Analysis of Different Approaches for the Selection of Reference Genes in RT-qPCR Experiments: A Case Study in Skeletal Muscle of Growing Mice
Abstract
:1. Introduction
2. Results
2.1. Primer Specificity and Amplification Efficiency
2.2. Selection of Potential Reference Genes for Studies in Skeletal Muscle of Normal and GH-Overexpressing Growing Mice
2.3. Evaluation of Potential Reference Gene Expression Stability in Skeletal Muscle of Normal and GH-Overexpressing Growing Mice
2.3.1. Analysis of Quantification Cycle (Cq) Values Dispersion and Determination of Expression Stability by Algorithms
2.3.2. Analysis of Relative Expression Levels
2.4. Determination of Potential Reference Gene Expression Levels in Isolated Skeletal Muscle mRNA from Normal Mice
2.5. Analysis of the Relative Expression Levels of Target Genes Normalized against the Selected Reference Genes
3. Discussion
4. Materials and Methods
4.1. Animals
4.2. Experimental Design
4.3. RNA Extraction and Reverse Transcription (RT)
4.4. Messenger RNA (mRNA) Isolation
4.5. Primer Design
4.6. Quantitative PCR (qPCR)
4.7. Data Analysis
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Gene Symbol | Efficiency (%) | R2 |
---|---|---|
ACTB | 101 | 0.982 |
B2M | 98 | 0.998 |
GAPDH | 95 | 0.999 |
GHR | 93 | 0.995 |
GSK3B | 91 | 0.995 |
HPRT1 | 105 | 0.991 |
IGF1 | 108 | 0.996 |
PPIA | 101 | 0.998 |
RN18S | 101 | 0.998 |
RPL13A | 100 | 0.998 |
YWHAZ | 99 | 0.980 |
Ranking | Bestkeeper | geNorm | Comparative ΔCq | NormFinder | ||||
---|---|---|---|---|---|---|---|---|
Gene Symbol | Stability Value | Gene Symbol | Stability Value | Gene Symbol | Stability Value | Gene Symbol | Stability Value | |
1 | GSK3B | 0.587 | RPL13A/YWHAZ | 0.458 | RPL13A | 1.073 | RPL13A | 0.202 |
2 | YWHAZ | 0.762 | GSK3B | 0.549 | YWHAZ | 1.092 | RN18S | 0.279 |
3 | RPL13A | 0.916 | RN18S | 0.698 | GSK3B | 1.146 | YWHAZ | 0.291 |
4 | RN18S | 0.962 | GAPDH | 0.757 | RN18S | 1.159 | PPIA | 0.398 |
5 | GAPDH | 1.194 | PPIA | 0.871 | PPIA | 1.279 | GSK3B | 0.409 |
6 | B2M | 1.263 | HPRTA | 0.989 | HPRT1 | 1.427 | HPRT1 | 0.420 |
7 | PPIA | 1.317 | B2M | 1.104 | GAPDH | 1.479 | GAPDH | 0.421 |
8 | HPRT1 | 1.399 | ACTB | 1.279 | B2M | 1.780 | B2M | 0.762 |
9 | ACTB | 1.958 | - | - | ACTB | 1.891 | ACTB | 0.822 |
Ranking | Bestkeeper | geNorm | Comparative ΔCq | NormFinder | ||||
---|---|---|---|---|---|---|---|---|
Gene Symbol | Stability Value | Gene Symbol | Stability Value | Gene Symbol | Stability Value | Gene Symbol | Stability Value | |
1 | GSK3B | 0.646 | RPL13A/YWHAZ | 0.518 | YWHAZ | 1.173 | RPL13A | 0.169 |
2 | YWHAZ | 0.796 | GSK3B | 0.593 | RPL13A | 1.188 | RN18S | 0.227 |
3 | RN18S | 0.952 | RN18S | 0.701 | RN18S | 1.197 | GAPDH | 0.233 |
4 | RPL13A | 1.010 | GAPDH | 0.722 | GSK3B | 1.240 | YWHAZ | 0.268 |
5 | GAPDH | 1.207 | PPIA | 0.900 | PPIA | 1.492 | GSK3B | 0.449 |
6 | B2M | 1.378 | B2M | 1.031 | HPRT1 | 1.558 | PPIA | 0.486 |
7 | HPRT1 | 1.488 | HPRT1 | 1.152 | GAPDH | 1.611 | HPRT1 | 0.498 |
