Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera
Abstract
:1. Introduction
2. Results
2.1. Determination of Primer Specificity and Amplification Efficiency
2.2. Ct Values of the Internal Reference Genes
2.3. geNorm Analysis
2.4. NormFinder Analysis
2.5. BestKeeper Analysis
2.6. RefFinder Analysis
2.7. Verification of the Expression Stability of the Internal Reference Genes
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Extraction and Detection of RNA
4.3. Synthesis of cDNA
4.4. Screening of Candidate Internal Reference Genes and Designing of Primers
4.5. RT-qPCR Reaction Conditions
4.6. Detection of Primer Specificity and Amplification Efficiency
4.7. The Stability of Candidate Internal Reference Genes
4.8. Verification of the Expression Stability of the Internal Reference Genes
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 ID | Primer sequence (5′-3′) | Amplicon Size (Bp) | Efficiency (%) | R2 |
---|---|---|---|---|---|
NADH | NADH-F | GGACAGGTGGAAGATCGTCTG | 111 | 97.18 | 0.987 |
NADH-R | GGAATCTTCAGAACCCCGGAA | ||||
L13 | L13-F | TGCCAGCCCTAACTTTCATGT | 126 | 92.17 | 0.999 |
L13-R | AGACCCGGAGAAGAATTGCTC | ||||
EIF3 | EIF3-F | GTCCACATCATTCGAAGCAGC | 130 | 106.31 | 0.997 |
EIF3-R | GATCTATGAAGTGCCTGCGGA | ||||
HIS | HIS-F | TGGCCTTGCATTCTCCAGTAG | 118 | 98.87 | 0.996 |
HIS-R | GACAAGCTGCGAGAGTGGTAT | ||||
Actin | Actin-F | TACGCATTGAAGACCCTCCAC | 148 | 90.26 | 0.998 |
Actin-R | TGGCCACACTTGCTTAGACAA | ||||
PP2A | PP2A-F | TCCTTTTGCGAGTCGATGGAA | 119 | 117.99 | 0.988 |
PP2A-R | CTTTGACGTTTGAAGCGAGCA | ||||
DOUB | DOUB-F | CCTGATCTTCGCCGGAAAACA | 194 | 97.48 | 0.999 |
DOUB-R | TGGAGAGGGTTGAAGAGAGCT | ||||
UBE2 | UBE2-F | TCTCTGCTTACGGACCCAAAC | 144 | 92.29 | 0.998 |
UBE2-R | GAGGAGGAGCTATTGGGCCTA | ||||
UBC | UBC-F | AGCATTACTTTCCGCTCCACA | 119 | 91.44 | 0.995 |
UBC-R | TGGCGAAAGTTTCTGTCCAGT | ||||
PTB | PTB-F | CTGGAAACCTGCTGCCTTTTC | 151 | 96.29 | 0.999 |
PTB-R | ATTGAGGGTGTAGAAGCTGGC | ||||
rRNA | rRNA-F | CAGGTTTCGATGTTGGGGAGA | 196 | 95.56 | 0.999 |
rRNA-R | CCAGCTTCCGAGAACATTCCT | ||||
GAPDH | GAPDH-F | CCATGGAAGGACTTGGGGATC | 156 | 90.43 | 0.995 |
GAPDH-R | GTTCACTCCCACCACGTATGT | ||||
HSP | HSP-F | CCAGCGCTGATGTTAGATTGC | 174 | 92.66 | 0.993 |
HSP-R | TTGCCATCAGAGCCTTTTCCT | ||||
RPL8 | RPL8-F | TGATCACCGACATCATCCACG | 185 | 90.55 | 0.992 |
RPL8-R | TCTGATCGGAAGGACATTGCC | ||||
TUA | TUA-F | TCGAAAGGCCAACATACACCA | 175 | 96.59 | 0.997 |
TUA-R | GAGATGACAGGGGCATACGAG | ||||
POD | POD-F | CTCCTGTGACCTCAACTGCAA | 136 | 91.71 | 0.987 |
POD-R | GAGTTGAACCATGGCGCAAAT | ||||
