Selection and Validation of Reliable Reference Genes for Liquidambar formosana Leaves with Different Leaf Colors
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials
2.2. Total RNA Extraction and cDNA First-Strand Synthesis
2.3. Primer Design and Specific Detection
2.4. Real-Time Fluorescence-Quantitative PCR of Candidate Reference Genes
2.5. Establishment of Reference Gene Primer Standard Curves
2.6. Data Analysis
2.7. Stability Verification of Reference Genes
3. Results
3.1. RNA Quality Testing and Primer Specificity Validation
3.2. Reference Primer Amplification Ct Value Analysis
3.3. Analysis of Gene Expression Stability
3.3.1. ΔCt Analysis
3.3.2. geNorm Analysis
3.3.3. BestKeeper Analysis
3.3.4. NormFinder Analysis
3.3.5. RefFinder Analysis
3.4. Reference Gene Stability Verification
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gene | Gene Description | Primer Sequence F/R (5′-3′) | Product Size (bp) | Efficiency (%) | R2 |
---|---|---|---|---|---|
α-TUB1 | Alpha-tubulin 1 | AGCAACTCATCAGCGGCAAGG | 264 | 101.21 | 0.9801 |
TGGCTCCACAACAGAGGTAGAAA | |||||
β-TUB | Tubulin | ACGAGGCTCTTTATGATATTTG | 264 | 107.22 | 0.9960 |
GTTGGGTGAGTTCAGGGACA | |||||
ACT | Actin | ATGTTCCCTGGCATTGCAGAC | 181 | 102.45 | 0.9916 |
ACTCATCATATTCACCCTTCG | |||||
ARF2 | ADP-ribosylation factor 2 | GTTGAATGAGGATGAGTTGAGGGAA | 177 | 105.14 | 0.9813 |
CGTTGACGGAGCGAGTGAAGA | |||||
CDC2 | Cyclin-dependent kinase-putative | TGGCATTGCTTATTGTCATTC | 123 | 106.40 | 0.9875 |
GGTGCTCTGTACCACAGGGTC | |||||
DnaJ | Chaperone protein DnaJ 49 | TGGGTCAGATGAGCCCGTTTA | 135 | 105.27 | 0.9923 |
AATTGGGTCGGTTGCATTCCT | |||||
EF1-α | Elongation factor 1-alpha | GATTGGTGGCATTGGAACTGT | 124 | 109.53 | 0.9903 |
GTGGTGCATCTCAACGGACTT | |||||
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase | GGTGGATTTGGCACATTTGGT | 108 | 105.90 | 0.9944 |
CACTCCTCATCAGCAGGGTTT | |||||
H2A | Histone H2A | GCTTGAGTTGGCGGGAAATGC | 168 | 103.12 | 0.9961 |
TTCGGAAGGAGAAGGTTGTGAATGT | |||||
HIS | Histone superfamily protein H3 | GAAGAAGCCTCACAGATACCG | 135 | 105.02 | 0.9950 |
GTCTTGAAGTCCTGGGCAAT | |||||
LTP | Seed storage/lipid transfer protein | CATCAGCAGCACAAGATTCAA | 102 | 117.49 | 0.9765 |
AAAGCATAACAGCACAGAGGC | |||||
SAND | SAND family protein | GGCTTCAGAGTTTCCATCACC | 157 | 107.50 | 0.9843 |
GACCCAGCAGAGTAGAACATAG | |||||
UBC | Ubiquitin conjugating enzyme | AGCCCTGCCCTTACCATTTCC | 141 | 108.51 | 0.9920 |
GCTCCTTGCGGTTGTTTCATA | |||||
UBQ | Ubiquitin family | AGGAGTGCCCTAATGCCGAGTG | 105 | 99.31 | 0.9947 |
CAGCCTTCTGATAAACGTAAGTCAA | |||||
Target gene | |||||
bHLH137 | Basic helix-loop-helix | AAGAAGCTCCAACAGGGTAC | 200 | 101.77 | 0.9972 |
