Evaluation and Validation of Reference Genes for Gene Expression Analysis Using qRT-PCR in the Sugarcane Stem Borer Chilo sacchariphagus (Lepidoptera: Pyralidae)
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Insect Rearing and Experimental Conditions
2.2. Selection of Reference Genes and Primer Design
2.3. Total RNA Isolation and First Strand cDNA Synthesis
2.4. Quantitative Real-Time PCR (qRT-PCR)
2.5. Evaluation of Reference Gene Expression Stability
2.6. Validation of Recommended Reference Genes
3. Results
3.1. Specificity and Efficiency of Primers
3.2. Expression Levels of Candidate Reference Genes
3.3. Expression Stability of Seven Reference Genes Across Different Tissues
3.4. Expression Stability of Seven Reference Genes Across Different Temperature Treatments
3.5. Expression Stability of Seven Reference Genes Across Different Sexes
3.6. Validation of Recommended Reference Genes
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene Symbol | Gene Name | Primer Sequence (5′-3′) | Length (bp) | Efficiency (%) | R2 |
---|---|---|---|---|---|
β-ACT | Beta-Actin | F: CAATCCTAAAGCCAACAGA | 180 | 95.06 | 0.9948 |
R: GCGTAGCCCTCGTAGAT | |||||
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase | F: CATGCCACTACTGCTACCC | 131 | 101.47 | 0.9922 |
R: GGAATGACTTTGCCTACGG | |||||
BTF3 | Basic transcription factor 3 | F: AAGAAGGTTGTTCACGCTAC | 155 | 91.75 | 0.9994 |
R: GCTTGTGCTTTCGGATTA | |||||
28S | 28S ribosomal | F: TCGCAGAATGTAGCAGGTT | 129 | 90.02 | 0.9990 |
R: AGCATTGATTCGGGTCCTC | |||||
RPL7 | Ribosomal protein L7 | F: TTTTGTTATCCGTATTCGTG | 131 | 95.61 | 0.9989 |
R: ACAGTCGCCTTGTTGAGA | |||||
EF1α | Elongation factor 1 alpha | F: GCTCTGCTCGCTTTCACC | 90 | 107.87 | 0.9994 |
R: TCGGGATTCACTGTATGG | |||||
SDHA | Succinate dehydrogenase complex subunit A | F: AGAGGTGATAACGCACTACAA | 86 | 105.35 | 0.9993 |
R: CGTGAACAGAGGCACAAGA | |||||
PBP1 | Pheromone binding protein 1 | F: CGCTGATTCGGACAC | 158 | 99.59 | 0.9995 |
R: TCACCTCTACACTGGGAT |
Conditions | Reference Gens | geNorm | Normfinder | BestKeeper | ΔCT | ||||
---|---|---|---|---|---|---|---|---|---|
Stability | Rank | Stability | Rank | Stability | Rank | Stability | Rank | ||
Tissues | β-ACT | 0.363 | 5 | 0.198 | 1 | 0.82 | 3 | 0.41 | 1 |
GAPDH | 0.254 | 3 | 0.325 | 4 | 1.14 | 7 | 0.44 | 4 | |
BTF3 | 0.151 | 1 | 0.315 | 3 | 1.10 | 6 | 0.43 | 3 | |
28S | 0.318 | 4 | 0.335 | 5 | 1.06 | 5 | 0.46 | 5 | |
RPL7 | 0.151 | 1 | 0.286 | 2 | 1.03 | 4 | 0.42 | 2 | |
EF1α | 0.428 | 6 | 0.406 | 6 | 0.59 | 2 | 0.49 | 6 | |
SDHA | 0.456 | 7 | 0.460 | 7 | 0.54 | 1 | 0.53 | 7 | |
Temperature | β-ACT | 0.161 | 1 | 0.390 | 4 | 0.59 | 2 | 0.51 | 3 |
GAPDH | 0.380 | 5 | 0.069 | 1 | 1.04 | 5 | 0.45 | 1 | |
BTF3 | 0.477 | 6 | 0.513 | 6 | 1.36 | 6 | 0.62 | 6 | |
28S | 0.544 | 7 | 0.650 | 7 | 1.43 | 7 | 0.71 | 7 | |
RPL7 | 0.278 | 3 | 0.179 | 2 | 0.86 | 4 | 0.45 | 1 | |
EF1α | 0.161 | 1 | 0.437 | 5 | 0.57 | 1 | 0.54 | 4 | |
SDHA | 0.326 | 4 | 0.386 | 3 | 0.83 | 3 | 0.54 | 4 | |
Sex | β-ACT | 0.199 | 5 | 0.274 | 3 | 0.51 | 5 | 0.45 | 5 |
GAPDH | 0.129 | 4 | 0.326 | 5 | 0.37 | 1 | 0.42 | 4 | |
BTF3 | 0.352 | 6 | 0.489 | 6 | 1.01 | 6 | 0.62 | 6 | |
28S | 0.483 | 7 | 0.777 | 7 | 1.19 | 7 | 0.81 | 7 | |
RPL7 | 0.109 | 3 | 0.287 | 4 | 0.38 | 2 | 0.39 | 3 | |
EF1α | 0.063 | 1 | 0.100 | 1 | 0.47 | 4 | 0.34 | 1 | |
SDHA | 0.063 | 1 | 0.145 | 2 | 0.46 | 3 | 0.35 | 2 |
Conditions | Recommended Reference Genes | |
---|---|---|
Tissues | β-ACT | RPL7 |
Temperature | β-ACT | RPL7 |
Sex | EF1α | SDHA |
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Wang, Z.; Shang, X.; Wei, J.; Tian, X.; Liu, Y.; Zhang, G. Evaluation and Validation of Reference Genes for Gene Expression Analysis Using qRT-PCR in the Sugarcane Stem Borer Chilo sacchariphagus (Lepidoptera: Pyralidae). Insects 2024, 15, 594. https://doi.org/10.3390/insects15080594
Wang Z, Shang X, Wei J, Tian X, Liu Y, Zhang G. Evaluation and Validation of Reference Genes for Gene Expression Analysis Using qRT-PCR in the Sugarcane Stem Borer Chilo sacchariphagus (Lepidoptera: Pyralidae). Insects. 2024; 15(8):594. https://doi.org/10.3390/insects15080594
Chicago/Turabian StyleWang, Zhixiong, Xiankun Shang, Jili Wei, Xiaoli Tian, Yi Liu, and Guohui Zhang. 2024. "Evaluation and Validation of Reference Genes for Gene Expression Analysis Using qRT-PCR in the Sugarcane Stem Borer Chilo sacchariphagus (Lepidoptera: Pyralidae)" Insects 15, no. 8: 594. https://doi.org/10.3390/insects15080594