Identification and Validation of Reference Genes for Expression Analysis Using qRT-PCR in Cimex hemipterus (Hemiptera: Cimicidae)
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Insects
2.2. Experimental Condition and Sample Collection
2.3. RNA Extraction and cDNA Synthesis
2.4. Reference Gene Selection, Primer Design, and qRT-PCR
2.5. Determination of Reference Gene Expression Stability
2.6. Validation of the Candidate Reference Genes
3. Results
3.1. Primer Specificity and Efficiency
3.2. Ct Values of Candidate Reference Genes
3.3. Stability of the 10 Reference Genes under Different Experimental Conditions
3.4. Validation of Candidate Reference Genes in Different Tissues
4. Discussion
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 Sequence a (5′–3′) | Amplicon Length (bp) | Efficiency b (%) | R2 c |
---|---|---|---|---|
RPL8 | F: AGGCACGGTTACATCAAAGG | 131 | 111.07 | 0.9952 |
R: TCGGGAGCAATGAAGAGTTC | ||||
RPL11 | F: GAAGAATGTCATGCGAGATGTCAGG | 129 | 92.28 | 0.9987 |
R: CCTTTGAGAAGACTGGCTGCTG | ||||
RPL13 | F: ATTGGCAGAGGTTCATTCGT | 131 | 100.91 | 0.9979 |
R: GCATCTCACTGCTGGTCTCA | ||||
RPS16 | F: AATGGTCGCCCACTTGAGAT | 158 | 87.46 | 0.9976 |
R: GCCTGCCTGATTGCGTAGAT | ||||
α-tubulin | F: TCAACTACCAACCACCCACT | 139 | 94.93 | 0.9990 |
R: TTGGCGTACATCAAGTCAAA | ||||
β-tubulin | F: CCTTTGCTGGACGCCTCATT | 84 | 103.67 | 0.9902 |
R: CGACCCGACTGGTGCATACC | ||||
GAPDH | F: GCATTTAGAGTCCCTGTCGC | 149 | 91.12 | 0.9806 |
R: ACTTCATCTTCGGTGTAGCC | ||||
EF1α | F: CGTTAGGACGTTTCGCTGTC | 103 | 106.65 | 0.9893 |
R: GCCTTTGTCACTTTGCCACT | ||||
Actin | F: GACTTCGAGCAGGAAATGGC | 114 | 98.08 | 0.9989 |
R: TTCTGGGCAACGGAACCTCT | ||||
NADH | F: AAAGACGAAGGTCGGGATTA | 76 | 87.11 | 0.9812 |
R: GCATCAAAGAAACACGCATC |
Candidate Gene | Experimental Conditions | All * | ||||
---|---|---|---|---|---|---|
Developmental Stage | Sex | Tissue | Gas Stimulation | Temperature | ||
RPL8 | 19.15 ± 0.16 | 18.50 ± 0.42 | 19.33 ± 0.11 | 27.61 ± 0.56 | 21.26 ± 0.23 | 20.68 ± 0.40 |
RPL11 | 18.23 ± 0.57 | 18.62 ± 0.16 | 19.29 ± 0.16 | 26.97 ± 0.49 | 22.39 ± 0.23 | 20.78 ± 0.41 |
RPL13 | 18.75 ± 0.24 | 19.40 ± 0.66 | 22.24 ± 1.03 | 28.34 ± 0.60 | 21.53 ± 0.23 | 21.60 ± 0.48 |
RPS16 | 17.94 ± 0.65 | 17.63 ± 0.15 | 22.73 ± 1.19 | 29.70 ± 0.87 | 30.44 ± 0.41 | 24.09 ± 0.83 |
α-tubulin | 15.74 ± 0.67 | 16.59 ± 0.61 | 16.58 ± 0.16 | 23.08 ± 0.47 | 17.67 ± 0.16 | 17.47 ± 0.33 |
β-tubulin | 20.61 ± 0.44 | 22.18 ± 0.97 | 19.50 ± 0.50 | 27.67 ± 0.65 | 20.95 ± 0.17 | 21.46 ± 0.38 |
