Differential expression of novel TARs in G85R motor neurons.

<p>A. RNA-Seq signal plots in reads per million mapped reads at the Limk1 gene from wild-type (black) and G85R (red) motor neurons. The canonical Limk1 gene map is shown at the top. The enlarged region shows a clear 3′-UTR extension in G85R motor neuron RNA. Similar data were observed for Gak (not shown). B. Validation of novel 3′-UTRs of Gak and Limk1 by qRT-PCR. Values are relative expression for each 3′-UTR in G85R versus wild-type motor neuron RNA.</p

RNA-Seq of laser capture microdissected spinal cord motor neurons from wtSOD1-YFP and G85R SOD1-YFP transgenic mice.

<p>A. Survival curve of G85R SOD1-YFP mice with copy number greater than 200. 80% of the mice were paralyzed (and euthanized) between ∼115 days and 205 days (red bar). N = 226. For the present study of motor neuron RNA, presymptomatic mice at ∼90 days of age were used. B.-D. Spinal cord from a 3 month old G85R SOD1-YFP mouse. Frozen section of right ventral horn region is shown, stained with Azure B dye, panel B (see Methods); incubated with anti-ChAT antibodies, panel C; or directly examined for YFP fluorescence, panel D. The large blue-stained cell bodies in panel B are motor neurons as indicated by anti-ChAT staining in panel C. Note that the same cells have YFP fluorescence in panel D. E. Large Azure B-stained cell bodies were laser captured directly into a guanidine thiocyanate solution (see Methods) and subsequent steps carried out as diagrammed (see Methods).</p

Differential expression in G85R motor neurons.

<p>A. Heatmap of selected genes from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053575#pone.0053575.s007" target="_blank">Table S2</a> significantly differentially expressed (raw p-value <0.005) between wild-type and G85R SOD1-YFP mice. For each gene listed, the ratio of RPKM values (G85R/WT) for individual pairs of biological replicates (Rep1, Rep2) is plotted according to the color code below. B. Validation of differentially expressed genes in G85R by qRT-PCR. Shown are box plots representing relative expression values of each gene in G85R versus wild-type motor neurons. Upper whisker represents top 25% of values, box represents the middle 50% of values, and lower whisker represents bottom 25% of values. Median value is indicated by horizontal dashed line. Statistical significance calculated by REST 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053575#pone.0053575-Pfaffl1" target="_blank">[37]</a> is indicated by *  =  p<0.05, **  =  p<0.005, ***  =  p<0.0005. RNAs from at least three different mouse pairs were compared for each gene. Note that the Hsp110 and B2m validations used one exon-junction-spanning and one non-spanning primer set; minus reverse transcriptase controls for these samples were negative for DNA contamination. The expression changes in the left nine genes were validated with 0.19 ng of total RNA, while the remaining six were validated with 1.5 ng of total RNA.</p