Identification of Reference Genes for Expression Studies in the Whole-Blood from Three Cattle Breeds under Two States of Livestock Weather Safety
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Study Population
2.3. Weather Data
2.4. Samples, RNA Extraction, and cDNA Synthesis
2.5. Gene Selection and Primer Design
2.6. End-Point PCR and Quantitative Polymerase Chain Reaction (qPCR)
2.7. Analysis of Reference Gene Expression Stability
3. Results
3.1. Primer Specificity
3.2. Expression Profiles of Reference Genes
3.3. Reference Gene Stability: geNorm
3.4. Reference Gene Stability: NormFinder
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Primer Sequence (5′–3′) | Primer Length (nt) | Tm (°C) | GC% | Annealing Temperature (°C) | Amplicon Size (bp) | Reference | |
---|---|---|---|---|---|---|---|---|
b2m | F | CTGCTATGTGTATGGGTTCC | 20 | 55.6 | 50 | 54 | 141 | [22] |
R | GGAGTGAACTCAGCGTG | 17 | 54.8 | 58.8 | ||||
sdha | F | TGCAGACCATCTACGGAGCGGA | 22 | 65.44 | 59.09 | 55 | 163 | This study |
R | ACGTAGGAGAGCGTGTGCTTCCTCC | 25 | 67.96 | 60.00 | ||||
ywhaz | F | AGCAGGCTGAGCGATATGAT | 20 | 59.02 | 50.00 | 55 | 180 | This study |
R | TCTCAGCACCTTCCGTCTTT | 20 | 58.95 | 50.00 | ||||
actb | F | GGGATGAGGCTCAGAGCAAGAGA | 23 | 63.65 | 56.52 | 60 | 118 | This study |
R | AGCTCGTTGTAGAAGGTGTGGTGCC | 25 | 66.91 | 56.00 | ||||
18S rRNA | F | TAGAGGGACAAGTGGCGTTC | 20 | 59.39 | 55.00 | 55 | 104 | This study |
R | CGCTGAGCCAGTCAGTGTAG | 20 | 60.46 | 60.00 |
Ranking | Brahman | Gyr | Romosinuano | |||
---|---|---|---|---|---|---|
Gene 1 | Stability Value | Gene 1 | Stability Value | Gene 1 | Stability Value | |
1 | actb | 0.009 | 18SrRNA | 0.009 | actb | 0.017 |
2 | 18SrRNA | 0.018 | b2m | 0.016 | b2m | 0.017 |
3 | b2m | 0.021 | ywhaz | 0.018 | ywhaz | 0.020 |
4 | ywhaz | 0.025 | actb | 0.021 | 18SrRNA | 0.022 |
5 | sdha | 0.043 | sdha | 0.032 | sdha | 0.029 |
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Lozano-Villegas, K.J.; Rodríguez-Hernández, R.; Herrera-Sánchez, M.P.; Uribe-García, H.F.; Naranjo-Gómez, J.S.; Otero-Arroyo, R.J.; Rondón-Barragán, I.S. Identification of Reference Genes for Expression Studies in the Whole-Blood from Three Cattle Breeds under Two States of Livestock Weather Safety. Animals 2021, 11, 3073. https://doi.org/10.3390/ani11113073
Lozano-Villegas KJ, Rodríguez-Hernández R, Herrera-Sánchez MP, Uribe-García HF, Naranjo-Gómez JS, Otero-Arroyo RJ, Rondón-Barragán IS. Identification of Reference Genes for Expression Studies in the Whole-Blood from Three Cattle Breeds under Two States of Livestock Weather Safety. Animals. 2021; 11(11):3073. https://doi.org/10.3390/ani11113073
Chicago/Turabian StyleLozano-Villegas, Kelly J., Roy Rodríguez-Hernández, María P. Herrera-Sánchez, Heinner F. Uribe-García, Juan S. Naranjo-Gómez, Rafael J. Otero-Arroyo, and Iang S. Rondón-Barragán. 2021. "Identification of Reference Genes for Expression Studies in the Whole-Blood from Three Cattle Breeds under Two States of Livestock Weather Safety" Animals 11, no. 11: 3073. https://doi.org/10.3390/ani11113073