Selection of Reliable Reference Genes for Analysis of Gene Expression in Spinal Cord during Rat Postnatal Development and after Injury
Abstract
:1. Introduction
2. Materials and Methods
2.1. Experimental Animals
Spinal Cord Injury Models and Tissue Isolation
2.2. Analysis of Gene Expression
2.2.1. RNA Isolation and cDNA Synthesis
2.2.2. Primer Design and Gene Selection
2.2.3. Quantitative RT-PCR and Normalized Gene Expression
2.2.4. Reference Gene Stability Testing
2.3. Data Representation and Statistics
3. Results
3.1. Comparison of the Expressions of the Tested Genes
3.2. Stability of RGs Expression during Postnatal Development
3.3. Stability of RGs Expression after SCI
3.3.1. Stability of RGs’ Expression after SCI Examined en bloc (cSCI and mSCI Altogether)
3.3.2. Stability of RGs’ Expression after mSCI and cSCI Examined Separately
4. Discussions
4.1. Selection of Candidate RG Set
4.2. Stability of Expressed Genes during Rat Postnatal Development
4.3. Stability of RGs Expression after SCI
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene Symbol | Gene Name | Main Function |
---|---|---|
Cds2 | CDP-diacylglycerol synthase 2 | Glycerophospholipid metabolism and phosphatidylinositol signaling system |
Eef1a1 | eukaryotic translation elongation factor 1 alpha 1 | Protein synthesis |
Eif2b2 | eukaryotic translation initiation factor 2B subunit beta | Protein synthesis |
Gapdh | glyceraldehyde-3-phosphate dehydrogenase | Glycolysis |
Gorasp1 | golgi reassembly stacking protein 1 | Golgi apparatus structure. |
Gpatch1 | G patch domain containing 1 | Nucleic acid-binding protein |
Hprt1 | hypoxanthine phosphoribosyltransferase 1 | Metabolic salvage of purines |
Mapk6 | mitogen-activated protein kinase 6 | Protein kinase |
Rpl29 | ribosomal protein L29 | Component of ribosomes (60S subunit) |
Gene Symbol | Acc. No | Sequence for | Sequence Rev | Length (bp) |
---|---|---|---|---|
Reference Genes | ||||
Cds2 | NM_053643 | TGGCTGGAAAACCATGAGGAT | GGTACTGGCAGTCAAAGCGA | 185 |
Eef1a1 | NM_175838 | TGCTGGAGCCAAGTGCTAAT | GTGCCAATGCCGCCAATTTT | 181 |
Eif2b2 | NM_032058 | ATCCGCAGAGAGGGTAGGAG | GTGCCTTCCAGTTCCACTAGC | 260 |
Gapdh1 | NM_017008 | AGACAGCCGCATCTTCTTGT | TGATGGCAACAATGTCCACT | 142 |
Gorasp1 | NM_019385 | CTGAAGGCTAATGTGGAGAAG | CCAACAATGTAGTCTGTGTAAGG | 239 |
Gpatch1 | NM_001106246 | GGACCAGCCATCTTCTTGGA | TCTCTCTCGGGTTCTTTGTGA | 226 |
Hprt1 | NM_012583 | TGCTGGTGAAAAGGACCTCTC | AGATTCAACTTGCCGCTGTCT | 192 |
Mapk62 | NM_031622 | TAAAGCCATTGACATGTGGG | TCGTGCACAACAGGGATAGA | 129 |
Rpl29 | NM_017150 | AGTCCAAGAACCACACCACA | ATTCGTATCTTTGTGACCGGGG | 84 |
Target Genes | ||||
Aif1 (Iba1) | NM_017196 | CCTCATCGTCATCTCCCCAC | CTCCATGTACTTCGTCTTGAAGG | 214 |
Cd68 (Ed1) | NM_001031638 | TGGTTCCCAGCCATGTGTTC | TCTGATGTCGGTCCTGTTTG | 209 |
Gfap | NM_017009 | CACTCAGTACGAGGCAGTGG | ACTCAAGGTCGCAGGTCAAG | 176 |
