Assessment of Common Housekeeping Genes As Reference for Gene Expression Studies Using RT-qPCR in Mouse Choroid Plexus

Overview

Journal Sci Rep

Specialty Science

Date 2021 Feb 9

PMID 33558629

Citations 21

Authors

Kim Hoa Ho

Annarita Patrizi

Affiliations

Soon will be listed here.

Abstract

Choroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

Citing Articles

Paralemmin-1 controls the nanoarchitecture of the neuronal submembrane cytoskeleton.

Macarron-Palacios V, Hubrich J, do Rego Barros Fernandes Lima M, Metzendorf N, Kneilmann S, Trapp M Sci Adv. 2025; 11(10):eadt3724.

PMID: 40053592 PMC: 11887803. DOI: 10.1126/sciadv.adt3724.

Amino Acid Hepatotoxicity Biomarkers in Human Hepatic Organoids: Promising Standardization of Drug Toxicity Evaluation.

Noh H, Choi S, Park K, Lee S, Seok D, Kim Y ACS Pharmacol Transl Sci. 2025; 8(2):510-521.

PMID: 39974651 PMC: 11833729. DOI: 10.1021/acsptsci.4c00612.

Evaluation of suitable reference genes for gene expression studies in the developing mouse cortex using RT-qPCR.

Uppalapati A, Wang T, Nguyen L BMC Neurosci. 2025; 26(1):12.

PMID: 39966711 PMC: 11837712. DOI: 10.1186/s12868-025-00934-y.

Lysate of prevents severe forms of experimental autoimmune encephalomyelitis by modulating the priming of T cell response.

Jiraskova Zakostelska Z, Kraus M, Coufal S, Prochazkova P, Slavickova Z, Thon T Front Immunol. 2024; 15:1475126.

PMID: 39737164 PMC: 11682988. DOI: 10.3389/fimmu.2024.1475126.

Effect of pandemic influenza A virus PB1 genes of avian origin on viral RNA polymerase activity and pathogenicity.

Williams S, Qi L, Sheng Z, Xiao Y, Freeman A, Matthews L Sci Adv. 2024; 10(50):eads5735.

PMID: 39671482 PMC: 11641000. DOI: 10.1126/sciadv.ads5735.

References

Ge R, Tornero D, Hirota M, Monni E, Laterza C, Lindvall O . Choroid plexus-cerebrospinal fluid route for monocyte-derived macrophages after stroke. J Neuroinflammation. 2017; 14(1):153. PMC: 5534106. DOI: 10.1186/s12974-017-0909-3. View

Scicchitano S, Giordano M, Lucchino V, Montalcini Y, Chiarella E, Aloisio A . The stem cell-associated transcription co-factor, ZNF521, interacts with GLI1 and GLI2 and enhances the activity of the Sonic hedgehog pathway. Cell Death Dis. 2019; 10(10):715. PMC: 6763495. DOI: 10.1038/s41419-019-1946-x. View

Lefever S, Hellemans J, Pattyn F, Przybylski D, Taylor C, Geurts R . RDML: structured language and reporting guidelines for real-time quantitative PCR data. Nucleic Acids Res. 2009; 37(7):2065-9. PMC: 2673419. DOI: 10.1093/nar/gkp056. View

Andersen C, Jensen J, Orntoft T . Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004; 64(15):5245-50. DOI: 10.1158/0008-5472.CAN-04-0496. View

Bustin S, Benes V, Garson J, Hellemans J, Huggett J, Kubista M . The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4):611-22. DOI: 10.1373/clinchem.2008.112797. View

Prasongchean W, Vernay B, Asgarian Z, Jannatul N, Ferretti P . The neural milieu of the developing choroid plexus: neural stem cells, neurons and innervation. Front Neurosci. 2015; 9:103. PMC: 4379892. DOI: 10.3389/fnins.2015.00103. View

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A . Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002; 3(7):RESEARCH0034. PMC: 126239. DOI: 10.1186/gb-2002-3-7-research0034. View