DREB | DREB-F | TAAACCAGCTCACCCAATCCC | 274 | 90.99 | 0.989 |
DREB-R | CGGTTCTTGGGGAGTCTGATC |
Rank | Drought Stress | Salt Stress | Heavy Metal Stress | Different Tissues | All Samples | |||||
---|---|---|---|---|---|---|---|---|---|---|
Gene | Stability | Gene | Stability | Gene | Stability | Gene | Stability | Gene | Stability | |
1 | DOUB | 0.152 | DOUB | 0.172 | HSP | 0.359 | rRNA | 0.051 | rRNA | 0.338 |
2 | rRNA | 0.162 | HSP | 0.396 | rRNA | 0.362 | Actin | 0.222 | HSP | 0.383 |
3 | Actin | 0.394 | NADH | 0.540 | NADH | 0.401 | EIF3 | 0.229 | NADH | 0.495 |
4 | HSP | 0.429 | rRNA | 0.569 | UBC | 0.513 | TUA | 0.343 | PP2A | 0.691 |
5 | UBC | 0.436 | PP2A | 0.630 | DOUB | 0.707 | DOUB | 0.346 | UBC | 0.738 |
6 | PTB | 0.520 | HIS | 0.635 | HIS | 0.745 | PP2A | 0.383 | Actin | 0.751 |
7 | EIF3 | 0.547 | UBC | 0.654 | Actin | 0.805 | PTB | 0.383 | DOUB | 0.753 |
8 | TUA | 0.586 | L13 | 0.720 | PP2A | 0.907 | HIS | 0.456 | PTB | 0.792 |
9 | PP2A | 0.596 | Actin | 0.742 | PTB | 0.969 | NADH | 0.484 | HIS | 0.966 |
10 | NADH | 0.616 | PTB | 0.817 | L13 | 0.992 | HSP | 0.493 | L13 | 1.028 |
11 | RPL8 | 0.729 | TUA | 1.133 | RPL8 | 1.265 | UBC | 0.503 | TUA | 1.090 |
12 | HIS | 0.917 | EIF3 | 1.461 | TUA | 1.400 | L13 | 0.557 | EIF3 | 1.277 |
13 | L13 | 0.998 | UBE2 | 1.791 | GAPDH | 1.511 | RPL8 | 0.836 | RPL8 | 1.598 |
14 | UBE2 | 1.090 | RPL8 | 1.924 | EIF3 | 1.672 | GAPDH | 1.178 | UBE2 | 1.652 |
15 | GAPDH | 4.183 | GAPDH | 2.216 | UBE2 | 1.921 | UBE2 | 1.727 | GAPDH | 2.786 |
Rank | Drought Stress | Salt Stress | Heavy Metal Stress | Different Tissues | All Samples | |||||
---|---|---|---|---|---|---|---|---|---|---|
Gene | Stability | Gene | Stability | Gene | Stability | Gene | Stability | Gene | Stability | |
1 | UBC | 0.398 | HSP | 0.453 | UBC | 0.382 | HSP | 0.215 | HSP | 0.518 |
2 | rRNA | 0.465 | NADH | 0.49 | NADH | 0.416 | RPL8 | 0.216 | UBC | 0.535 |
3 | HSP | 0.481 | DOUB | 0.521 | HSP | 0.439 | DOUB | 0.256 | NADH | 0.607 |
4 | DOUB | 0.513 | UBC | 0.561 | rRNA | 0.493 | UBC | 0.475 | DOUB | 0.615 |
5 | PP2A | 0.515 | HIS | 0.596 | DOUB | 0.524 | NADH | 0.489 | Actin | 0.643 |
6 | Actin | 0.530 | PP2A | 0.609 | Actin | 0.607 | Actin | 0.514 | PP2A | 0.647 |
7 | EIF3 | 0.557 | rRNA | 0.723 | PP2A | 0.645 | PP2A | 0.566 | rRNA | 0.672 |
8 | PTB | 0.634 | L13 | 0.729 | HIS | 0.741 | L13 | 0.588 | TUA | 0.886 |
9 | RPL8 | 0.639 | Actin | 0.772 | L13 | 0.768 | EIF3 | 0.622 | L13 | 0.913 |
10 | TUA | 0.671 | TUA | 0.784 | PTB | 0.866 | TUA | 0.641 | PTB | 0.917 |
11 | NADH | 0.727 | PTB | 0.871 | RPL8 | 0.972 | rRNA | 0.650 | HIS | 1.018 |