Slc1a3 (Glast) | NM_019225 | GACCTCCTCAAGTTCTGCCA | ATCTGGTGATGCGTTTGTCC | 83 |
geNorm | NormFinder | BestKeeper | ||||||
---|---|---|---|---|---|---|---|---|
Gene Rank | Gene | geNorm M | geNorm V | Gene | S Value | Gene | Coeff of Corr. [r] | Std Dev [±Cq] |
1 | Eef1a1/Gapdh | 0.378 | --- | Gapdh | 0.196 | Eef1a1 | 0.935 | 0.57 |
2 | Eef1a1 | 0.222 | Gapdh | 0.891 | 0.49 | |||
3 | Rpl29 | 0.431 | 0.141 | Gpatch1 | 0.231 | Rpl29 | 0.889 | 0.58 |
4 | Hprt1 | 0.474 | 0.113 | Hprt1 | 0.270 | Hprt1 | 0.789 | 0.36 |
5 | Gpatch1 | 0.502 | 0.095 | Rpl29 | 0.290 | Gorasp1 | 0.767 | 0.56 |
6 | Gorasp1 | 0.537 | 0.088 | Gorasp1 | 0.312 | Mapk6 | 0.703 | 0.65 |
7 | Eif2b2 | 0.573 | 0.082 | Cds2 | 0.355 | Gpatch1 | 0.669 | 0.33 |
8 | Cds2 | 0.610 | 0.079 | Mapk6 | 0.400 | Eif2b2 | 0.646 | 0.58 |
9 | Mapk6 | 0.643 | 0.073 | Eif2b2 | 0.407 | Cds2 | 0.590 | 0.49 |
geNorm | NormFinder | BestKeeper | ||||||
---|---|---|---|---|---|---|---|---|
Gene Rank | Gene | geNorm M | geNorm V | Gene | S Value | Gene | Coeff of Corr. [r] | Std Dev [±Cq] |
1 | Eif2b2/Gapdh | 0.340 | --- | Eif2b2 | 0.199 | Gapdh | 0.881 | 0.36 |
2 | Gapdh | 0.201 | Eif2b2 | 0.879 | 0.38 | |||
3 | Gorasp1 | 0.453 | 0.164 | Gorasp1 | 0.313 | Gorasp1 | 0.825 | 0.67 |
4 | Hprt1 | 0.498 | 0.118 | Gpatch1 | 0.313 | Gpatch1 | 0.813 | 0.70 |
5 | Gpatch1 | 0.538 | 0.104 | Hprt1 | 0.319 | Cds2 | 0.720 | 0.63 |
6 | Mapk6 | 0.555 | 0.080 | Mapk6 | 0.352 | Mapk6 | 0.717 | 0.67 |
7 | Cds2 | 0.565 | 0.069 | Cds2 | 0.379 | eEF1a1 | 0.690 | 0.78 |
8 | Eef1a1 | 0.625 | 0.091 | Eef1a1 | 0.421 | Hprt1 | 0.658 | 0.53 |
9 | Rpl29 | 0.689 | 0.094 | Rpl29 | 0.457 | Rpl29 | 0.212 | 0.55 |
geNorm | NormFinder | BestKeeper | ||||||
---|---|---|---|---|---|---|---|---|
Gene Rank | Gene | geNorm M | geNorm V | Gene | S Value | Gene | Coeff of Corr. [r] | Std Dev [±Cq] |
1 | Eif2b2/Gapdh | 0.261 | --- | Eif2b2 | 0.162 | Gorasp1 | 0.951 | 0.81 |
2 | Eef1a1 | 0.187 | Eif2b2 | 0.945 | 0.46 | |||
3 | Eef1a1 | 0.281 | 0.087 | Gapdh | 0.211 | eEF1a1 | 0.916 | 0.52 |
4 | Hprt1 | 0.361 | 0.104 | Mapk6 | 0.266 | Gpatch1 | 0.915 | 0.80 |
5 | Mapk6 | 0.410 | 0.085 | Hprt1 | 0.277 | Gapdh | 0.888 | 0.37 |
6 | Gorasp1 | 0.454 | 0.080 | Gorasp1 | 0.299 | Mapk6 | 0.845 | 0.59 |
7 | Gpatch1 | 0.485 | 0.070 | Gpatch1 | 0.307 | Cds2 | 0.844 | 0.67 |
8 | Cds2 | 0.508 | 0.061 | Cds2 | 0.328 | Hprt1 | 0.821 | 0.49 |
9 | Rpl29 | 0.576 | 0.084 | Rpl29 | 0.428 | Rpl29 | 0.151 | 0.40 |
geNorm | NormFinder | BestKeeper | ||||||
---|---|---|---|---|---|---|---|---|
Gene Rank | Gene | geNorm M | geNorm V | Gene | S Value | Gene | Coeff of Corr. [r] | Std Dev [±Cq] |
1 | Hprt1/Gorasp1 | 0.358 | --- | Gapdh | 0.202 | Gapdh | 0.845 | 0.27 |
2 | Eif2b2 | 0.260 | Mapk6 | 0.838 | 0.78 | |||
3 | Eif2b2 | 0.405 | 0.131 | Hprt1 | 0.268 | Gpatch1 | 0.817 | 0.81 |
4 | Gpatch1 | 0.491 | 0.133 | Gpatch1 | 0.334 | Cds2 | 0.787 | 0.81 |
