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View additional product information for TaqMan™ hPSC Scorecard™ Panel, Fast 96-well - FAQs (A15876)
15 product FAQs found
You can load samples using a single channel pipette, but this method is time-consuming and may increase pipetting error. We strongly recommend that you use an 8- or 16-channel multichannel pipette. You also can dispense samples using automated systems, with the understanding that additional sample will be required to compensate for the dead volume.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center
Setting up your cDNA synthesis in 8 wells of a 96-well plate or in 8-well PCR strips facilitates sample loading of the 96-well and 384-well hPSC Scorecard Panel plates.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center
Somatic non-pluripotent primary cells, such as the parental lines used for iPSC generation, are not pluripotent in nature and their scores will be low. However, the expression of lineage markers will largely rely on homogeneity of the cells. Note that the markers in the panel are designed to evaluate early germ layer specification and not any particular terminally differentiated state.
Minor differences in gene expression profiles are sometimes observed based on the reprogramming method, but in general the hPSC Scorecard analysis results do not change significantly for lines derived using various reprogramming methods. This was tested with ESC and iPS derived using episomal or Sendai-based reprogramming systems before and after seven days of spontaneous differentiation. Cells were grown in KSR-based media on irradiated MEFs prior to removal of FGF for EB formation.
The presence or absence of Sendai virus in established iPSC clones does not have an impact on pluripotency and hence on TaqMan hPSC Scorecard analysis results.
Sendai virus (SEV) is included in the TaqMan hPSC Scorecard kit and can detect the presence of the Sendai virus backbone. Note that this method will not distinguish between the different reprogramming factors but just the presence or absence of the residual virus in the cells. If an unexpected signal is detected, check the amplification curve to determine if it is a false positive. If it's a false positive, you may ignore the flag.
We recommend that you culture iPSC clones to at least passage 8-10 until they are stable and homogeneous, prior to hPSC Scorecard analysis. Early passage iPSC clones may give low self-renewal ("pluri" in v1.1 of the analysis software) scores or show higher expression of lineage genes.
H9 cells were differentiated into NSCs using Gibco PSC Neural Induction Medium (NIM) and hPSC Scorecard analysis was performed at various time points. The control sample was undifferentiated H9 ESC. Cells were seen to become positive for ectoderm by day 5.
You can perform directed differentiation according to your own methods. However, the time point when expression is noticeable will largely depend on the robustness of the methods. We recommend testing a few time points to monitor differentiation with time.
You can differentiate cells using any of the established methods. When using suspension embryoid bodies, we recommend that you allow at least 7 days for differentiation prior to analysis.
The TaqMan hPSC Scorecard kit/panel measures self-renewal and trilineage differentiation potential of PSCs and is not restricted by culture conditions. You should be aware that using novel media systems may have particular effects on the cells in terms of pluripotency and/or their differentiation. We recommend designing experiments that use novel media by including a control condition that utilizes traditional media or media in which expression patterns have been tested and confirmed. The most recent version of the hPSC Scorecard Analysis application offers a differentiation index plot for viewing gene expression in embryoid bodies (EBs) relative to the undifferentiated state.
We recommend removing feeders from your culture before proceeding with Scorecard analysis. To remove feeders, harvest your PSCs with collagenase, allow the colonies to settle by gravity sedimentation to reduce feeder-carryover, and re-seed cells in feeder-free conditions on Geltrex matrix-coated dishes and MEF conditioned medium for 1 to 2 passages. The presence of feeders can contribute to gene expression measured in the assay, thus altering the gene-signature pattern.
Yes. The TaqMan hPSC Scorecard kit/panel measures the potential for self-renewal and trilineage differentiation of PSCs grown on feeders or in feeder-free conditions.
While we recommend that you use about 0.5 million cells per experiment, or the number of cells equivalent to 1 well of a 6-well dish, you can use as few as 100,000 cells when performing RNA purification. If you perform a lysis protocol using Cells-to-CT or CellsDirect One-Step qPCR kits, as few as 15,000 cells is sufficient. Please note, however, that reducing cell number can compromise the quality of the results.
Panel configurations:
- Two 96-well (2 x 96w FAST) plates with optical plate covers
- One 384-well (384w) plate with optical plate covers
Kit configurations:
- Two 96-well (2 x 96w FAST) plates with optical plate covers & TaqMan Gene Expression Master Mix
- One 384-well (384w) plate with optical plate covers & TaqMan Gene Expression Master Mix