Thomas K, Zheng X, Suarez F, Raftery J, Quinlan K, Yang N . Evidence based selection of commonly used RT-qPCR reference genes for the analysis of mouse skeletal muscle. PLoS One. 2014; 9(2):e88653. PMC: 3921188. DOI: 10.1371/journal.pone.0088653. View

Chutake Y, Costello W, Lam C, Parikh A, Hughes T, Michalopulos M . FXN Promoter Silencing in the Humanized Mouse Model of Friedreich Ataxia. PLoS One. 2015; 10(9):e0138437. PMC: 4579136. DOI: 10.1371/journal.pone.0138437. View

10.

Massimino L, Flores-Garcia L, Di Stefano B, Colasante G, Icoresi-Mazzeo C, Zaghi M . TBR2 antagonizes retinoic acid dependent neuronal differentiation by repressing Zfp423 during corticogenesis. Dev Biol. 2018; 434(2):231-248. PMC: 7032051. DOI: 10.1016/j.ydbio.2017.12.020. View

11.

Yoshikawa A, Nakamachi T, Shibato J, Rakwal R, Shioda S . Comprehensive analysis of neonatal versus adult unilateral decortication in a mouse model using behavioral, neuroanatomical, and DNA microarray approaches. Int J Mol Sci. 2014; 15(12):22492-517. PMC: 4284721. DOI: 10.3390/ijms151222492. View

12.

Gasic I, Boswell S, Mitchison T . Tubulin mRNA stability is sensitive to change in microtubule dynamics caused by multiple physiological and toxic cues. PLoS Biol. 2019; 17(4):e3000225. PMC: 6474637. DOI: 10.1371/journal.pbio.3000225. View

13.

Hruz T, Wyss M, Docquier M, Pfaffl M, Masanetz S, Borghi L . RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization. BMC Genomics. 2011; 12:156. PMC: 3072958. DOI: 10.1186/1471-2164-12-156. View

14.

Zhao T, Mu X, You Q . Succinate: An initiator in tumorigenesis and progression. Oncotarget. 2017; 8(32):53819-53828. PMC: 5581152. DOI: 10.18632/oncotarget.17734. View

15.

Akhtar W, Veenstra G . TBP-related factors: a paradigm of diversity in transcription initiation. Cell Biosci. 2011; 1(1):23. PMC: 3142196. DOI: 10.1186/2045-3701-1-23. View

16.

Khimani A, Mhashilkar A, Mikulskis A, OMalley M, Liao J, Golenko E . Housekeeping genes in cancer: normalization of array data. Biotechniques. 2005; 38(5):739-45. DOI: 10.2144/05385ST04. View

17.

Rydbirk R, Folke J, Winge K, Aznar S, Pakkenberg B, Brudek T . Assessment of brain reference genes for RT-qPCR studies in neurodegenerative diseases. Sci Rep. 2016; 6:37116. PMC: 5112547. DOI: 10.1038/srep37116. View

18.

Musilli S, Nicolas N, El Ali Z, Orellana-Moreno P, Grand C, Tack K . DNA damage induced by Strontium-90 exposure at low concentrations in mesenchymal stromal cells: the functional consequences. Sci Rep. 2017; 7:41580. PMC: 5278504. DOI: 10.1038/srep41580. View

19.

Herbert J, Wilcox J, Pham K, Fremeau Jr R, Zeviani M, Dwork A . Transthyretin: a choroid plexus-specific transport protein in human brain. The 1986 S. Weir Mitchell award. Neurology. 1986; 36(7):900-11. DOI: 10.1212/wnl.36.7.900. View

20.

Moy J, Khoutorsky A, Asiedu M, Dussor G, Price T . eIF4E Phosphorylation Influences mRNA Translation in Mouse Dorsal Root Ganglion Neurons. Front Cell Neurosci. 2018; 12:29. PMC: 5808250. DOI: 10.3389/fncel.2018.00029. View