5 | Mapk6 | 0.531 | 0.104 | Mapk6 | 0.358 | Eif2b2 | 0.779 | 0.35 |
6 | Gapdh | 0.555 | 0.086 | Gorasp1 | 0.373 | Gorasp1 | 0.773 | 0.71 |
7 | Cds2 | 0.585 | 0.083 | Eef1a1 | 0.475 | Hprt1 | 0.768 | 0.53 |
8 | Eef1a1 | 0.660 | 0.102 | Cds2 | 0.484 | eEF1a1 | 0.496 | 0.67 |
9 | Rpl29 | 0.744 | 0.110 | Rpl29 | 0.494 | Rpl29 | −0.112 | 0.60 |
Experimental Condition | Tissue | Species | Tested Genes | Best Rated RGs | Reference |
---|---|---|---|---|---|
development | spinal cord, brain (cerebellum) | mouse, C57BL/6J | Actb, Gapdh, Hsp60, Mrpl10, Pgk1, Ppia, Rpl13a, Rps26, Sdha, Tbp | Mrpl10, Ppia | [15] |
development | brain (somatosensory cortex, visual cortex) | rat, Wistar | Gapdh, Hprt1, Kif5c, Ospb, Rn18s, Rps18, Tfr1, Uqcrfs1, Ywhaz | Ywhaz, Uqcrfs1 Gapdh, Tfr1 Osbp | [35] |
development, in vitro differentiation | brain (neocortex), cell culture—mESC | mouse, C57BL/6 | 18S rRNA, Actb, Gapdh, Hprt1, RpII | Gapdh Hprt1 | [36] |
development | brain (different parts) | mouse, CD-1 | 18s rRNA, B2m, Gapdh, Gusb, Pgk1, Tfrc | Pgk1 | [38] |
aging, dietary restriction, glucocorticoid treatment | brain (cortex, hippocampus) | rat, Wistar | 18S rRNA, Actb, Gapdh, Cypb | Actb, Gapdh | [37] |
PNI | dorsal root ganglia | rat, Sprague-Dawley | 18s rRNA, Act, Gapdh, Hprt1, Mapk6, Tubb3, Tubb5 | Mapk6, Gapdh | [24] |
PNI | sciatic nerve, dorsal root ganglia | rat, Sprague-Dawley | 18S rRNA, Actb, Ankrd27, CypA, Gapdh, Hprt1, Mrpl10, Pgk1, Rictor, Tbp, Ubc, Ubxn11, Ywhaz | Mrpl10, Tbp | [42] |
SNI | spinal cord, dorsal root ganglia | rat, Sprague-Dawley | 18S rRNA, Actb, Gapdh, Hmbs, Hprt1, Rpl13a, Rpl29 | Rpl29, Rpl13a Hprt1, Actb | [16] |
neuropathic pain | dorsal root ganglia | rat, Sprague-Dawley | Actb, Gapdh, Hmbs, Rpl3, Rpl19, Rpl29 | Rpl29, Rpl3 | [39] |
inflammatory injury | spinal cord | rat, Sprague-Dawley | Actb, B2m, Hprt1, Mapk6 | Actb, B2m, Hprt1, Mapk6 | [18] |
TBI | brain | mouse, CD-1 | 18S rRNA, Actb, B2m, Gapd, S100b | 18S rRNA, Gapdh | [43] |
TBI | brain (cortex, hippocampus) | rat, Sprague-Dawley | B2m, Gapdh, Gusb, Hprt1, Tbp, Sdha | Hprt1, Sdha, Gusb B2m, Tbp, Gapdh | [44] |
TBI | brain (hippocampus, parietotemporal cortex) | rat, Sprague-Dawley | 18S rRNA, Actb, Cyca, Gapdh | Actb, Ppia | [45] |
TBI, aging | brain (hemispheres) | mouse, C57BL/6N | 18S rRNA, Actb, B2m, Gapdh, Hprt1, Pbgd, Ppia, S100b | Hprt1, Ppia | [46] |
cancer (astrocytoma) | cell culture—astrocytoma | human | B2M, CYC1, GAPDH, HMBS, HPRT1, RPL13a, SDHA, TBA, YWHAZ | GAPDH, RPL13A, CYC1 | [48] |
cancer (gliomas) | cell culture—glioma | human | ACTB, GAPDH, POLR2A, RPL13A, SDHA, TBP | ACTB, SDHA | [49] |
in vitro (differentiation0 | cell culture—Oligodendrocytes | rat, Wistar | 18S rRNA, Actb, Cyca, Gapdh, Hmbs, Hprt1, Pgk1, Rpl13A, Tbp, Ywhaz | Cyca, Pgk1, Rpl13A, Ywhaz | [50] |
