Real-Time qPCR Identifies Suitable Reference Genes for Borna Disease Virus-Infected Rat Cortical Neurons
Abstract
:1. Introduction
2. Results
2.1. Immunofluorescence Assay
2.2. Evaluation of Expression Stability
2.3. Candidate Reference Gene Ranking
Symbol | Accession | Name | Forward and Reverse Primer (5'-3') | Amplicon Size | Primer Efficiency | Cq Value (Average ± SD) | Reference a | Mean Relative Quantification b(9, 12 Days) |
---|---|---|---|---|---|---|---|---|
HPRT | NM012583 | Hypoxanthine phosphoribosyl-transferase | CTCATGGACTGATTATGGACAGGACGCAGGTCAGCAAAGAACTTATAGCC | 123 | 93 | 23.36 ± 0.78 | [10] | 0.83; 1.16 |
YWHAZ | NM013011 | Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide | GATGAAGCCATTGCTGAACTTGGTCTCCTTGGGTATCCGATGTC | 117 | 100 | 19.04 ± 0.66 | [10] | 0.78; 0.91 |
TPB | NM001004198 | TATA box binding protein | TGGGATTGTACCACAGCTCCACTCATGATGACTGCAGCAAACC | 131 | 93 | 23.94 ± 0.65 | [10] | 0.91; 1.25 |
Rpl13A | NM173340 | Ribosomal protein L13A | GGATCCCTCCACCCTATGACACTGGTACTTCCACCCGACCTC | 132 | 94 | 18.67 ± 1.12 | [10] | 0.99; 1.28 |
GAPDH | NM017008 | Glyceraldehyde-3-phosphate dehydrogenase | CAACTCCCTCAAGATTGTCAGCAAGGCATGGACTGTGGTCATGA | 118 | 105 | 20.35 ± 0.67 | [14] | 0.80; 0.78 |
ACTB | NM031144 | Beta-actin | CAGGGTGTGATGGTGGGTATGGAGTTGGTGACAATGCCGTGTTC | 115 | 103 | 17.88 ± 0.85 | [15] | 1.00; 0.99 |
PPIA | NM017101 | peptidylprolylisomerase A | GTCAACCCCACCGTGTTCTTCATCCTTTCTCCCCAGTGCTCAG | 133 | 93 | 17.06 ± 0.82 | [15] | 0.85; 0.97 |
ARBP | NM022402 | Acidic ribosomal phosphoprotein P0 | TAGAGGGTGTCCGCAATGTGCAGTGGGAAGGTGTAGTCAGTC | 137 | 102 | 22.02 ± 0.62 | [16] | 0.81; 0.73 |
B2M | NM012512 | Beta-2 microglobulin | CGAGACCGATGTATATGCTTGCGTCCAGATGATTCAGAGCTCCA | 114 | 92 | 18.16 ± 1.15 | [14] | 2.51; 3.24 |
18s rRNA | M11188 | 18s subunit ribosomal RNA | ACGGACCAGAGCGAAAGCATTGTCAATCCTGTCCGTGTCC | 310 | 107 | 18.07±0.76 | [17] | 0.72; 0.69 |
BDNF | NM001270630 | Brain-derived neurotrophic factor | ACAGTATTAGCGAGTGGGATTGGGTAGTTCGGCATT | 213 | 96 | 25.55 ± 0.30 | a | - |
AldoC | NM012497 | Aldolase C, fructose bisphosphate | ACCTGGAAGGGACTCTCCTCAAAGTCACCCCTGGGACAGCT | 141 | 103 | 20.86 ± 0.77 | [18] | - |
Genorm | 9 Days (C + V, n = 12) | 12 Days (C + V, n = 12) | ||
---|---|---|---|---|
Rank | Gene | M-Value | Gene | M-Value |
1 | ARBP | 0.2859 | GAPDH | 0.2159 |
2 | HPRT | 0.2859 | YWHAZ | 0.2159 |
3 | YWHAZ | 0.3152 | ACTB | 0.2617 |
4 | TPB | 0.3539 | ARBP | 0.3249 |
5 | Rpl13A | 0.3771 | ppIA | 0.3529 |
6 | ACTB | 0.3928 | TPB | 0.3645 |
7 | GAPDH | 0.4035 | HPRT | 0.387 |
8 | ppIA | 0.4103 | Rpl13A | 0.537 |
NormFinder | 9 Days (C + V, n = 12) | 12 Days (C + V, n = 12) | ||
Rank | Gene | Variability | Gene | Variability |
1 | ARBP | 0.0565 | ACTB | 0.0709 |
2 | ACTB | 0.0627 | YWHAZ | 0.1131 |
3 | TPB | 0.0713 | ppIA | 0.1363 |
4 | YWHAZ | 0.0796 | TPB | 0.1521 |
5 | HPRT | 0.0815 | HPRT | 0.1626 |
6 | Rpl13A | 0.0821 | GAPDH | 0.1643 |
7 | GAPDH | 0.0873 | ARBP | 0.199 |
8 | ppIA | 0.1055 | Rpl13A | 0.3008 |
BestKeeper | 9 Days (C + V, n = 12) | 12 Days (C + V, n = 12) | ||
Rank | Gene | (r) | Gene | (r) |
1 | ppIA | 0.879 | ppIA | 0.874 |
2 | ARBP | 0.868 | TPB | 0.809 |
