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

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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Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Journal of Visualized Experiments ◽

10.3791/50308 ◽

2013 ◽

Cited By ~ 7

Author(s):

Deborah R. Boone ◽

Stacy L. Sell ◽

Helen Lee Hellmich

Keyword(s):

Gene Expression ◽

Traumatic Brain Injury ◽

Brain Injury ◽

Expression Analysis ◽

Laser Capture Microdissection ◽

Gene Expression Analysis ◽

Single Neurons ◽

Laser Capture

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Selection and evaluation of reference genes for quantitative gene expression analysis in broomcorn millet (Panicum miliaceum L.)

Journal of Plant Biology ◽

10.1007/s12374-016-0024-5 ◽

2016 ◽

Vol 59 (5) ◽

pp. 435-443 ◽

Cited By ~ 2

Author(s):

Hong Yue ◽

Pingchuan Deng ◽

Siyan Liu ◽

Meng Wang ◽

Weining Song ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Panicum Miliaceum ◽

Quantitative Gene Expression ◽

Broomcorn Millet

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Gene Expression Analysis of the Activating Factor 3/Nuclear Protein 1 Axis in a Non-alcoholic Steatohepatitis Mouse Model

Yonago Acta Medica ◽

10.33160/yam.2019.03.006 ◽

2019 ◽

Vol 62 (1) ◽

pp. 036-046 ◽

Cited By ~ 1

Author(s):

Ran Nagahara ◽

Tomomitsu Matono ◽

Takaaki Sugihara ◽

Yukako Matsuki ◽

Masafumi Yamane ◽

...

Keyword(s):

Gene Expression ◽

Mouse Model ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Nuclear Protein ◽

Alcoholic Steatohepatitis

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Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR

BMC Molecular Biology ◽

10.1186/1471-2199-11-15 ◽

2010 ◽

Vol 11 (1) ◽

pp. 15 ◽

Cited By ~ 149

Author(s):

Asad Maroufi ◽

Erik Van Bockstaele ◽

Marc De Loose

Keyword(s):

Gene Expression ◽

Real Time ◽

Expression Analysis ◽

Real Time Pcr ◽

Reference Genes ◽

Gene Expression Analysis ◽

Cichorium Intybus ◽

Quantitative Real Time Pcr

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Validation of Internal Control Genes for Quantitative Real-Time PCR Gene Expression Analysis in Morchella

Molecules ◽

10.3390/molecules23092331 ◽

2018 ◽

Vol 23 (9) ◽

pp. 2331 ◽

Cited By ~ 10

Author(s):

Qianqian Zhang ◽

Wei Liu ◽

Yingli Cai ◽

A-Feng Lan ◽

Yinbing Bian

Keyword(s):

Gene Expression ◽

Candidate Genes ◽

Expression Analysis ◽

Internal Control ◽

Reference Genes ◽

Gene Expression Analysis ◽

Stable Gene ◽

Experimental Conditions ◽

The Stability ◽

Vacuolar Protein

The reliability of qRT-PCR results depend on the stability of reference genes used for normalization, suggesting the necessity of identification of reference genes before gene expression analysis. Morels are edible mushrooms well-known across the world and highly prized by many culinary kitchens. Here, several candidate genes were selected and designed according to the Morchella importuna transcriptome data. The stability of the candidate genes was evaluated with geNorm and NormFinder under three different experimental conditions, and several genes with excellent stability were selected. The extensive adaptability of the selected genes was tested in ten Morchella species. Results from the three experimental conditions revealed that ACT1 and INTF7 were the most prominent genes in Morchella, CYC3 was the most stable gene in different development stages, INTF4/AEF3 were the top-ranked genes across carbon sources, while INTF3/CYC3 pair showed the robust stability for temperature stress treatment. We suggest using ACT1, AEF3, CYC3, INTF3, INTF4 and INTF7 as reference genes for gene expression analysis studies for any of the 10 Morchella strains tested in this study. The stability and practicality of the gene, vacuolar protein sorting (INTF3), vacuolar ATP synthase (INTF4) and14-3-3 protein (INTF7) involving the basic biological processes were validated for the first time as the candidate reference genes for quantitative PCR. Furthermore, the stability of the reference genes was found to vary under the three different experimental conditions, indicating the importance of identifying specific reference genes for particular conditions.

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