Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues
Abstract
:1. Introduction
2. Results and Discussion
2.1. Stability of Candidate Reference Genes in C. sativa Stem Tissues
2.2. Optimal Number of Reference Genes in C. sativa Stem Tissues
2.3. Validation of the Reference Genes in Hemp Stem Tissues
3. Materials and Methods
3.1. Plant Material and Growth Conditions
3.2. Gene Identification and Primer Design
3.3. RNA Extraction, cDNA Synthesis and RT-qPCR
4. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
RT-qPCR | Real-Time PCR |
G-layer | Gelatinous layer |
References
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Name | Sequence (5′→3′) | Amplicon Length (bp) | Amplicon Tm (°C) | PCR Efficiency (%) | Regression Coefficient (R2) |
---|---|---|---|---|---|
ActinFwd | TTGCTGGTCGTGATCTTACTG | 148 | 83 | 90.8 | 0.993 |
ActinRev | GTCTCCATCTCCTGCTCAAAG | ||||
eTIF4EFwd | AGTGGGAAGATCCTGAATGTGC | 150 | 81 | 95.1 | 0.997 |
eTIF4ERev | TTGCGACCACACCACAAATC | ||||
EF2Fwd | ACGCAACAGCTATCAGGAAC | 113 | 80.3 | 92.3 | 0.998 |
EF2Rev | TGCAAAGACACGACCAAAGG | ||||
GAPDHFwd | AATCGCAACCCAAACTCTGC | 123 | 81.1 | 99.4 | 0.995 |
GAPDHRev | AGTGGCCGTTGCTTTAATGG | ||||
CycloFwd | ACAACATGTCGAACCCCAAG | 106 | 81.4 | 93.8 | 0.998 |
CycloRev | TCAGCGGTTTTTGGCGTAAC | ||||
RANFwd | TTTGGAGACTTCAGCACTGG | 129 | 81.8 | 97.8 | 0.998 |
RANRev | GCAGGGTTACCATTTCCTTG | ||||
F-boxFwd | TATCGGCGGAGAGATTTGAG | 77 | 78.4 | 99.5 | 0.975 |
F-boxRev | TAAGCCCTTCCCTTGATTCC | ||||
ClathrinFwd | TGTCAGTTTTGTGCCACCAG | 139 | 80.3 | 98.3 | 0.998 |
ClathrinRev | TCCATGCGTGTTCTACCAAG | ||||
Histone3Fwd | TGAAGAAGCCTCATCGGTTC | 127 | 82.9 | 96.1 | 0.998 |
Histone3Rev | TCTTGAGCGATTTCCCTGAC | ||||
TIP41Fwd | TGAACAGTGGGGAGAAAAGC | 144 | 80.3 | 100.2 | 0.989 |
TIP41Rev | GCTTCCTGTTTCCATCCAAG | ||||
TubulinFwd | ATAACTGTACTGGGCTTCAAGG | 110 | 84 | 97.5 | 0.999 |
TubulinRev | CCTGTGGAGATGGGTAAACTG | ||||
CDPKFwd | GGTGGCTTTGCTTCTCTTTG | 86 | 78.7 | 97 | 0.986 |
CDPKRev | GTCAAACCCCTTTTCACACC | ||||
TA1Fwd | TTCGAGAAGTTCCCTCCAAC | 118 | 81.5 | 92.8 | 0.999 |
TA1Rev | AGCCATATCCACAGCATTCC | ||||
TA2Fwd | CTAGCAACCCAGCGATTTTC | 126 | 81.3 | 97 | 0.998 |
TA2Rev | ACCACAAGCTCCCAATATGC | ||||
DHS1Fwd | TGAGACTTTCCCTCCGATTG | 144 | 84.6 | 96.8 | 0.998 |
DHS1Rev | TCAGCACAATCTCCACCTTG | ||||
DHS2Fwd | TATCAAGGCTGTTCGTGGAG | 129 | 81.8 | 103 | 0.997 |
DHS2Rev | AGGTGCTTTGATGGTGTTCC |
GeNormPLUS | NormFinder | BestKeeper | Comparative delta-Ct | RefFinder | |||||
---|---|---|---|---|---|---|---|---|---|
Gene | Stability Coeff. | Gene | Stability Coeff. | Gene | Stability Coeff. | Gene | Stability Coeff. | Gene | Stability Coeff. |
Histone3 | 1.22 | Histone3 | 0.812 | Histone3 | 1.58 | Histone3 | 1.678 | Histone3 | 12 |
EF2 | 1.135 | EF2 | 0.743 | RAN | 1.085 | EF2 | 1.549 | EF2 | 10.462 |
Actin | 1.061 | Tubulin | 0.579 | Tubulin | 1.059 | Actin | 1.343 | Actin | 8.409 |
Cyclo | 1.028 | Cyclo | 0.523 | EF2 | 0.926 | GAPDH | 1.335 | Cyclo | 6.701 |
GAPDH | 0.959 | Actin | 0.518 | Clathrin | 0.926 | Clathrin | 1.33 | Clathrin | 6.447 |
eTIF4E | 0.857 | Clathrin | 0.504 | eTIF4E | 0.888 | Cyclo | 1.319 | Tubulin | 5.948 |
TIP41 | 0.798 | TIP41 | 0.502 | F-box | 0.885 | RAN | 1.302 | GAPDH | 5.635 |
Tubulin | 0.703 | GAPDH | 0.462 | Actin | 0.87 | Tubulin | 1.299 | RAN | 4.461 |
CDPK | 0.599 | RAN | 0.453 | Cyclo | 0.718 | TIP41 | 1.241 | eTIF4E | 3.742 |
RAN | 0.503 | Fbox | 0.452 | CDPK | 0.699 | F-box | 1.234 | TIP41 | 2.913 |
Clathrin | 0.47 | CDPK | 0.272 | GAPDH | 0.681 | eTIF4E | 1.155 | F-box | 2.913 |
F-box | 0.45 | eTIF4E | 0.257 | TIP41 | 0.601 | CDPK | 1.091 | CDPK | 1.861 |
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Mangeot-Peter, L.; Legay, S.; Hausman, J.-F.; Esposito, S.; Guerriero, G. Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues. Int. J. Mol. Sci. 2016, 17, 1556. https://doi.org/10.3390/ijms17091556
Mangeot-Peter L, Legay S, Hausman J-F, Esposito S, Guerriero G. Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues. International Journal of Molecular Sciences. 2016; 17(9):1556. https://doi.org/10.3390/ijms17091556
Chicago/Turabian StyleMangeot-Peter, Lauralie, Sylvain Legay, Jean-Francois Hausman, Sergio Esposito, and Gea Guerriero. 2016. "Identification of Reference Genes for RT-qPCR Data Normalization in Cannabis sativa Stem Tissues" International Journal of Molecular Sciences 17, no. 9: 1556. https://doi.org/10.3390/ijms17091556