantitative gene expression analysis and functional genomics studies in I. sexdentatus. Twelve commonly used reference genes in Coleopterans were screened under different experimental conditions to obtain accurate and reliable normalization of gene expression data. The majority of the 12 reference genes showed a relatively stable expression pattern among developmental stages, tissue-specific, and sex-specific stages; however, some variabilities were observed during varied temperature incubation. Under developmental conditions, the Tubulin beta-1 chain (β-Tubulin) was the most stable reference gene, followed by translation elongation factor (eEF2) and ribosomal protein S3 (RPS3). In sex-specific conditions, RPS3, β-Tubulin, and eEF2 were the most stable reference genes. In contrast, different sets of genes were shown higher stability in terms of expression under tissue-specific conditions, i.e., RPS3 and eEF2 in head tissue, V-ATPase-A and eEF2 in the fat body, V-ATPase-A and eEF2 in the gut. Under varied temperatures, β-Tubulin and V-ATPase-A were most stable, whereas ubiquitin (UbiQ) and V-ATPase-A displayed the highest expression stability after Juvenile Hormone III treatment. The findings were validated further using real-time quantitative reverse transcription PCR (RT-qPCR)-based target gene expression analysis. Nevertheless, the present study delivers a catalog of reference genes under varied experimental conditions for the coleopteran forest pest I. sexdentatus and paves the way for future gene expression and functional genomic studies on this species.

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Validation of reference genes for gene expression analysis following experimental traumatic brain injury in a pediatric mouse model

Brain Research Bulletin ◽

10.1016/j.brainresbull.2019.12.015 ◽

2020 ◽

Vol 156 ◽

pp. 43-49 ◽

Cited By ~ 2

Author(s):

Akram Zamani ◽

Kim L. Powell ◽

Ashleigh May ◽

Bridgette D. Semple

Keyword(s):

Gene Expression ◽

Traumatic Brain Injury ◽

Brain Injury ◽

Mouse Model ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Experimental Traumatic Brain Injury

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Expression Stability of Internal Reference Gene in Response to Trichoderma polysporum Infection in Avena fatua L.

10.21203/rs.3.rs-507940/v1 ◽

2021 ◽

Author(s):

Haixia Zhu ◽

Yongqiang Ma ◽

Liang Cheng

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Avena Fatua ◽

Expression Stability ◽

Internal Reference ◽

Qpcr Analysis ◽

Suitable Combination

Abstract In order to construct a RT-qPCR system suitable for response of Avena fatua L. to Trichoderma polysporum , and screen stable internal reference genes, GeNorm, NormFinder, BestKeeper and RefFinde were used to perform SYBR Green-based RT-qPCR analysis on 8 candidate internal reference genes ( 18S , 28S , TUA , UBC , ACT , GAPDH , TBP and EF-1 ) in A. fatua samples after inoculation of T. polysporum Strain HZ-31. The results showed that TBP , 18S and UBC were the most stable internal reference genes, TBP and TUA , TBP and GAPDH , 18S and TBP , UBC and 18S were the most suitable combination of the two internal reference genes, which could be used as the internal reference genes for functional gene expression analysis during the interaction between T. polysporum and A. fatua .

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Reference genes for gene expression analysis in the fungal pathogen Neonectria ditissima and their use demonstrating expression up-regulation of candidate virulence genes

10.1101/2020.08.12.247601 ◽

2020 ◽

Author(s):

Liz M. Florez ◽

Reiny W. A. Scheper ◽

Brent M. Fisher ◽

Paul W. Sutherland ◽

Matthew D. Templeton ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reference Genes ◽

Time Course ◽

Gene Expression Analysis ◽

Virulence Genes ◽

Functional Characterization ◽

Post Inoculation ◽

In Planta ◽

Qrt Pcr

AbstractEuropean canker, caused by the necrotrophic fungal phytopathogen Neonectria ditissima, is one of the most damaging apple diseases worldwide. An understanding of the molecular basis of N. ditissima virulence is currently lacking. Identification of genes with an up-regulation of expression during infection, which are therefore probably involved in virulence, is a first step towards this understanding. Real-time quantitative reverse transcription PCR (qRT-PCR) can be used to identify these candidate virulence genes, but relies on the use of reference genes for relative gene expression data normalisation. However, no report that addresses selecting appropriate fungal reference genes for use in the N. ditissima-apple pathosystem has been published to date. In this study, eight N. ditissima genes were selected as candidate qRT-PCR reference genes for gene expression analysis. A subset of the primers (six) designed to amplify regions from these genes were specific for N. ditissima, failing to amplify PCR products with template from other fungal pathogens present in the apple orchard. The efficiency of amplification of these six primer sets was satisfactory, ranging from 81.8 to 107.53%. Analysis of expression stability when a highly pathogenic N. ditissima isolate was cultured under 10 regimes, using the statistical algorithms geNorm, NormFinder and BestKeeper, indicated that actin and myo-inositol-1-phosphate synthase (mips), or their combination, could be utilised as the most suitable reference genes for normalisation of N. ditissima gene expression. As a test case, these reference genes were used to study expression of three candidate virulence genes during a time course of infection. All three, which shared traits with fungal effector genes, had up-regulated expression in planta compared to in vitro with expression peaking between five and six weeks post inoculation (wpi). Thus, these three genes may well be involved in N. ditissima pathogenicity and are priority candidates for further functional characterization.

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