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Fig. 6 | Molecular Brain

Fig. 6

From: Cacna1b alternative splicing impacts excitatory neurotransmission and is linked to behavioral responses to aversive stimuli

Fig. 6

E37a-Cacna1b mRNA is more enriched in CaMKIIα+PNs relative to CCK+INs. a Schematic depicting the work flow to quantify 37a-CaV2.2 mRNA in CamKIIα+PNs and CCK+INs, this includes genetic labeling using Cre/loxP and Flpe/FRT systems, neuronal dissociation, FACS and RT-qPCR. b Quantification of Gad2 mRNA in total RNA isolated from CaMKIIα+PNs and CCK+INs. Data are shown as mean ± SE of Gad2 fold change. Gad2 mRNA expression was normalized to Gapdh mRNA levels. c Upper panel. Approximate location of primers (arrows) to amplify a sequence spanning e35–36 and e36-37a. Lower panel. Representative image to show the specificity of both sets of primers. Note the lack of amplification for e36-37a set of primers in the presence of the e37b-Cacna1b clone. As expected, e35–36 primers amplified both e37a-Cacna1b and e37b-Cacna1b clones. d Upper panel. Melting curve for e36-37a and e35–36 sets of primers. The derivative of fluorescence as a function of temperature was plotted (−dF/dT) against temperature. One single peak in each plot strongly suggest the presence of one product of amplification for both e36-37a and e35–36 sets of primers. Lower panel. Standard curves to assess the PCR efficiency for e36-37a and e35–36 sets of primers. Open circles indicate individual measurements of Ct values at a given dilution. All points for each sets of primers were considered to calculate slope of standard curve and PCR efficiency. e Quantification of e37a-Cacna1b mRNA in RNA isolated from CaMKIIα+PNs and CCK+INs. Data are shown as mean (filled symbols) ± SE, and individual values for each mouse (empty symbols). * p < 0.05

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