Microarray data were validated using qPCR.

<p><i>A</i>, Gene expression fold changes indicated by microarray and qPCR. Eighteen genes (listed on the figure's left) were selected from the synapse, FGF, and unclustered categories for qPCR validation. Array and qPCR fold change values were calculated for each gene for the comparisons, <i>5-day-Reach</i> versus <i>0-day, 5-day-Reach</i> versus <i>5-day-Sham</i>, and <i>5-day-Reach</i> versus <i>12-day-Reach</i>, resulting into a total of 18×3 = 54 comparisons. The extent of agreement between the direction of fold change values derived from the two methods was assessed by the ratio of qPCR-fold change to the array-fold change. A positive ratio indicates an agreement, and a negative ratio, a disagreement. The heat map on the left shows this ratio of each of the 54 comparisons using a color map with green showing the highest (positive) value, and red, the lowest (negative) value. Four genes (<i>Prkci</i>, <i>Rab11fip4</i>, <i>Ctnnd1</i>, and <i>Cask</i>) showed a disagreement in at least one of the three comparisons. These four genes showing mismatches between the array- and qPCR-fold change directions (*) were also the ones whose qPCR-▵C_T values of the <i>5-day-Reach</i> group were not significantly different from those of the other groups (p>0.05), and whose qPCR-▵C_T values did not correlate well with the microarray expression intensities (p>0.05). To the right of the heat map is a table indicating to which functional categories each gene belonged (S, synapse; D, development; I, intracellular signaling; T, transforming growth factor β receptor activity; F, fibroblast growth factor family; U, unclustered). <i>B</i>, For some of the genes, we observed an excellent correlation between the sample qPCR ▵C_T values and the sample microarray gene expression intensities. They included <i>Fgf2</i> (top panel; <i>r</i> = 0.64. p<0.01) and <i>Adcy1</i> (bottom panel; <i>r</i> = 0.73, p<0.01). In both graphs here, the qPCR C_T values were normalized with respect to those of the <i>Ywhaz</i> gene.