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Tissue Specific Reference Genes for MicroRNA Expression Analysis in a Mouse Model of Peripheral Nerve Injury

Kalpachidou, Theodora, Kummer, Kai K., Mitrić, Miodrag, Kress, Michaela·2019·Frontiers in Molecular Neuroscience
DOI 10.3389/fnmol.2019.00283PMC PMC6883285
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FIGURE 1 Raw Cq values and average standard deviations of technical duplicates for all evaluated ncRNAs in DRG (A,D), dhSC (B,E), and mPFC (C,F) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. Values are presented as boxplots, representing upper quartile, median, and lower quartile, whereas whiskers depict minimum and maximum values. The dotted line in A–C is set at Cq = 30 and in D–F at 0.167. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex, Cq: quantitation cycle, SD: standard deviation.
FIGURE 1 Raw Cq values and average standard deviations of technical duplicates for all evaluated ncRNAs in DRG (A,D), dhSC (B,E), and mPFC (C,F) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. Values are presented as boxplots, representing upper quartile, median, and lower quartile, whereas whiskers depict minimum and maximum values. The dotted line in A–C is set at Cq = 30 and in D–F at 0.167. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex, Cq: quantitation cycle, SD: standard deviation.CC BY · PubMed Central
FIGURE 2 Total ranking obtained from Bestkeeper for each evaluated ncRNA (A), dhSC (B), and mPFC (C) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. Total ranking was calculated by averaging the rankings achieved by each ncRNA in SD and BKI correlation coefficient. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex.
FIGURE 2 Total ranking obtained from Bestkeeper for each evaluated ncRNA (A), dhSC (B), and mPFC (C) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. Total ranking was calculated by averaging the rankings achieved by each ncRNA in SD and BKI correlation coefficient. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex.CC BY · PubMed Central
FIGURE 3 Average standard deviation (SD) of the Cq differences (ΔCq) computed for each evaluated ncRNA vs all the remaining ncRNAs (comparative delta-Cq method) in DRG (A), dhSC (B), and mPFC (C) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex, SD: standard deviation.
FIGURE 3 Average standard deviation (SD) of the Cq differences (ΔCq) computed for each evaluated ncRNA vs all the remaining ncRNAs (comparative delta-Cq method) in DRG (A), dhSC (B), and mPFC (C) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex, SD: standard deviation.CC BY · PubMed Central
FIGURE 4 Average expression stability of the remaining ncRNAs after stepwise exclusion of the least stable candidate (upper panel) and pairwise variation analysis (lower panel), as calculated by geNorm for all ncRNAs in DRG (A,D), dhSC (B,E), and mPFC (C,F) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. The dotted line represents a pairwise variability of 0.15. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex.
FIGURE 4 Average expression stability of the remaining ncRNAs after stepwise exclusion of the least stable candidate (upper panel) and pairwise variation analysis (lower panel), as calculated by geNorm for all ncRNAs in DRG (A,D), dhSC (B,E), and mPFC (C,F) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. The dotted line represents a pairwise variability of 0.15. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex.CC BY · PubMed Central
FIGURE 5 Stability value ρ (upper panel), inter- (box) and intra- (whiskers) group variability (lower panel), as evaluated by NormFinder for all evaluated ncRNAs in DRG (A,B), dhSC (C,D), and mPFC (E,F) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex.
FIGURE 5 Stability value ρ (upper panel), inter- (box) and intra- (whiskers) group variability (lower panel), as evaluated by NormFinder for all evaluated ncRNAs in DRG (A,B), dhSC (C,D), and mPFC (E,F) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex.CC BY · PubMed Central
FIGURE 6 Overall ranking of all evaluated ncRNAs, as obtained by calculating the mean of the separate rankings achieved in BestKeeper, comparative delta-Cq method, geNorm, and NormFinder, in DRG (A), dhSC (B), and mPFC (C) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex.
FIGURE 6 Overall ranking of all evaluated ncRNAs, as obtained by calculating the mean of the separate rankings achieved in BestKeeper, comparative delta-Cq method, geNorm, and NormFinder, in DRG (A), dhSC (B), and mPFC (C) of non-treated (n = 5), sham (n = 6), and SNI (n = 5) mice, as estimated by setting a common threshold (0.1) and by using LinRegPCR software. DRG: dorsal root ganglia, dhSC: dorsal horn of the spinal cord, mPFC: medial prefrontal cortex.CC BY · PubMed Central
FIGURE 7 miR-21a-5p quantification in the DRG of non-treated (nt), sham and SNI mice using (A) three (sno420/sno429/sno202, left panel) or two (sno420/sno429, right panel) most stable ncRNAs from the overall ranking list, the most stable pair of ncRNAs as computed by (B) geNorm (sno202/sno420) and (C) NormFinder (sno234/sno429), (D) U6, and (E) sno135. miR-21a-5p levels were expressed as fold changes relative to the respective expression levels in the non-treated group using the 2– ΔΔCq method. Values are presented as boxplots, representing upper quartile, median, and lower quartile, whereas whiskers depict minimum and maximum values.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.
FIGURE 7 miR-21a-5p quantification in the DRG of non-treated (nt), sham and SNI mice using (A) three (sno420/sno429/sno202, left panel) or two (sno420/sno429, right panel) most stable ncRNAs from the overall ranking list, the most stable pair of ncRNAs as computed by (B) geNorm (sno202/sno420) and (C) NormFinder (sno234/sno429), (D) U6, and (E) sno135. miR-21a-5p levels were expressed as fold changes relative to the respective expression levels in the non-treated group using the 2– ΔΔCq method. Values are presented as boxplots, representing upper quartile, median, and lower quartile, whereas whiskers depict minimum and maximum values.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.CC BY · PubMed Central

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Authors
Kalpachidou, T., Kummer, K. K., Mitrić, M., & Kress, M.
Venue
Frontiers in Molecular Neuroscience
Publisher
Frontiers Media SA
Published
2019-11-22
Volume
12
ISSN
1662-5099
DOI
10.3389/fnmol.2019.00283
PMC
PMC6883285
Cited by
10
References
1
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • Austrian Science Fund (DK-SPIN W1206-06, P25345, P28611, P30809)
  • Österreichische Forschungsförderungsgesellschaft (D-150700-013-017)
  • Seventh Framework Programme (602133)

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Kalpachidou, T., Kummer, K. K., Mitrić, M., & Kress, M. (2019). Tissue Specific Reference Genes for MicroRNA Expression Analysis in a Mouse Model of Peripheral Nerve Injury. Frontiers in Molecular Neuroscience. https://doi.org/10.3389/fnmol.2019.00283