An example of the display of transcript analysis data with biosynthetic pathways. A schematic representation of the biosynthesis of various classes of glycolipids is shown in the upper panel (including a key for glycan components in the pathway). Linkages are shown for each step of the biosynthetic pathway and the numbers in the ovals designate the pathway steps in the upper panel that link to the transcript abundance data in the corresponding numbered step in the lower panel (plotted as a histogram on a log₁₀ scale). Relative transcript abundances for undifferentiated mES and differentiated EB or ExE cell types are presented as a clustered set of histograms above the corresponding pathway step number (numbered oval) and gene names. Multiple genes for a given pathway step are listed in cases where multiple distinct subunits contribute to catalysis or where several genes within a common family encode enzymes capable of creating the specified linkage. Error bars represent one standard deviation from the mean for four independent biological replicates. Asterisks identify instances where changes in transcript abundance between mES and EB or ExE were statistically significant (p < 0.05) using a Mann–Whitney test. The center panel represents fold change in transcript abundance relative to undifferentiated mES. The center horizontal black bar represents no change between samples and positive values indicate an increase in transcript abundance in differentiated cells while negative values indicate a decrease.

Relative transcript abundance for stem cell marker genes in BG01 human ESCs during a time course differentiation into a mesoderm lineage. Relative transcript abundance for stem cell pluripotency, ME and mesoderm marker genes (noted by brackets) is plotted on a log₁₀ scale. Pluripotent BG01 line human ESCs were treated with specific cytokines and growth factors (Dr Steve Dalton, personal communication) to induce differentiation toward a mesoderm lineage. Cells were harvested at various time points, flash-frozen and isolated RNA was converted into cDNA to use as template for qRT-PCR analysis with gene-specific primers. Error bars represent one standard deviation from the mean for triplicate reactions.

Primer validation for qRT-PCR. Panel A: Primer pairs were analyzed by qRT-PCR using genomic DNA (gDNA) as template and Ct values were compared to Rpl4 as reference. Primer sets that generated Ct values with ±2 Ct units of Rpl4 (shaded area) were considered acceptable for further validation. Primer pairs falling outside the acceptable range are indicated with an asterisk. Panel B: Typical amplification curve generated with gene-specific primers and mouse gDNA as template. The threshold for determining Ct values is indicated by the dotted line. The baseline fluorescence trace expected from a “no template control” (NTC) is also indicated on the graph. Panel C: Typical standard curve generated by qRT-PCR of a given primer pair to determine amplification efficiency using a dilution series of mouse gDNA as template. The solid line indicates the linear regression for the data points at each template concentration. The dashed and dotted lines indicate the lower (90%) and upper (110%) efficiency limits, respectively, for primer validation. Panel D: Melt curve analysis of qRT-PCR amplimers generated following the standard curve reactions in shown in Panel C indicating a single sharp peak (single product) formed during the amplification. Panel E: Melt curve analysis of standard curve reactions for primers that failed our quality control validation illustrating the presence of multiple products (peaks) in an amplification reaction. This research was originally published as a supplementary figure in Nairn et al. (2008)©. The American Society of Biochemistry and Molecular Biology.

Relative transcript abundance for stem cell marker genes in four different human embryonic stem cell lines (BG01, BG02, H7, and H9). Relative transcript abundance for stem cell pluripotency and differentiation marker genes (noted by brackets) is plotted on a log₁₀ scale. Cells were harvested, flash-frozen and isolated RNA was converted into cDNA to use as template for qRT-PCR analysis with gene-specific primers. Error bars represent one standard deviation from the mean for triplicate reactions.