Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Experimental Materials and Treatment Methods
2.2. RNA Isolation and Reverse Transcription
2.3. Selection of Reference Genes and Primer Formulation
2.4. Fluorescent Quantitative PCR Reaction Conditions
2.5. Assessment of Reference Gene Stability
2.6. Validation of Selected Reference Genes
3. Results
3.1. Assessment of Primer Efficiency and Specificity in Amplification
3.2. Expression Profile of Reference Genes
3.3. Identifying Reference Genes with Consistent Expression Stability
3.4. Optimal Number of Reference Genes
3.5. The Influence of Housekeeping Genes on Quantitative PCR Data Interpretation
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene | Primer Sequences (5′-3′, F/R) | Efficieny (E) (%) | Regression Coefficient (R2) |
---|---|---|---|
GAPDH | AGTCGCCGTAAATGATCCCT | 110.68 | 0.9971 |
CGTCGACTGTGCCATTGAAT | |||
RPL32 | GCAAAAACCCGTCATATGCT | 110.17 | 0.998 |
TGATACGCCATGTGCAATTT | |||
RPL7A | TGCAAATACAATCCGAGCTG | 107.71 | 0.9916 |
GGCAGGAAGAGCACAAGTTC | |||
RPS18 | CGTGCTGGAGAATGTTCTGA | 105.82 | 0.9972 |
GCTGGCTATATTTGCCATCAA | |||
RPL13a | GGCGTACCTCCACCTTATGA | 106.76 | 0.9978 |
CAGAATTTGCGACCAGGTTT | |||
RPL18 | CATTACGCTCACCAACAGGA | 111.19 | 0.998 |
CTGGAGCAGGACCAAAGTGT | |||
Actin | TGTCACTGTATGCCTCTGGT | 109.67 | 0.9978 |
TACCAGCCAAATCCAAACGC | |||
RPS7 | CGCGAGCTTGAGAAGAAGTT | 109.18 | 0.9994 |
AGAACGTGGACGCTTCTGTT | |||
RPS3 | ATCCACTCAGGTGACCCTTG | 114.35 | 0.9956 |
AACGGCCTCTTAGGTCCAAT | |||
VATB | GGTCTACCGCACAACGAAAT | 126.92 | 0.9933 |
ACCTAGCGGTTTCCATGTTG | |||
EF1A | GCCAGAAGCTGTACCTGGAG | 106.76 | 0.9994 |
TGTCACCGGCTACATAACCA | |||
Target gene | |||
VgR | TGGCGAAGACGAGAAAAACT | - | - |
TCGTCCGACAAATCGTAACA | |||
FoxL 2 | CAGCAGCCTATACGCAACAA | - | - |
AGGAGGCCAATAAGCTGGAT |
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Gong, Z.; Zhang, J.; Chen, Q.; Li, H.; Zhang, Z.; Duan, Y.; Jiang, Y.; Li, T.; Miao, J.; Wu, Y. Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses. Insects 2024, 15, 661. https://doi.org/10.3390/insects15090661
Gong Z, Zhang J, Chen Q, Li H, Zhang Z, Duan Y, Jiang Y, Li T, Miao J, Wu Y. Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses. Insects. 2024; 15(9):661. https://doi.org/10.3390/insects15090661
Chicago/Turabian StyleGong, Zhongjun, Jing Zhang, Qi Chen, Huiling Li, Ziqi Zhang, Yun Duan, Yueli Jiang, Tong Li, Jin Miao, and Yuqing Wu. 2024. "Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses" Insects 15, no. 9: 661. https://doi.org/10.3390/insects15090661
APA StyleGong, Z., Zhang, J., Chen, Q., Li, H., Zhang, Z., Duan, Y., Jiang, Y., Li, T., Miao, J., & Wu, Y. (2024). Comprehensive Screening and Validation of Stable Internal Reference Genes for Accurate qRT-PCR Analysis in Holotrichia parallela under Diverse Biological Conditions and Environmental Stresses. Insects, 15(9), 661. https://doi.org/10.3390/insects15090661