Identification of Reference Genes for Circadian Studies on Brain Microvessels and Choroid Plexus Samples Isolated from Rats
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals and Tissue Collection
2.2. RNA Extraction, cDNA Preparation, and qPCR Analysis
2.3. Verification of the Tissue Isolation Process
2.4. Identification and qPCR Verification of Optimal Reference Genes
Gene | (Forward/Reverse) Sequence 5′→3′ | Amplicon Size (bp) | References/Sources | ||
---|---|---|---|---|---|
Reference genes | Actb | F: TGTCACCAACTGGGACGATA | R: GGGGTGTTGAAGGTCTCAAA | 165 | [26] |
Apoe | F: CCTGAACCGCTTCTGGGATT | R: GCTCTTCCTGGACCTGGTCA | 65 | [27] | |
Hmbs | F: TCCTGGCTTTACCATTGGAG | R: TGAATTCCAGGTGAGGGAAC | 176 | [26] | |
Hprt1 | F: CCTGTTGATGTGGCCAGTAAAGA | R: ATCAAAAGGGACGCAGCAAC | 137 | [28] | |
Pgk1 | F: ATGCAAAGACTGGCCAAGCTAC | R: AGCCACAGCCTCAGCATATTTC | 104 | [29] | |
Ppia | F: TATCTGCACTGCCAAGACTGAGTG | R: CTTCTTGCTGGTCTTGCCATTCC | 126 | [29] | |
Rplp2 | F: ATTGAGGATGTCATCGCTCAGG | R: TCTTTCTTCTCCTCTGCTGCAG | 137 | [28] | |
Rps18 | F: ACGGACCAGAGCGAAAGCAT | R: TGTCAATCCTGTCCGTGTCC | 310 | [26] | |
Tbp | F: TAATCCCAAGCGGTTTGCTG | R: TTCTTCACTCTTGGCTCCTGTG | 111 | [30] | |
Ywhaz | F: TTCGCAGCCAGAAAGCAAAG | R: TTGTCATCACCAGCAGCAAC | 87 | [28] | |
Marker genes | Plvap | F: ATAATCGGTTCATCGCCGCT | R: GCTTGAAGAGCAAGGCTTCG | 96 | NM_020086.1 |
Cldn-5 | F: CTACAGGCTCTTGTGAGGACTTGAC | R: AGTAGGAACTGTTAGCGGCAGTTTG | 121 | [31] | |
Pecam1 | F: GCCTCACCAAGAGAACGGAA | R: AATTGGATGGCTTGGCCTGA | 191 | NM_031591.1 | |
Bmal1 | F: TGGACTGCAACCGCAAGAG | R: CCTTCCATGAGGGTCATCTTTG | 137 | [28] |
2.5. Digital PCR
2.6. Statistical Analysis
3. Results
3.1. Characterization of the Isolated BrMV Fractions and ChP
3.2. Selection of Reference Genes and Efficiency of Primers
3.3. Selection and Verification of the Most Stable Reference Genes for Brain Microvessels
3.4. Selection and Verification of the Most Stable Reference Genes for Choroid Plexus
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene Symbol | Full Name | Function | Primers Efficiency | BrMV Cq Mean ± SEM | BrMV Cq Range | ChP Cq Mean ± SEM | ChP Cq Range |
---|---|---|---|---|---|---|---|
Actb | Beta actin | cytoskeletal structure protein | 85% | 19.56 ± 0.13 | 3.25 | 18.93 ± 0.08 | 2.12 |
Apoe | Apolipoprotein E | transport of lipids, fat soluble vitamins, and cholesterol | 85% | 19.58 ± 0.08 | 1.95 | 22.77 ± 0.2 | 5.06 |
Hmbs | Hydroxymethylbilane synthase | the third enzyme in the heme biosynthetic pathway | 85% | 26.5 ± 0.13 | 3.16 | 23.92 ± 0.09 | 2.36 |
Hprt1 | Hypoxanthine-guanine phosphoribosyltransferase | recycle purines in cells | 86% | 23.43 ± 0.14 | 3.74 | 20.66 ± 0.07 | 2.20 |
Pgk1 | Phosphoglycerate kinase 1 | enzyme involved in glycolysis process | 86% | 22.42 ± 0.13 | 3.02 | 19.63 ± 0.1 | 3.08 |
Ppia | Peptidylprolyl isomerase A | catalyse the cis–trans isomerisation of peptide bonds N-terminal to proline residues in polypeptide chains | 84% | 18.82 ± 0.12 | 3.33 | 16.37 ± 0.07 | 2.15 |
Rplp2 | Ribosomal protein lateral stalk subunit P2 | ribosomal phosphoprotein—component of the 60S subunit | 76% | 22.27 ± 0.14 | 3.91 | 21.71 ± 0.08 | 2.27 |
Rps18 | Ribosomal protein S18 | ribosomal subunit | 83% | 17.36 ± 0.07 | 1.77 | 19.22 ± 0.08 | 1.92 |
Tbp | TATA-box binding protein | transcription factor | 87% | 25.74 ± 0.15 | 2.78 | 24.08 ± 0.08 | 2.37 |
Ywhaz | Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta | involved in signal transduction pathways and role in tumor progression | 90% | 20.74 ± 0.11 | 2.56 | 20.41 ± 0.08 | 1.86 |
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Szczepkowska, A.; Harazin, A.; Barna, L.; Deli, M.A.; Skipor, J. Identification of Reference Genes for Circadian Studies on Brain Microvessels and Choroid Plexus Samples Isolated from Rats. Biomolecules 2021, 11, 1227. https://doi.org/10.3390/biom11081227
Szczepkowska A, Harazin A, Barna L, Deli MA, Skipor J. Identification of Reference Genes for Circadian Studies on Brain Microvessels and Choroid Plexus Samples Isolated from Rats. Biomolecules. 2021; 11(8):1227. https://doi.org/10.3390/biom11081227
Chicago/Turabian StyleSzczepkowska, Aleksandra, András Harazin, Lilla Barna, Mária A. Deli, and Janina Skipor. 2021. "Identification of Reference Genes for Circadian Studies on Brain Microvessels and Choroid Plexus Samples Isolated from Rats" Biomolecules 11, no. 8: 1227. https://doi.org/10.3390/biom11081227
APA StyleSzczepkowska, A., Harazin, A., Barna, L., Deli, M. A., & Skipor, J. (2021). Identification of Reference Genes for Circadian Studies on Brain Microvessels and Choroid Plexus Samples Isolated from Rats. Biomolecules, 11(8), 1227. https://doi.org/10.3390/biom11081227