GAPDH | 17.88 ± 0.53 | 18.30 ± 0.32 | 20.25 ± 0.20 | 27.77 ± 0.48 | 22.67 ± 0.19 | 21.06 ± 0.45 |
EF1α | 15.54 ± 0.54 | 15.52 ± 0.25 | 16.70 ± 0.17 | 24.27 ± 0.46 | 17.94 ± 0.23 | 17.55 ± 0.39 |
Actin | 15.67 ± 1.20 | 16.87 ± 0.88 | 14.92 ± 0.31 | 23.29 ± 0.63 | 15.42 ± 0.12 | 16.46 ± 0.40 |
NADH | 21.26 ± 0.35 | 26.60 ± 2.67 | 23.12 ± 0.52 | 32.34 ± 0.60 | 24.61 ± 0.10 | 24.61 ± 0.56 |
Condition | Rank * | geNorm | NormFinder | BestKeeper | ∆Ct | ||||
---|---|---|---|---|---|---|---|---|---|
Gene | Stability | Gene | Stability | Gene | Stability | Gene | Stability | ||
Developmental stage | 1 | RPL8 | 0.118 | EF1α | 0.274 | NADH | 0.283 | RPL8 | 0.622 |
2 | RPL11 | 0.118 | NADH | 0.278 | β-tubulin | 0.349 | EF1α | 0.623 | |
3 | EF1α | 0.240 | RPL8 | 0.334 | EF1α | 0.410 | RPL11 | 0.636 | |
4 | RPS16 | 0.269 | GAPDH | 0.378 | GAPDH | 0.428 | RPS16 | 0.675 | |
5 | RPL13 | 0.356 | RPL11 | 0.382 | RPL8 | 0.443 | NADH | 0.707 | |
6 | GAPDH | 0.434 | RPS16 | 0.433 | RPL11 | 0.457 | GAPDH | 0.722 | |
7 | NADH | 0.497 | β-tubulin | 0.588 | RPS16 | 0.490 | RPL13 | 0.837 | |
8 | α-tubulin | 0.560 | RPL13 | 0.685 | α-tubulin | 0.622 | β-tubulin | 0.865 | |
9 | β-tubulin | 0.629 | α-tubulin | 0.708 | RPL13 | 0.672 | α-tubulin | 0.896 | |
10 | Actin | 0.813 | Actin | 1.513 | Actin | 0.963 | Actin | 1.551 | |
Sex | 1 | RPS16 | 0.328 | RPL8 | 0.364 | RPS16 | 0.290 | RPL8 | 1.709 |
2 | EF1α | 0.328 | RPL13 | 0.428 | RPL11 | 0.317 | RPL11 | 1.794 | |
3 | RPL11 | 0.640 | RPL11 | 0.709 | EF1α | 0.385 | GAPDH | 1.914 | |
4 | GAPDH | 0.749 | GAPDH | 1.133 | GAPDH | 0.520 | RPS16 | 1.944 | |
5 | RPL8 | 0.881 | α-tubulin | 1.192 | RPL8 | 0.691 | α-tubulin | 1.947 | |
6 | α-tubulin | 1.002 | Actin | 1.217 | α-tubulin | 1.253 | EF1α | 1.948 | |
7 | RPL13 | 1.183 | RPS16 | 1.380 | RPL13 | 1.272 | RPL13 | 2.039 | |
8 | Actin | 1.325 | β-tubulin | 1.428 | Actin | 1.771 | Actin | 2.091 | |
9 | β-tubulin | 1.421 | EF1α | 1.448 | β-tubulin | 1.986 | β-tubulin | 2.241 | |
10 | NADH | 2.388 | NADH | 6.188 | NADH | 5.589 | NADH | 6.256 | |
Tissue | 1 | RPL8 | 0.355 | RPL8 | 0.178 | RPL8 | 0.329 | EF1α | 1.595 |
2 | RPL11 | 0.355 | RPL11 | 0.178 | α-tubulin | 0.415 | RPL8 | 1.600 | |
3 | EF1α | 0.484 | EF1α | 0.247 | RPL11 | 0.422 | RPL11 | 1.667 | |
4 | GAPDH | 0.556 | α-tubulin | 0.312 | EF1α | 0.490 | α-tubulin | 1.723 | |
5 | α-tubulin | 0.632 | GAPDH | 0.579 | GAPDH | 0.524 | GAPDH | 1.775 | |
6 | Actin | 0.847 | Actin | 0.830 | Actin | 0.808 | Actin | 1.915 | |
7 | β-tubulin | 1.057 | β-tubulin | 1.165 | β-tubulin | 1.173 | β-tubulin | 2.158 | |
