Figure 1｜ Immunostaining of rat spinal cord segments at the caudal region at 1, 4, 7, 14, and 21 days following spinal cord injury.
Few astrocytes were present at 1 day after spinal cord injury by immunostaining with the astrocyte marker GFAP (Zhang et al., 2017). GFAP immunopositivity was elevated at later time points, reaching its peak at 7 days after injury and gradually reduced toward the basal value at 14 and 21 days after spinal cord injury (Figure 1A). Immunostaining from the astrocyte marker GFAP was mainly localized in the gray matter, whereas immunostaining from the microglia marker IBA1 (Waller et al., 2019) was observed in the gray and white matter. The immunopositivity of IBA1 was elevated at 4 days after spinal cord injury and recovered to the basal level at 21 days after injury (Figure 1B), similar to GFAP results. These observations showed that there are robust morphological and molecular changes after spinal cord hemisection. These remarkable cellular changes indicate that many genes, including reference genes, might be differentially expressed after spinal cord injury.
Figure 3｜geNorm-determined expression stability of candidate reference genes.
Figure 4｜NormFinder-determined gene expression stability of candidate reference genes.
The raw Ct values of the 13 reference genes were analyzed using geNorm to determine gene expression stability, and the M-value, which negatively correlates with gene stability, was calculated. Actb had the highest M-value, which suggests that the stability of the expression of Actb was relatively low. Conversely, Ankrd27 and Ubc had the lowest M-values and the highest gene expression stabilities (Figure 3A). Calculation of pairwise variation (Vn/n+1), a normalization factor that advises the ideal number of internal control genes, showed that the pairwise variation of V2/3 was low (Figure 3B). Bioinformatic analysis using NormFinder indicated that Tbp, Ankrd27, and Ubc were the most stable genes, while Rn18s and Actb were the least stable genes in spinal cord segments following rat spinal cord injury (Figure 4). Therefore, the reference genes Tbp, Ankrd27, and Ubc can be used as internal controls for the determination of gene expressions in injured spinal cord segments.