{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T04:57:55Z","timestamp":1783745875457,"version":"3.55.0"},"reference-count":60,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2021,8,17]],"date-time":"2021-08-17T00:00:00Z","timestamp":1629158400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"KNOW Consortium&quot; Healthy Animal - Safe Food&quot;, MS&amp;HE","award":["05-1\/KNOW2\/2015"],"award-info":[{"award-number":["05-1\/KNOW2\/2015"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Delivery of putative compounds of therapeutic value to the brain is limited by brain barriers: the blood\u2013brain barrier located in the endothelium of the brain microvessels (BrMVs) and the blood\u2013cerebrospinal fluid barrier located in the epithelium of the choroid plexus (ChP). Understanding their function and modulation by the circadian clock may enhance the efficacy of brain-targeting therapies. The aim of the present study was to evaluate the stability of 10 reference genes in the BrMV and ChP, isolated from male and female rats at six time points (ZT1, 5, 9, 13, 17, and 21). Gene evaluations were performed by qPCR, analyzed by RefFinder tool, and verified by analyzing the expression of the brain and muscle ARNT-like 1 (Bmal1) using the qPCR and digital PCR methods. We identified as the most stable genes for circadian studies tyrosine 3-monooxygenase\/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and apolipoprotein E (Apoe) for BrMV, and beta actin (Actb) and hypoxanthine-guanine phosphoribosyltransferase (Hprt1) for ChP. After verification, ribosomal protein (Rps18) was also included as a sufficient reference gene. Additionally, the observed gender difference in the Bmal1 oscillations in both BrMV and ChP suggests that separate studies for each gender are recommended.<\/jats:p>","DOI":"10.3390\/biom11081227","type":"journal-article","created":{"date-parts":[[2021,8,17]],"date-time":"2021-08-17T21:27:40Z","timestamp":1629235660000},"page":"1227","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Identification of Reference Genes for Circadian Studies on Brain Microvessels and Choroid Plexus Samples Isolated from Rats"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5578-3063","authenticated-orcid":false,"given":"Aleksandra","family":"Szczepkowska","sequence":"first","affiliation":[{"name":"Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-748 Olsztyn, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0904-5606","authenticated-orcid":false,"given":"Andr\u00e1s","family":"Harazin","sequence":"additional","affiliation":[{"name":"Institute of Biophysics, Biological Research Centre, 6726 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lilla","family":"Barna","sequence":"additional","affiliation":[{"name":"Institute of Biophysics, Biological Research Centre, 6726 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6084-6524","authenticated-orcid":false,"given":"M\u00e1ria A.","family":"Deli","sequence":"additional","affiliation":[{"name":"Institute of Biophysics, Biological Research Centre, 6726 Szeged, Hungary"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5900-6472","authenticated-orcid":false,"given":"Janina","family":"Skipor","sequence":"additional","affiliation":[{"name":"Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-748 Olsztyn, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.nbd.2009.07.030","article-title":"Structure and function of the blood-brain barrier","volume":"37","author":"Abbott","year":"2010","journal-title":"Neurobiol. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1083\/jcb.201412147","article-title":"Brain barriers: Crosstalk between complex tight junctions and adherens junctions","volume":"209","author":"Tietz","year":"2015","journal-title":"J. Cell Biol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"39","DOI":"10.29245\/2572.942X\/2018\/4.1194","article-title":"Choroid Plexus Blood-CSF Barrier: Major Player in Brain Disease Modeling and Neuromedicine","volume":"3","author":"Johanson","year":"2018","journal-title":"J. Neurol. Neuromed."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1481","DOI":"10.7150\/thno.21254","article-title":"Current Strategies for Brain Drug Delivery","volume":"8","author":"Dong","year":"2018","journal-title":"Theranostics"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Santa-Maria, A.R., Heymans, M., Walter, F.R., Culot, M., Gosselet, F., Deli, M.A., and Neuhaus, W. (2020). Transport Studies Using Blood-Brain Barrier In Vitro Models: A Critical Review and Guidelines. Handb. Exp. Pharm.","DOI":"10.1007\/164_2020_394"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1177\/1073858416639005","article-title":"The Blood-Brain Barrier: Regulatory Roles in Wakefulness and Sleep","volume":"23","author":"Pan","year":"2017","journal-title":"Neuroscientist"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1062","DOI":"10.1038\/s41467-018-03507-2","article-title":"The