3 | GAPDH | 0.784 | ACTB | 0.749 |
4 | YWHAZ | 0.72 | ARBP | 0.734 |
5 | HPRT | 0.703 | HPRT | 0.584 |
6 | ACTB | 0.7 | YWHAZ | 0.583 |
7 | Rpl13A | 0.527 | GAPDH | 0.511 |
8 | TPB | 0.497 | Rpl13A | 0.146 |
Delta-Ct | 9 Days (C + V, n = 12) | 12 Days (C + V, n = 12) | ||
Rank | Gene | Mean SD | Gene | Mean SD |
1 | ARBP | 0.34 | ACTB | 0.42 |
2 | TPB | 0.4 | YWHAZ | 0.45 |
3 | GAPDH | 0.4 | TPB | 0.45 |
4 | HPRT | 0.41 | GAPDH | 0.46 |
5 | ACTB | 0.41 | ARBP | 0.49 |
6 | Rpl13A | 0.42 | ppIA | 0.5 |
7 | ppIA | 0.42 | HPRT | 0.52 |
8 | YWHAZ | 0.43 | Rpl13A | 1 |
Day 9 | |||||
Ranking | geNorm | NormFinder | BestKeeper | Delta-Ct | Consensus |
1 | ARBP and HPRT | ARBP | ppIA | ARBP | ARBP |
2 | - | ACTB | ARBP | TPB | ACTB |
3 | YWHAZ | TPB | GAPDH | GAPDH | TPB |
4 | TPB | YWHAZ | YWHAZ | HPRT | YWHAZ |
5 | Rpl13A | HPRT | HPRT | ACTB | HPRT |
6 | ACTB | Rpl13A | ACTB | Rpl13A | GAPDH |
7 | GAPDH | GAPDH | Rpl13A | ppIA | RpI13A |
8 | ppIA | ppIA | TPB | YWHAZ | ppIA |
Day 12 | |||||
Ranking | geNorm | NormFinder | BestKeeper | Delta-Ct | Consensus |
1 | GAPDH and YWHAZ | ACTB | ppIA | ACTB | ACTB |
2 | - | YWHAZ | TPB | YWHAZ | YWHAZ |
3 | ACTB | ppIA | ACTB | TPB | GAPDH |
4 | ARBP | TPB | ARBP | GAPDH | TPB |
5 | ppIA | HPRT | HPRT | ARBP | ARBP |
6 | TPB | GAPDH | YWHAZ | ppIA | ppIA |
7 | HPRT | ARBP | GAPDH | HPRT | HPRT |
8 | Rpl13A | Rpl13A | Rpl13A | Rpl13A | RpI13A |
Two Time Points | |||||
Ranking | Day 9 | Day 12 | Consensus | ||
1 | ARBP | ACTB | ACTB | ||
2 | ACTB | YWHAZ | ARBP | ||
3 | TPB | GAPDH | TPB | ||
4 | YWHAZ | TPB | YWHAZ | ||
5 | HPRT | ARBP | GAPDH | ||
6 | GAPDH | ppIA | HPRT | ||
7 | RpI13A | HPRT | ppIA | ||
8 | ppIA | RpI13A | RpI13A |
2.4. Assessment of the Validity
Reference Genes at Day 12 | BDNF | AldoC |
---|---|---|
(p-value, n) | (p-value, n) | |
ACTB and YWHAZ | 0.74 | 0.086 |
(0.002, n = 12) | (0.612, n = 12) | |
RpI13A | 1.37 | 1.95 |
(0.075, n = 12) | (0.873, n = 12) |
3. Discussion
4. Experimental Section
4.1. Primary Culture of Neurons and Viral Infections
4.2. Immunofluorescence
4.3. RNA Isolation and Reverse Transcription
4.4. Quantitative Real-Time PCR
4.5. Data Analysis
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Zhang, L.; Liu, S.; Zhang, L.; You, H.; Huang, R.; Sun, L.; He, P.; Chen, S.; Zhang, H.; Xie, P. Real-Time qPCR Identifies Suitable Reference Genes for Borna Disease Virus-Infected Rat Cortical Neurons. Int. J. Mol. Sci. 2014, 15, 21825-21839. https://doi.org/10.3390/ijms151221825
Zhang L, Liu S, Zhang L, You H, Huang R, Sun L, He P, Chen S, Zhang H, Xie P. Real-Time qPCR Identifies Suitable Reference Genes for Borna Disease Virus-Infected Rat Cortical Neurons. International Journal of Molecular Sciences. 2014; 15(12):21825-21839. https://doi.org/10.3390/ijms151221825
Chicago/Turabian StyleZhang, Lujun, Siwen Liu, Liang Zhang, Hongmin You, Rongzhong Huang, Lin Sun, Peng He, Shigang Chen, Hong Zhang, and Peng Xie. 2014. "Real-Time qPCR Identifies Suitable Reference Genes for Borna Disease Virus-Infected Rat Cortical Neurons" International Journal of Molecular Sciences 15, no. 12: 21825-21839. https://doi.org/10.3390/ijms151221825