8 | NADH | 1.313 | NADH | 2.099 | NADH | 1.524 | NADH | 2.656 | |
9 | RPL13 | 1.838 | RPL13 | 3.835 | RPL13 | 3.203 | RPL13 | 4.031 | |
10 | RPS16 | 2.353 | RPS16 | 4.235 | RPS16 | 3.629 | RPS16 | 4.412 | |
Gas stimulation | 1 | Actin | 0.325 | RPL8 | 0.241 | EF1α | 0.893 | RPL8 | 0.689 |
2 | β-tubulin | 0.325 | β-tubulin | 0.400 | GAPDH | 0.958 | β-tubulin | 0.737 | |
3 | RPL8 | 0.440 | RPL11 | 0.435 | α-tubulin | 0.998 | RPL11 | 0.764 | |
4 | RPL11 | 0.491 | Actin | 0.453 | RPL11 | 1.004 | Actin | 0.766 | |
5 | α-tubulin | 0.588 | NADH | 0.579 | RPL8 | 1.100 | EF1α | 0.834 | |
6 | EF1α | 0.624 | EF1α | 0.616 | RPL13 | 1.105 | α-tubulin | 0.846 | |
7 | GAPDH | 0.644 | α-tubulin | 0.651 | NADH | 1.180 | NADH | 0.857 | |
8 | NADH | 0.696 | GAPDH | 0.718 | Actin | 1.231 | GAPDH | 0.892 | |
9 | RPL13 | 0.778 | RPL13 | 0.855 | β-tubulin | 1.281 | RPL13 | 1.041 | |
10 | RPS16 | 0.864 | RPS16 | 1.089 | RPS16 | 1.767 | RPS16 | 1.207 | |
Temperature | 1 | RPL8 | 0.230 | RPL8 | 0.279 | NADH | 0.345 | RPL8 | 0.616 |
2 | RPL13 | 0.230 | β-tubulin | 0.343 | Actin | 0.390 | NADH | 0.653 | |
3 | EF1α | 0.360 | NADH | 0.361 | α-tubulin | 0.446 | β-tubulin | 0.660 | |
4 | RPL11 | 0.416 | GAPDH | 0.407 | β-tubulin | 0.554 | EF1α | 0.664 | |
5 | GAPDH | 0.464 | EF1α | 0.426 | GAPDH | 0.563 | GAPDH | 0.679 | |
6 | NADH | 0.512 | RPL13 | 0.447 | RPL11 | 0.726 | RPL13 | 0.688 | |
7 | β-tubulin | 0.528 | RPL11 | 0.462 | RPL8 | 0.764 | RPL11 | 0.702 | |
8 | Actin | 0.573 | α-tubulin | 0.568 | EF1α | 0.776 | α-tubulin | 0.791 | |
9 | α-tubulin | 0.605 | Actin | 0.593 | RPL13 | 0.797 | Actin | 0.795 | |
10 | RPS16 | 0.766 | RPS16 | 1.341 | RPS16 | 1.211 | RPS16 | 1.411 |
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Kong, D.; Shi, D.; Wang, C.; Zhai, R.; Lyu, L.; He, Y.; Wang, D. Identification and Validation of Reference Genes for Expression Analysis Using qRT-PCR in Cimex hemipterus (Hemiptera: Cimicidae). Insects 2022, 13, 784. https://doi.org/10.3390/insects13090784
Kong D, Shi D, Wang C, Zhai R, Lyu L, He Y, Wang D. Identification and Validation of Reference Genes for Expression Analysis Using qRT-PCR in Cimex hemipterus (Hemiptera: Cimicidae). Insects. 2022; 13(9):784. https://doi.org/10.3390/insects13090784
Chicago/Turabian StyleKong, Delong, Daxia Shi, Changlu Wang, Ruyue Zhai, Lingling Lyu, Yurong He, and Desen Wang. 2022. "Identification and Validation of Reference Genes for Expression Analysis Using qRT-PCR in Cimex hemipterus (Hemiptera: Cimicidae)" Insects 13, no. 9: 784. https://doi.org/10.3390/insects13090784