choroid plexus is an important circadian clock component","volume":"9","author":"Myung","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3195","DOI":"10.1111\/j.1460-9568.2007.05581.x","article-title":"Challenging the omnipotence of the suprachiasmatic timekeeper: Are circadian oscillators present throughout the mammalian brain?","volume":"25","author":"Guilding","year":"2007","journal-title":"Eur. J. Neurosci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1038\/nrn3208","article-title":"The circadian clock and pathology of the ageing brain","volume":"13","author":"Kondratova","year":"2012","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1251","DOI":"10.1007\/s00429-014-0720-1","article-title":"Gender associated circadian oscillations of the clock genes in rat choroid plexus","volume":"220","author":"Quintela","year":"2015","journal-title":"Brain Struct. Funct."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"102129","DOI":"10.1016\/j.pneurobio.2021.102129","article-title":"The Role of Circadian Rhythm in Choroid Plexus Functions","volume":"31","author":"Quintela","year":"2021","journal-title":"Prog. Neurobiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"C989","DOI":"10.1152\/ajpcell.00401.2013","article-title":"Rhythmic expression of cytochrome P450 epoxygenases CYP4x1 and CYP2c11 in the rat brain and vasculature","volume":"307","author":"Carver","year":"2014","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"10052","DOI":"10.1523\/JNEUROSCI.3639-16.2017","article-title":"Disruption of Bmal1 Impairs Blood-Brain Barrier Integrity via Pericyte Dysfunction","volume":"37","author":"Nakazato","year":"2017","journal-title":"J. Neurosci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.cell.2018.02.017","article-title":"A Circadian Clock in the Blood-Brain Barrier Regulates Xenobiotic Efflux","volume":"173","author":"Zhang","year":"2018","journal-title":"Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"234","DOI":"10.2174\/138920207781386960","article-title":"Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes","volume":"8","author":"Deepak","year":"2007","journal-title":"Curr. Genom."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"609","DOI":"10.1590\/S1519-69842002000400008","article-title":"Determination of the estrous cycle phases of rats: Some helpful considerations","volume":"62","author":"Marcondes","year":"2002","journal-title":"Braz. J. Biol."},{"key":"ref_17","first-page":"e4285","article-title":"A visual description of the dissection of the cerebral surface vasculature and associated meninges and the choroid plexus from rat brain","volume":"69","author":"Bowyer","year":"2012","journal-title":"J. Vis. Exp."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.brainres.2006.11.089","article-title":"Expression of drug transporters at the blood-brain barrier using an optimized isolated rat brain microvessel strategy","volume":"1134","author":"Yousif","year":"2007","journal-title":"Brain Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1038\/nprot.2006.83","article-title":"The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: Twenty-something years on","volume":"1","author":"Chomczynski","year":"2006","journal-title":"Nat. Protoc."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1047","DOI":"10.1089\/cmb.2005.12.1047","article-title":"Comprehensive algorithm for quantitative real-time polymerase chain reaction","volume":"12","author":"Zhao","year":"2005","journal-title":"J. Comput. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Dai, M., Lin, Y., El-Amouri, S.S., Kohls, M., and Pan, D. (2018). Comprehensive evaluation of blood-brain barrier-forming micro-vasculatures: Reference and marker genes with cellular composition. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0197379"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"RESEARCH0034","DOI":"10.1186\/gb-2002-3-7-research0034","article-title":"Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes","volume":"3","author":"Vandesompele","year":"2002","journal-title":"Genome Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"5245","DOI":"10.1158\/0008-5472.CAN-04-0496","article-title":"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","volume":"64","author":"Andersen","year":"2004","journal-title":"Cancer Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1023\/B:BILE.0000019559.84305.47","article-title":"Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper\u2014excel-based tool using pair-wise correlations","volume":"26","author":"Pfaffl","year":"2004","journal-title":"Biotechnol. Lett."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Silver, N., Best, S., Jiang, J., and Thein, S.L. (2006). Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol. Biol., 7.","DOI":"10.1186\/1471-2199-7-33"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"302","DOI":"10.1002\/syn.20496","article-title":"Reference genes for normalization: A study of rat brain tissue","volume":"62","author":"Bonefeld","year":"2008","journal-title":"Synapse"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1262","DOI":"10.1016\/j.neurobiolaging.2009.07.005","article-title":"Liver X receptor activation restores memory in aged AD mice without reducing amyloid","volume":"32","author":"Vanmierlo","year":"2011","journal-title":"Neurobiol. Aging"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3215","DOI":"10.3892\/mmr.2017.6375","article-title":"Hypothalamo-hypophysial system in rats with autotransplantation of the adrenal cortex","volume":"15","author":"Takizawa","year":"2017","journal-title":"Mol. Med. Rep."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Langnaese, K., John, R., Schweizer, H., Ebmeyer, U., and Keilhoff, G. (2008). Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model. Bmc Mol. Biol., 9.","DOI":"10.1186\/1471-2199-9-53"},{"key":"ref_30","first-page":"4377","article-title":"Identification of suitable reference genes for gene expression studies in rat skeletal muscle following sciatic nerve crush injury","volume":"19","author":"Wang","year":"2019","journal-title":"Mol. Med. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1186\/s12987-015-0015-9","article-title":"Transfection of brain capillary endothelial cells in primary culture with defined blood-brain barrier properties","volume":"12","author":"Burkhart","year":"2015","journal-title":"Fluids Barriers CNS"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"pyw061","DOI":"10.1093\/ijnp\/pyw061","article-title":"Altered Expression Pattern of Clock Genes in a Rat Model of Depression","volume":"19","author":"Christiansen","year":"2016","journal-title":"Int. J. Neuropsychopharmacol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"325","DOI":"10.2478\/jomb-2014-0001","article-title":"Gene expression studies: How to obtain accurate and reliable data by quantitative real-time RT PCR","volume":"32","author":"Nestorov","year":"2013","journal-title":"J. Med. Biochem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1063","DOI":"10.1038\/nmeth.2697","article-title":"The need for transparency and good practices in the qPCR literature","volume":"10","author":"Bustin","year":"2013","journal-title":"Nat. Methods"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"32","DOI":"10.17221\/6\/2017-CJAS","article-title":"Effect of Melatonin from Slow-Release Implants on Aquaporins (AQP1 and AQP4) in the Ovine Choroid Plexus","volume":"63","author":"Szczepkowska","year":"2018","journal-title":"Czech. J. Anim. Sci."},{"key":"ref_36","first-page":"249","article-title":"Melatonin from slow-release implants upregulates claudin-2 in the ovine choroid plexus","volume":"70","author":"Szczepkowska","year":"2019","journal-title":"J. Physiol. Pharm."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.rvsc.2017.09.003","article-title":"The effect of melatonin from slow-release implants on basic and TLR-4-mediated gene expression of inflammatory cytokines and their receptors in the choroid plexus in ewes","volume":"113","author":"Kowalewska","year":"2017","journal-title":"Res. Vet. Sci."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.smallrumres.2016.11.018","article-title":"Melatonin from slow-release implants did not influence the gene expression of the lipopolysaccharide receptor complex in the choroid plexus of seasonally anoestrous adult ewes subjected or not to a systemic inflammatory stimulus","volume":"147","author":"Kowalewska","year":"2017b","journal-title":"Small Rumin Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1007\/BF03195635","article-title":"Evaluation of reference genes for studies of gene expression in the bovine liver, kidney, pituitary, and thyroid","volume":"49","author":"Lisowski","year":"2008","journal-title":"J. Appl. Genet."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"11484","DOI":"10.3390\/ijms140611484","article-title":"Reference gene selection for quantitative PCR studies in sheep neutrophils","volume":"14","author":"Vorachek","year":"2013","journal-title":"Int. J. Mol. Sci."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1186\/s12958-016-0176-7","article-title":"mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages","volume":"14","author":"Sharbati","year":"2016","journal-title":"Reprod. Biol. Endocrinol."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Chapman, J.R., and Waldenstrom, J. (2015). With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0141853"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Liu, Y.J., Zheng, D., Balasubramanian, S., Carriero, N., Khurana, E., Robilotto, R., and Gerstein, M.B. (2009). Comprehensive analysis of the pseudogenes of glycolytic enzymes in vertebrates: The anomalously high number of GAPDH pseudogenes highlights a recent burst of retrotrans-positional activity. Bmc Genom., 10.","DOI":"10.1186\/1471-2164-10-480"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1373\/clinchem.2008.112797","article-title":"The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments","volume":"55","author":"Bustin","year":"2009","journal-title":"Clin. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"391","DOI":"10.1007\/s13353-013-0173-x","article-title":"Reference genes in real-time PCR","volume":"54","author":"Kozera","year":"2013","journal-title":"J. Appl Genet."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"617","DOI":"10.1038\/s41467-020-20795-9","article-title":"A circadian clock regulates efflux by the blood-brain barrier in mice and human cells","volume":"12","author":"Zhang","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1006\/exmp.2002.2424","article-title":"Expression of the endothelial markers PECAM-1, vWf, and CD34 in vivo and in vitro","volume":"72","author":"Hermanns","year":"2002","journal-title":"Exp. Mol. Pathol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1186\/s12987-019-0123-z","article-title":"Claudin-5: Gatekeeper of neurological function","volume":"16","author":"Greene","year":"2019","journal-title":"Fluids Barriers CNS"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1186\/2045-8118-8-10","article-title":"Transcriptome signature of the adult mouse choroid plexus","volume":"8","author":"Marques","year":"2011","journal-title":"Fluids Barriers CNS"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"398","DOI":"10.3389\/fphys.2020.00398","article-title":"The Isolated Brain Microvessel: A Versatile Experimental Model of the Blood-Brain Barrier","volume":"11","author":"Pardridge","year":"2020","journal-title":"Front. Physiol."},{"key":"ref_51","first-page":"9605265","article-title":"The Appropriate Marker for Astrocytes: Comparing the Distribution and Expression of Three Astrocytic Markers in Different Mouse Cerebral Regions","volume":"2019","author":"Zhang","year":"2019","journal-title":"Biomed. Res. Int."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1186\/1750-1326-5-32","article-title":"Pericyte-specific expression of PDGF beta receptor in mouse models with normal and deficient PDGF beta receptor signaling","volume":"5","author":"Winkler","year":"2010","journal-title":"Mol. Neurodegener"},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Quan, P.L., Sauzade, M., and Brouzes, E. (2018). dPCR: A technology review. Sensors, 18.","DOI":"10.3390\/s18041271"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"190","DOI":"10.1016\/j.ymgme.2017.04.001","article-title":"Determination of reference genes that are independent of feeding rhythms for circadian studies of mouse metabolic tissues","volume":"121","author":"Nakao","year":"2017","journal-title":"Mol. Genet. Metab."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"R19","DOI":"10.1186\/gb-2007-8-2-r19","article-title":"qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data","volume":"8","author":"Hellemans","year":"2007","journal-title":"Genome Biol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1373\/clinchem.2014.221366","article-title":"Considerations for digital PCR as an accurate molecular diagnostic tool","volume":"61","author":"Huggett","year":"2015","journal-title":"Clin. Chem."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"1510","DOI":"10.1016\/j.cmi.2019.06.009","article-title":"Digital PCR: A new technology for diagnosis of parasitic infections","volume":"25","author":"Pomari","year":"2019","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1007\/s11523-019-00645-5","article-title":"Digital PCR-Based Detection of EGFR Mutations in Paired Plasma and CSF Samples of Lung Adenocarcinoma Patients with Central Nervous System Metastases","volume":"14","author":"Huang","year":"2019","journal-title":"Target. Oncol."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1007\/s12020-009-9154-6","article-title":"Estrogen modulates expression of putative housekeeping genes in the mouse uterus","volume":"35","author":"Schroder","year":"2009","journal-title":"Endocrine"},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"2492","DOI":"10.1210\/en.2014-1890","article-title":"Measurement of a Comprehensive Sex Steroid Profile in Rodent Serum by High-Sensitive Gas Chromatography-Tandem Mass Spectrometry","volume":"156","author":"Nilsson","year":"2015","journal-title":"Endocrinology"}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/11\/8\/1227\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:45:49Z","timestamp":1760165149000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/11\/8\/1227"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,17]]},"references-count":60,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["biom11081227"],"URL":"https:\/\/doi.org\/10.3390\/biom11081227","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,17]]}}}