Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood
Abstract
:1. Introduction
2. Results and Discussion
2.1. Expression Level of Candidate Reference Genes
2.2. GeNorm Analysis
2.3. NormFinder Analysis
2.4. Evaluation of the Analysed Reference Genes
3. Experimental Section
3.1. Sample Collection, Nucleic Acid Extraction and cDNA Synthesis
3.2. Selection of Genes and qRT-PCR Primer Design
3.3. Quantitative RT-PCR
3.4. Data Analysis
4. Conclusions
Acknowledgments
References
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Control sheep (n = 18) | Disease-stressed sheep (n = 10) | Combined groups (n = 10 + 10) | |||
---|---|---|---|---|---|
Gene symbol | Stability value (M) | Gene symbol | Stability value (M) | Gene symbol | Stability value (M) |
SDHA | 0.316 | YWHAZ | 0.624 | SDHA | 0.591 |
YWHAZ | 0.342 | GAPDH | 0.634 | YWHAZ | 0.593 |
HPRT | 0.344 | SDHA | 0.667 | ACTB | 0.596 |
RPL19 | 0.361 | ACTB | 0.701 | G6PD | 0.666 |
B2M | 0.362 | B2M | 0.720 | GAPDH | 0.681 |
ACTB | 0.377 | G6PD | 0.740 | GYPC | 0.698 |
GYPC | 0.412 | GYPC | 0.848 | RPL19 | 0.746 |
TFRC | 0.455 | RPL19 | 1.000 | B2M | 0.760 |
G6PD | 0.486 | HPRT | 1.188 | HPRT | 0.882 |
PGK1 | 0.498 | PGK1 | 1.466 | TFRC | 1.158 |
GAPDH | 0.579 | TFRC | 1.485 | PGK1 | 1.295 |
Control sheep (n = 18) | Disease-stressed sheep (n = 10) | Combined groups (n = 10 + 10) | |||
---|---|---|---|---|---|
Gene symbol | Stability value (ρ) | Gene symbol | Stability value (ρ) | Gene symbol | Stability value (ρ) |
SDHA | 0.068 | YWHAZ | 0.046 | SDHA | 0.093 |
YWHAZ | 0.125 | GAPDH | 0.064 | YWHAZ | 0.096 |
HPRT | 0.132 | SDHA | 0.188 | ACTB | 0.099 |
B2M | 0.146 | ACTB | 0.220 | GAPDH | 0.167 |
RPL19 | 0.155 | B2M | 0.229 | RPL19 | 0.175 |
ACTB | 0.174 | G6PD | 0.248 | G6PD | 0.184 |
GYPC | 0.201 | GYPC | 0.397 | HPRT | 0.188 |
TFRC | 0.245 | RPL19 | 0.540 | GYPC | 0.190 |
G6PD | 0.271 | HPRT | 0.702 | B2M | 0.212 |
PGK1 | 0.282 | PGK1 | 0.915 | TFRC | 0.274 |
GAPDH | 0.354 | TFRC | 0.941 | PGK1 | 0.280 |
Gene name | Primers sequences (forward/reverse) | Spanned exons | Amplicon size (bp) | PCR efficiency (%) | R2 |
---|---|---|---|---|---|
ACTB | CCAACCGTGAGAAGATGACC | 2nd | 97 | 102.1 | 0.999 |
CCAGAGGCGTACAGGGACAG | 3th | ||||
GYPC | ATCAACATCGCTGTCATTGC | 3th | 117 | 106.7 | 0.994 |
CTCGTTGGTGTGGTATGTGC | 4th | ||||
RPL19 | AGCCTGTGACTGTCCATTCC | 2nd | 126 | 102.0 | 0.998 |
ACGTTACCTTCTCGGGCATT | 3th | ||||
GAPDH | CTGGCCAAGGTCATCCAT | 7th | 86 | 104.1 | 0.997 |
ACAGTCTTCTGGGTGGCAGT | 8th | ||||
YWHAZ | AGACGGAAGGTGCTGAGAAA | 2nd | 123 | 100.0 | 0.998 |
CGTTGGGGATCAAGAACTTT | 3th | ||||
PGK1 | ACTCCTTGCAGCCAGTTGCT | 3th | 101 | 109.9 | 0.991 |
AGCACAAGCCTTCTCCACTTCT | 4th | ||||
HPRT | TTTATTCCTCATGGACTAATTATGGA | 2nd | 71 | 99.8 | 0.987 |
CCACCCATCTCCTTCATCAC | 3th | ||||
TFRC | TTCTGGGCAGACCTCAAATC | 4th | 106 | 100.9 | 0.991 |
CAGCTTCACGTGGGACATAA | 5th | ||||
SDHA | CATCCACTACATGACGGAGCA | 4th | 90 | 99.8 | 0.996 |
ATCTTGCCATCTTCAGTTCTGCTA | 5th | ||||
G6PD | TGACCTATGGCAACCGATACAA | 10th | 76 | 103.0 | 0.990 |
CCGCAAAAGACATCCAGGAT | 11th | ||||
B2M | CTGTCGCTGTCTGGACTGG | 1st | 86 | 98.9 | 0.997 |
TTTGGCTTTCCATCTTCTGG | 2nd |
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Peletto, S.; Bertuzzi, S.; Campanella, C.; Modesto, P.; Maniaci, M.G.; Bellino, C.; Ariello, D.; Quasso, A.; Caramelli, M.; Acutis, P.L. Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood. Int. J. Mol. Sci. 2011, 12, 7732-7747. https://doi.org/10.3390/ijms12117732
Peletto S, Bertuzzi S, Campanella C, Modesto P, Maniaci MG, Bellino C, Ariello D, Quasso A, Caramelli M, Acutis PL. Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood. International Journal of Molecular Sciences. 2011; 12(11):7732-7747. https://doi.org/10.3390/ijms12117732
Chicago/Turabian StylePeletto, Simone, Simone Bertuzzi, Chiara Campanella, Paola Modesto, Maria Grazia Maniaci, Claudio Bellino, Dario Ariello, Antonio Quasso, Maria Caramelli, and Pier Luigi Acutis. 2011. "Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood" International Journal of Molecular Sciences 12, no. 11: 7732-7747. https://doi.org/10.3390/ijms12117732