12 | UBE2 | 0.844 | EIF3 | 1.336 | TUA | 1.088 | HIS | 0.651 | EIF3 | 1.023 |
13 | L13 | 0.928 | UBE2 | 1.356 | GAPDH | 1.137 | PTB | 0.819 | RPL8 | 1.353 |
14 | HIS | 0.982 | RPL8 | 1.551 | EIF3 | 1.303 | GAPDH | 1.213 | UBE2 | 1.477 |
15 | GAPDH | 3.496 | GAPDH | 1.554 | UBE2 | 1.593 | UBE2 | 1.619 | GAPDH | 2.123 |
Rank | Drought Stress | Salt Stress | Heavy Metal Stress | Different Tissues | All Samples | |||||
---|---|---|---|---|---|---|---|---|---|---|
Gene | Stability | Gene | Stability | Gene | Stability | Gene | Stability | Gene | Stability | |
1 | rRNA | 2.115 | DOUB | 1.316 | HSP | 1.316 | EIF3 | 2.28 | HSP | 1.414 |
2 | Actin | 2.449 | HSP | 1.414 | NADH | 2.06 | Actin | 3.13 | rRNA | 1.627 |
3 | UBC | 2.590 | NADH | 3.31 | rRNA | 2.632 | PP2A | 3.742 | NADH | 3 |
4 | DOUB | 3.130 | rRNA | 3.984 | UBC | 2.828 | rRNA | 4.031 | UBC | 3.761 |
5 | EIF3 | 3.956 | HIS | 4.949 | DOUB | 5.477 | DOUB | 4.787 | PP2A | 5.091 |
6 | HSP | 4.899 | PP2A | 5.733 | HIS | 5.886 | HSP | 5.477 | Actin | 5.477 |
7 | PP2A | 6.160 | UBC | 6.293 | Actin | 6.735 | NADH | 6.344 | DOUB | 5.856 |
8 | PTB | 7.416 | L13 | 7.737 | PP2A | 7.737 | UBC | 6.477 | PTB | 8.459 |
9 | NADH | 9.124 | Actin | 9 | PTB | 9.487 | L13 | 7.502 | L13 | 9.24 |
10 | TUA | 9.685 | PTB | 10.241 | L13 | 9.487 | TUA | 7.933 | HIS | 9.975 |
11 | RPL8 | 10.215 | TUA | 10.741 | RPL8 | 11 | RPL8 | 8.142 | TUA | 10.158 |
12 | HIS | 12.471 | EIF3 | 12 | TUA | 12.243 | PTB | 8.596 | EIF3 | 12 |
13 | L13 | 13 | UBE2 | 13 | GAPDH | 13.243 | HIS | 10.843 | RPL8 | 13 |
14 | UBE2 | 13.471 | RPL8 | 14 | EIF3 | 13.741 | GAPDH | 14 | UBE2 | 14 |
15 | GAPDH | 15 | GAPDH | 15 | UBE2 | 15 | UBE2 | 15 | GAPDH | 15 |
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Chen, M.; Wang, Z.; Hao, Z.; Li, H.; Feng, Q.; Yang, X.; Han, X.; Zhao, X. Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera. Int. J. Mol. Sci. 2023, 24, 15087. https://doi.org/10.3390/ijms242015087
Chen M, Wang Z, Hao Z, Li H, Feng Q, Yang X, Han X, Zhao X. Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera. International Journal of Molecular Sciences. 2023; 24(20):15087. https://doi.org/10.3390/ijms242015087
Chicago/Turabian StyleChen, Mengdi, Zhengbo Wang, Ziyuan Hao, Hongying Li, Qi Feng, Xue Yang, Xiaojiao Han, and Xiping Zhao. 2023. "Screening and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR under PEG, NaCl and ZnSO4 Treatments in Broussonetia papyrifera" International Journal of Molecular Sciences 24, no. 20: 15087. https://doi.org/10.3390/ijms242015087