Skip to main content
. 2016 Oct 14;6:35406. doi: 10.1038/srep35406

Figure 1. Design and test of a CD46-isoform real-time PCR assay.

Figure 1

(a) Schematic representation of the mRNA for the four CD46 isoforms BC1, BC2, C1, and C2. The localization of primers and probes in the CD46-isoform real-time PCR assay is indicated on the figure. The fw primer is located in exon 6 and amplifies all isoforms, whereas the rev primers are located in either the exons 12-13 junction amplifying CYT-1-expressing isoforms or in the exons 12–14 junction amplifying the CYT-2-expressing isoforms. The hybridization probe detecting the BC isoforms is located in the exons 6–8 junction and the probe detecting the C isoforms is located in the exons 6–9 junction. (b) Efficiency of the real-time PCR assay. Equivalent amounts of cDNA from CHO cell lines each stably expressing the ORF from each of the four CD46 isoforms (BC1, BC2, C1 and C2) was mixed with cDNA from CHO wt cells (“Single isoform”) or cDNA from CHO cells expressing each of the other isoforms (“Pool isoforms”) and analysed by the real-time PCR assay to test the efficiency of the assay for each of the single isoform upon presence of all the other isoforms. The y-axis indicates the Ct value for the isoforms. Error bars indicate +SEM of two technical replicates and the data is representative of two independent experiments. (c) Specificity of the CD46-isoform real-time PCR assay. The four CHO cell lines each stably expressing the ORF from each of the four CD46 isoforms (BC1, BC2, C1, and C2, as indicated on the x-axes) were analysed by the real-time PCR assay to test for the specificity of the assay. The y-axes indicate the relative frequency of the four isoforms. Error bars indicate +SEM of three technical replicates.