Comprehensive Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Tamarixia radiata, a Predominant Parasitoid of Diaphorina citri
Abstract
:1. Introduction
2. Materials and Methods
2.1. Insect Rearing
2.2. Sample Collection of T. radiata
2.3. Total RNA Extraction and cDNA Synthesis
2.4. Gene Cloning and Primer Design
2.5. RT-qPCR with SYBR Green
2.6. Determination of Reference Gene Expression Stability
2.7. Determination of Gene Expression Levels based on Different Reference Genes
3. Results
3.1. PCR Amplification and Performance of Candidate Reference Genes in T. radiata
3.2. Ct Values of Reference Genes
3.3. Stability of the Reference Genes under Specific Experimental Conditions
3.4. The Overall RefFinder Ranking for Reference Gene Expression Stability
3.5. Validation of the Selected Reference Genes
4. Discussion
5. Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Gene | Primer Sequences (5′–3′) | Length (bp) | Efficiency (%) | R2 | Linear Regression |
---|---|---|---|---|---|
18S-F | CTTTCAAATGTCTGCCTTAT | 134 | 104.26 | 0.9907 | y = −3.224x + 7.286 |
18S-R | GCTGCCTTCCTTGGATGTGGT | ||||
Actin-F | GTGTGGTGCCATATCTTCTCC | 137 | 107.28 | 0.9912 | y = −3.159x + 19.938 |
Actin-R | ATGGTGGGTATGGGACAGAA | ||||
ArgK-F | AGGTCTTGTCCTCGTTGTGG | 148 | 115.94 | 0.9940 | y = −2.991x + 18.848 |
ArgK-R | CCATTAACCGGAATGAGCAA | ||||
EF1A -F | TTCCTTCAAGTATGCCTGGG | 139 | 107.90 | 0.9898 | y = −3.146x + 17.486 |
EF1A -R | GAAATCTCTGTGTCCAGGGG | ||||
RPL13-F | TAGCCTTTTTGACCGGCAC | 90 | 91.82 | 0.9963 | y = −3.535x + 18.426 |
RPL13-R | GCACAAGGGTCTCAGAAAGG | ||||
RPS18-F | ACAAAAGGACATTGTTGACGG | 137 | 97.99 | 0.9863 | y = −3.371x + 17.824 |
RPS18-R | GAACACGGAGACCCCAGTAG | ||||
β-tubulin-F | GAGCAGATGCTCAACATCCA | 134 | 83.68 | 0.9922 | y = −3.787x + 19.628 |
β-tubulin-R | GTGCTGTTTCCGATGAAGGT |
Conditions | Rank | geNorm | NormFinder | BestKeeper | ΔCt | ||||
---|---|---|---|---|---|---|---|---|---|
Gene | Stability | Gene | Stability | Gene | Stability | Gene | Stability | ||
Developmental stage | 1 | EF1A | 0.306 | EF1A | 0.054 | 18S | 0.12 | 18S | 0.66 |
2 | RPS18 | 0.306 | 18S | 0.182 | RPL13 | 0.24 | EF1A | 0.68 | |
3 | 18S | 0.35 | RPL13 | 0.268 | EF1A | 0.26 | RPS18 | 0.73 | |
4 | RPL13 | 0.374 | RPS18 | 0.337 | RPS18 | 0.27 | RPL13 | 0.73 | |
5 | Actin | 0.492 | Actin | 0.699 | Actin | 0.39 | Actin | 0.91 | |
6 | β-tubulin | 0.696 | β-tubulin | 1.066 | β-tubulin | 0.88 | β-tubulin | 1.21 | |
7 | ArgK | 0.907 | ArgK | 1.358 | ArgK | 1.23 | ArgK | 1.44 | |
Sex | 1 | EF1A | 0.07 | RPS18 | 0.311 | 18S | 0.06 | RPS18 | 0.69 |
2 | β-tubuulin | 0.07 | EF1A | 0.607 | Actin | 0.09 | EF1A | 0.73 | |
3 | RPL13 | 0.098 | 18S | 0.626 | ArgK | 0.41 | β-tubulin | 0.74 | |
4 | RPS18 | 0.182 | β-tubulin | 0.629 | RPS18 | 0.83 | RPL13 | 0.8 | |
5 | 18S | 0.57 | Actin | 0.692 | EF1A | 1.02 | 18S | 0.85 | |
6 | Actin | 0.707 | RPL13 | 0.745 | β-tubulin | 1.04 | Actin | 0.88 | |
7 | ArgK | 0.837 | ArgK | 1.132 | RPL13 | 1.12 | ArgK | 1.16 | |
Tissue | 1 | RPL13 | 0.179 | Actin | 0.218 | 18S | 0.14 | RPS18 | 0.93 |