8 | PPIA | 1.516 | ACTB | 1.344 | B2M | 1.996 | B2M | 0.718 |
9 | ACTB | 2.273 | - | - | ACTB | 2.029 | ACTB | 0.813 |
Gene Symbol | Gene Name | GenBank Accession Number | Primer Sequence (5′–3′) a |
---|---|---|---|
ACTB | Actin, β | NM_007393.5 | F: GTGCCCATCTACGAGGGCTATGCT |
R: TACCCAAGAAGGAAGGCTGGAAAA | |||
B2M | β-2 microglobulin | NM_009735.3 | F: AAGTATACTCACGCCACCCA |
R: AAGACCAGTCCTTGCTGAAG | |||
GAPDH | Glyceraldehyde-3-phosphate dehydrogenase | NM_008084.3 | F: AGTGCCAGCCTCGTCCCGTAG |
R: GTGCCGTTGAATTTGCCGTGAGTG | |||
GHR | Growth hormone receptor | NM_001286370.1 | F: CCAACTCGCCTCTACACCG |
R: GGGAAAGGACTACACCACCTG | |||
GSK3B | Glycogen synthase kinase 3β | NM_019827.6 | F: CCACCATCCTTATCCCTCCAC |
R: GTATCTGAGGCTGCTGTGGC | |||
HPRT1 | Hypoxanthine guanine phosphoribosyl transferase | NM_013556.2 | F: CAGTCCCAGCGTCGTGATTA |
R: TCGAGCAAGTCTTTCAGTCCT | |||
IGF1 | Insulin-like growth factor 1 | NM_010512.4 | F: CCAAACACAATTCTCCTTCC |
R: GCTACAGCAACCTGTGATTG | |||
PPIA | Peptidylprolyl isomerase A | NM_008907.1 | F: GCGTCTCCTTGAGCTGTT |
R: AAGTCACCACCCTGGCAC | |||
RN18S | 18S ribosomal RNA | NR_003278.3 | F: ACGGACAGGATTGACAGATT |
R: GCCAGAGTCTCGTTCGTTAT | |||
RPL13A | Ribosomal protein L13A | NM_009438.5 | F: TGACAAGAAAAAGCGGATGGTG |
R: GCTGTCACTGCCTGGTACTT | |||
YWHAZ | Tyr 3-monooxygenase/Trp 5-monooxygenase activation protein, Z polypeptide | NM_011740.3 | F: CCAGGACCTAAAAGGGTCGG |
R: ACACACCGAACTGTTGTCGT |
Gene Symbol | Exon–Exon Junction a,b | Tm (°C) | Product Length (bp) |
---|---|---|---|
ACTB | F: no | 63 | 319 |
R: yes | 58 | ||
B2M | F: no | 56 | 162 |
R: no | 55 | ||
GAPDH | F: yes | 64 | 171 |
R: no | 61 | ||
GHR | F: no | 58 | 104 |
R: no | 57 | ||
GSK3B | F: no | 57 | 79 |
R: yes | 58 | ||
HPRT1 | F: yes | 62 | 142 |
R: yes | 59 | ||
IGF1 | F: no | 57 | 97 |
R: no | 57 | ||
PPIA | F: no | 55 | 145 |
R: yes | 58 | ||
RN18S | F: no | 57 | 118 |
R: no | 57 | ||
RPL13A | F: yes | 64 | 126 |
R: no | 58 | ||
YWHAZ | F: no | 63 | 115 |
R: no | 59 |
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Piazza, V.G.; Bartke, A.; Miquet, J.G.; Sotelo, A.I. Analysis of Different Approaches for the Selection of Reference Genes in RT-qPCR Experiments: A Case Study in Skeletal Muscle of Growing Mice. Int. J. Mol. Sci. 2017, 18, 1060. https://doi.org/10.3390/ijms18051060
Piazza VG, Bartke A, Miquet JG, Sotelo AI. Analysis of Different Approaches for the Selection of Reference Genes in RT-qPCR Experiments: A Case Study in Skeletal Muscle of Growing Mice. International Journal of Molecular Sciences. 2017; 18(5):1060. https://doi.org/10.3390/ijms18051060
Chicago/Turabian StylePiazza, Verónica G., Andrzej Bartke, Johanna G. Miquet, and Ana I. Sotelo. 2017. "Analysis of Different Approaches for the Selection of Reference Genes in RT-qPCR Experiments: A Case Study in Skeletal Muscle of Growing Mice" International Journal of Molecular Sciences 18, no. 5: 1060. https://doi.org/10.3390/ijms18051060