in vitro (Borna disease virus infection) | cell culture—primary cortical neurons | rat, Sprague-Dawley | 18S rRNA, Actb, Arbp, B2m, Gapdh, Hprt1, Ppia, Rpl13a, Tpp, Ywhaz | Arbp, Actb | [51] |
in vitro (treatment with carbon monoxide) | cell culture—cortical astrocytes | mouse, C57BL/6 | Actg1, Gapdh, Hprt1, Pgk1, Ppia, Rn18s, Sdha, Tbp | Gapdh, Ppia | [52] |
Disease—neurodegenerative diseases | brain (prefrontal cortex, cerebellum) | human | ACTB, ATP5B, B2M, CYC1, EIF4A2, GAPDH, HMBS, HPRT1, PPIA, PUM1, RPL13, SDHA, TBP, TOP1, UBE2D2, UBC | UBE2D2, CYC1, RPL13 | [7] |
Disease—epilepsy | brain (neocortex temporal lobe) | human | ACTB, B2M, CYPA, GAPDH, HPRT1, MAP-2, MRPL, NNE, SDHA, SYP, TBP, UBC | SYP, NSE, MRPLl | [53] |
Disease—neurodegenerative disorders | CNS (brain, spinal cord) | human | AARS, ATP5E, BECN1, CSNK2B, DCTN2, GAPDH, GAPVD1, OSBP, QARS, NAT5, TUBB, XPNPEP1 | XPNPEP1 | [54] |
Neuroplasticity—morphine addiction | brain (caudate putamen, hippocampus) | mouse, C57BL/6J | Actb, B2m, Gapdh, Hmbs, Hprt1, Oaz1, Rps6, Tbp | Tbp Tbp, Oaz1 | [55] |
Neuroplasticity—methamphetamine | brain (striatum, substantia nigra) | rat, Sprague-Dawley | 18S rRNA, B2m, Actb, Gapdh, Hmbs, Hprt1, Oaz1, Rps6, Tbp, Ubc | Actb,, Gapdh, Hprt1, Rps6 | [56] |
Neuroplasticity—alcoholism, estrogen | brain, hearth | rat, Sprague-Dawley | U2, U5a, U6, U87, Z39, 5S rRNA, 18S rRNA, Actb, B2m, Gadd45af, Gapdh, Hprt1, Tbp, Tnks, Ubc | U87, 5S rRNA, Gapdh, U5a | [57] |
autism | brain (prefrontal cortex, hippocampus) | rat, Sprague-Dawley | Actb, Gapdh, Hmbs, Hprt1, Ppia, Rpl13a, Rps18, Tbp, Ywhaz | Hprt1 Hmbs, Tbp | [58] |
testosterone treatment | brain (hypothalamus), kidney | rat, Sprague-Dawley | Actb, B2m, Gapdh, Hmbs, Hprt1, Ppia | Hmbs, Ppia Hmbs, Gapdh | [59] |
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Košuth, J.; Farkašovská, M.; Mochnacký, F.; Daxnerová, Z.; Ševc, J. Selection of Reliable Reference Genes for Analysis of Gene Expression in Spinal Cord during Rat Postnatal Development and after Injury. Brain Sci. 2020, 10, 6. https://doi.org/10.3390/brainsci10010006
Košuth J, Farkašovská M, Mochnacký F, Daxnerová Z, Ševc J. Selection of Reliable Reference Genes for Analysis of Gene Expression in Spinal Cord during Rat Postnatal Development and after Injury. Brain Sciences. 2020; 10(1):6. https://doi.org/10.3390/brainsci10010006
Chicago/Turabian StyleKošuth, Ján, Martina Farkašovská, Filip Mochnacký, Zuzana Daxnerová, and Juraj Ševc. 2020. "Selection of Reliable Reference Genes for Analysis of Gene Expression in Spinal Cord during Rat Postnatal Development and after Injury" Brain Sciences 10, no. 1: 6. https://doi.org/10.3390/brainsci10010006
APA StyleKošuth, J., Farkašovská, M., Mochnacký, F., Daxnerová, Z., & Ševc, J. (2020). Selection of Reliable Reference Genes for Analysis of Gene Expression in Spinal Cord during Rat Postnatal Development and after Injury. Brain Sciences, 10(1), 6. https://doi.org/10.3390/brainsci10010006