TTGTAGGGACTGGACATAGTT |
Rank | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Spe | Gene | EF1-α | DnaJ | SAND | H2A | CDC2 | α-TUB | UBQ | HIS | UBC | β-TUB | ACT | GAPDH | ARF2 | LTP |
Stability | 0.63 | 0.65 | 0.67 | 0.68 | 0.69 | 0.74 | 0.75 | 0.76 | 0.78 | 0.8 | 0.92 | 1.02 | 1.1 | 1.45 | |
Dec | Gene | DnaJ | EF1-α | SAND | HIS | β-TUB | H2A | CDC2 | UBQ | α-TUB | UBC | ACT | ARF2 | GAPDH | LTP |
Stability | 0.6 | 0.64 | 0.64 | 0.65 | 0.66 | 0.66 | 0.69 | 0.75 | 0.77 | 0.79 | 0.82 | 0.96 | 1.03 | 1.6 | |
Total | Gene | EF1-α | ACT | HIS | UBQ | SAND | H2A | DnaJ | β-TUB | GAPDH | α-TUB | UBC | ARF2 | CDC2 | LTP |
Stability | 0.41 | 0.42 | 0.44 | 0.46 | 0.51 | 0.58 | 0.69 | 0.87 | 0.93 | 1.04 | 1.25 | 1.68 | 2.58 | 2.67 |
Rank | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Spe | Gene | EF1-α | ACT | β-TUB | DnaJ | UBQ | HIS | LTP | SAND | α-TUB | H2A | CDC2 | UBC | GAPDH | ARF2 |
SD | 0.69 | 0.76 | 0.78 | 0.86 | 0.91 | 0.92 | 1.08 | 1.19 | 1.27 | 1.34 | 1.51 | 1.57 | 1.71 | 1.93 | |
CV | 2.07 | 2.36 | 2.52 | 3.21 | 3.41 | 3.47 | 3.88 | 4.87 | 4.98 | 5.37 | 5.62 | 6.02 | 7.03 | 8.41 | |
Dec | Gene | EF1-α | ACT | DnaJ | β-TUB | α-TUB | UBQ | SAND | HIS | CDC2 | H2A | LTP | UBC | ARF2 | GAPDH |
SD | 0.61 | 0.64 | 0.72 | 0.81 | 2.47 | 2.55 | 2.58 | 2.64 | 2.76 | 2.89 | 3.05 | 3.09 | 3.32 | 3.33 | |
CV | 5.81 | 8.55 | 8.86 | 9.43 | 11.49 | 11.69 | 9.94 | 11.65 | 10.88 | 11.61 | 12.42 | 13.29 | 13.61 | 14.12 | |
Total | Gene | EF1-α | SAND | ACT | β-TUB | DnaJ | HIS | UBQ | H2A | LTP | α-TUB | GAPDH | UBC | CDC2 | ARF2 |
SD | 0.70 | 0.77 | 0.80 | 0.82 | 0.91 | 0.94 | 1.16 | 1.21 | 1.48 | 1.62 | 1.94 | 1.96 | 2.21 | 2.48 | |
CV | 2.38 | 2.55 | 2.76 | 3.15 | 3.21 | 3.48 | 4.16 | 4.85 | 5.41 | 6.21 | 8.65 | 8.98 | 9.85 | 10.81 |
Rank | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Spe | Gene | EF1-α | SAND | DnaJ | CDC2 | H2A | α-TUB | HIS | UBQ | UBC | β-TUB | ACT | GAPDH | ARF2 | LTP |
Stability | 0.158 | 0.249 | 0.267 | 0.34 | 0.34 | 0.425 | 0.467 | 0.496 | 0.537 | 0.544 | 0.74 | 0.899 | 0.988 | 1.381 | |
Dec | Gene | DnaJ | SAND | EF1-α | HIS | β-TUB | H2A | CDC2 | α-TUB | UBQ | UBC | ACT | ARF2 | GAPDH | LTP |
Stability | 0.114 | 0.242 | 0.264 | 0.285 | 0.292 | 0.329 | 0.387 | 0.525 | 0.527 | 0.583 | 0.588 | 0.84 | 0.941 | 1.560 | |
Total | Gene | ACT | EF1-α | SAND | UBQ | HIS | H2A | DnaJ | β-TUB | GAPDH | α-TUB | UBC | ARF2 | CDC2 | LTP |
Stability | 0.176 | 0.263 | 0.274 | 0.378 | 0.411 | 0.467 | 0.482 | 0.497 | 0.896 | 1.063 | 1.250 | 1.453 | 1.533 | 1.874 |
Rank | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Ranking order of leaves in September (better average) | ||||||||||||||