2 | RPS18 | 0.179 | RPS18 | 0.514 | Actin | 0.49 | RPL13 | 0.99 | |
3 | EF1A | 0.207 | RPL13 | 0.753 | ArgK | 1.08 | EF1A | 1.04 | |
4 | β-tubulin | 0.308 | 18S | 0.779 | RPS18 | 1.12 | Actin | 1.1 | |
5 | Actin | 0.601 | EF1A | 0.821 | β-tubulin | 1.24 | β-tubulin | 1.11 | |
6 | 18S | 0.846 | β-tubulin | 0.863 | RPL13 | 1.25 | 18S | 1.3 | |
7 | ArgK | 1.244 | ArgK | 2.205 | EF1A | 1.32 | ArgK | 2.24 | |
Population | 1 | EF1A | 0.06 | ArgK | 0.057 | EF1A | 0.01 | EF1A | 0.33 |
2 | RPS18 | 0.06 | 18S | 0.059 | RPS18 | 0.06 | ArgK | 0.34 | |
3 | 18S | 0.108 | EF1A | 0.09 | ArgK | 0.08 | 18S | 0.34 | |
4 | ArgK | 0.124 | RPS18 | 0.191 | 18S | 0.1 | RPS18 | 0.35 | |
5 | Actin | 0.199 | β-tubulin | 0.231 | Actin | 0.27 | β-tubulin | 0.48 | |
6 | β-tubulin | 0.286 | Actin | 0.502 | β-tubulin | 0.37 | Actin | 0.53 | |
7 | RPL13 | 0.471 | RPL13 | 0.926 | RPL13 | 0.87 | RPL13 | 0.93 | |
Temperature | 1 | EF1A | 0.138 | β-tubulin | 0.12 | 18S | 0.14 | β-tubulin | 0.26 |
2 | RPS18 | 0.138 | Actin | 0.123 | β-tubulin | 0.18 | EF1A | 0.28 | |
3 | β-tubulin | 0.187 | 18S | 0.133 | EF1A | 0.22 | 18S | 0.28 | |
4 | RPL13 | 0.193 | EF1A | 0.163 | Actin | 0.24 | RPL13 | 0.28 | |
5 | 18S | 0.212 | RPL13 | 0.188 | RPL13 | 0.24 | Actin | 0.29 | |
6 | Actin | 0.233 | RPS18 | 0.254 | RPS18 | 0.29 | RPS18 | 0.31 | |
7 | ArgK | 0.322 | ArgK | 0.526 | ArgK | 0.42 | ArgK | 0.54 | |
Diet | 1 | ArgK | 0.063 | ArgK | 0.032 | ArgK | 0.08 | RPS18 | 0.28 |
2 | RPS18 | 0.063 | RPS18 | 0.032 | RPS18 | 0.1 | EF1A | 0.3 | |
3 | EF1A | 0.1 | EF1A | 0.052 | EF1A | 0.12 | ArgK | 0.3 | |
4 | Actin | 0.197 | Actin | 0.328 | Actin | 0.16 | Actin | 0.42 | |
5 | β-tubulin | 0.247 | RPL13 | 0.381 | β-tubulin | 0.19 | RPL13 | 0.45 | |
6 | RPL13 | 0.335 | β-tubulin | 0.437 | RPL13 | 0.46 | β-tubulin | 0.48 | |
7 | 18S | 0.396 | 18S | 0.524 | 18S | 0.56 | 18S | 0.55 |
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Guo, C.-F.; Pan, H.-P.; Zhang, L.-H.; Ou, D.; Lu, Z.-T.; Khan, M.M.; Qiu, B.-L. Comprehensive Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Tamarixia radiata, a Predominant Parasitoid of Diaphorina citri. Genes 2020, 11, 1178. https://doi.org/10.3390/genes11101178
Guo C-F, Pan H-P, Zhang L-H, Ou D, Lu Z-T, Khan MM, Qiu B-L. Comprehensive Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Tamarixia radiata, a Predominant Parasitoid of Diaphorina citri. Genes. 2020; 11(10):1178. https://doi.org/10.3390/genes11101178
Chicago/Turabian StyleGuo, Chang-Fei, Hui-Peng Pan, Li-He Zhang, Da Ou, Zi-Tong Lu, Muhammad Musa Khan, and Bao-Li Qiu. 2020. "Comprehensive Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Tamarixia radiata, a Predominant Parasitoid of Diaphorina citri" Genes 11, no. 10: 1178. https://doi.org/10.3390/genes11101178
APA StyleGuo, C.-F., Pan, H.-P., Zhang, L.-H., Ou, D., Lu, Z.-T., Khan, M. M., & Qiu, B.-L. (2020). Comprehensive Assessment of Candidate Reference Genes for Gene Expression Studies Using RT-qPCR in Tamarixia radiata, a Predominant Parasitoid of Diaphorina citri. Genes, 11(10), 1178. https://doi.org/10.3390/genes11101178