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  • Microarray data was validated using quantitative PCR

    2018-10-22

    Microarray data was validated using quantitative PCR (qPCR). This was first performed on a set of genes chosen for their wide variation in gene tnf alpha inhibitor between the three cell types (Prl2c2, GHR, Zyx, Des) (Supplementary Figure S2). qPCR was then performed to validate the expression of genes regarded as classical MSC (Wagner et al., 2005; Phinney et al., 2006) and/or pericyte markers (da Silva Meirelles et al., 2008; Covas et al., 2008) (Twist1, CCL7, MMP2, MIF, HoxA5, Tagln) (Muller et al., 2008) (Supplementary Figure S3). This demonstrated good validation of relative expression levels between microarray and qPCR. Microarray data was validated using quantitative PCR (qPCR). This was first performed on a set of genes chosen for their wide variation in gene expression between the three cell types (Prl2c2, GHR, Zyx, Des) (Supplementary Figure S2). qPCR was then performed to validate the expression of genes regarded as classical MSC (Wagner et al., 2005; Phinney et al., 2006) and/or pericyte markers (da Silva Meirelles et al., 2008; Covas et al., 2008) (Twist1, CCL7, MMP2, MIF, HoxA5, Tagln) (Muller et al., 2008) (Supplementary Figure S3). This demonstrated good validation of relative expression levels between microarray and qPCR.
    Enrichment for stem cell networks in all three MSC-like populations To investigate the stem cell nature of these three populations, the expression of murine orthologs of components of the recently described Plurinet was examined in these cell lines (Muller et al., 2008). Murine orthologs of the human Plurinet genes were examined for their mean normalised expression and 66% of these orthologs displayed an Illumina detection score >0 in at least one population (Supplementary Table 6). In addition, hierarchical clustering showed strong congruence between the three CFU-F populations (Figure 3B). Once again, a closer relationship was evident between bmCFU-F and kCFU-F. Figure 3C illustrates the subcellular localisation and network relationships of those Plurinet genes expressed by these populations and qPCR was used to validated these results for a subset of Plurinet genes detected as present in the three populations (Myc, AnxA2 and Smarcad1) (Supplementary Figure S4). We also investigated the expression of genes critical for induced pluripotency. Expression of Klf4 and Sox2 (not in the Plurinet), whilst above the threshold for detection by microarray, was very low (Supplementary Figure S4). Expression of both Nanog and Oct4 (Pou5f1) fell below the Illumina detection threshold for all cell lines. qPCR for these genes also indicated extremely low levels of mRNA (Supplementary Figure S4). Despite this, the extensive expression of other Plurinet genes implies an active stem cell state in all three MSC-like populations. To investigate the stem cell nature of these three populations, the expression of murine orthologs of components of the recently described Plurinet was examined in these cell lines (Muller et al., 2008). Murine orthologs of the human Plurinet genes were examined for their mean normalised expression and 66% of these orthologs displayed an Illumina detection score >0 in at least one population tnf alpha inhibitor (Supplementary Table 6). In addition, hierarchical clustering showed strong congruence between the three CFU-F populations (Figure 3B). Once again, a closer relationship was evident between bmCFU-F and kCFU-F. Figure 3C illustrates the subcellular localisation and network relationships of those Plurinet genes expressed by these populations and qPCR was used to validated these results for a subset of Plurinet genes detected as present in the three populations (Myc, AnxA2 and Smarcad1) (Supplementary Figure S4). We also investigated the expression of genes critical for induced pluripotency. Expression of Klf4 and Sox2 (not in the Plurinet), whilst above the threshold for detection by microarray, was very low (Supplementary Figure S4). Expression of both Nanog and Oct4 (Pou5f1) fell below the Illumina detection threshold for all cell lines. qPCR for these genes also indicated extremely low levels of mRNA (Supplementary Figure S4). Despite this, the extensive expression of other Plurinet genes implies an active stem cell state in all three MSC-like populations.