Delta Ct | EF1-α | DnaJ | SAND | H2A | CDC2 | α-TUB | UBQ | HIS | UBC | β-TUB | ACT | GAPDH | ARF2 | LTP |
geNorm | DnaJ | SAND | H2A | EF1-α | ACT | GAPDH | HIS | CDC2 | UBQ | UBC | α-TUB | ARF2 | β-TUB | LTP |
BestKeeper | EF1-α | ACT | β-TUB | DnaJ | UBQ | HIS | LTP | SAND | α-TUB | H2A | CDC2 | UBC | GAPDH | ARF2 |
NormFinder | EF1-α | SAND | DnaJ | CDC2 | H2A | α-TUB | HIS | UBQ | UBC | β-TUB | ACT | GAPDH | ARF2 | LTP |
Comprehensive | EF1-α | SAND | DnaJ | H2A | CDC2 | α-TUB | HIS | UBQ | UBC | β-TUB | ACT | GAPDH | ARF2 | LTP |
Ranking order of leaves in December (better average) | ||||||||||||||
Delta Ct | DnaJ | EF1-α | SAND | HIS | β-TUB | H2A | CDC2 | UBQ | α-TUB | UBC | ACT | ARF2 | GAPDH | LTP |
geNorm | GAPDH | EF1-α | H2A | ACT | SAND | DnaJ | HIS | CDC2 | α-TUB | UBQ | UBC | β-TUB | ARF2 | LTP |
BestKeeper | EF1-α | ACT | DnaJ | β-TUB | α-TUB | UBQ | SAND | HIS | CDC2 | H2A | LTP | UBC | ARF2 | GAPDH |
NormFinder | DnaJ | SAND | EF1-α | HIS | β-TUB | H2A | CDC2 | α-TUB | UBQ | UBC | ACT | ARF2 | GAPDH | LTP |
Comprehensive | DnaJ | EF1-α | ACT | SAND | β-TUB | H2A | CDC2 | HIS | α-TUB | UBQ | UBC | ARF2 | GAPDH | LTP |
Ranking order under total samples (better average) | ||||||||||||||
Delta Ct | EF1-α | ACT | HIS | UBQ | SAND | H2A | DnaJ | β-TUB | GAPDH | α-TUB | UBC | ARF2 | CDC2 | LTP |
geNorm | ACT | EF1-α | GAPDH | H2A | SAND | HIS | DnaJ | CDC2 | UBQ | UBC | α-TUB | ARF2 | β-TUB | LTP |
BestKeeper | EF1-α | SAND | ACT | β-TUB | DnaJ | HIS | UBQ | H2A | LTP | α-TUB | GAPDH | UBC | CDC2 | ARF2 |
NormFinder | ACT | EF1-α | SAND | UBQ | HIS | H2A | DnaJ | β-TUB | GAPDH | α-TUB | UBC | ARF2 | CDC2 | LTP |
Comprehensive | EF1-α | ACT | SAND | UBQ | HIS | H2A | DnaJ | β-TUB | GAPDH | α-TUB | UBC | ARF2 | CDC2 | LTP |
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Zhou, F.; Xu, L.; Shi, C.; Yang, S.; Chen, Y. Selection and Validation of Reliable Reference Genes for Liquidambar formosana Leaves with Different Leaf Colors. Curr. Issues Mol. Biol. 2024, 46, 9449-9462. https://doi.org/10.3390/cimb46090560
Zhou F, Xu L, Shi C, Yang S, Chen Y. Selection and Validation of Reliable Reference Genes for Liquidambar formosana Leaves with Different Leaf Colors. Current Issues in Molecular Biology. 2024; 46(9):9449-9462. https://doi.org/10.3390/cimb46090560
Chicago/Turabian StyleZhou, Fangwei, Liang Xu, Congguang Shi, Shaozong Yang, and Yahui Chen. 2024. "Selection and Validation of Reliable Reference Genes for Liquidambar formosana Leaves with Different Leaf Colors" Current Issues in Molecular Biology 46, no. 9: 9449-9462. https://doi.org/10.3390/cimb46090560