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View additional product information for TaqMan™ hPSC Scorecard™ Kit, 384-well - FAQs (A15872)
38 product FAQs found
No software update is required. Because the hPSC Scorecard Analysis Software is cloud based, you will always have access to the most up-to-date version of the software every time you log in.
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The only way to view the Ct values in the current version is through the expression plot. We expect the software's Excel export to include the gene names and Ct values in a future version of the software.
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A proprietary algorithm compares the Ct values for each marker set to the values in the reference database and calculates the score based on how well the expression correlates. In general, scores close to 0 indicate comparable expression to that of the reference standard using undifferentiated cells. Scores higher than 1 indicates up regulation relative to undifferentiated cells and less than -1 indicate down regulation relative to undifferentiated cells.
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Thirteen pluripotent stem cell lines were used to generate the reference standard including:
The hPSC Scorecard Analysis Software performs a set of quality control checks upon data import to ensure the quality of the results.
You may see one of the following red flag warnings:
No. The software analyzes and retains all of the qPCR data from the plate upon import. If you want to look at a different sample type, simply select the desired sample type from the drop down list, and the analysis software will automatically update the results.
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The software allows you to easily evaluate pluripotency and/or inductive differentiation one germ layer at a time. To make it a little easier, a set of common sample types have been already built into the software to allow you to get to your results faster:
The analysis software validates the data file upon import. If your file is not imported (or you receive an error), click the pencil icon to edit the project, confirm that you have the correct Instrument Type and Block designated in the project window, and verify that you have used one of the following TaqMan hPSC Scorecard catalog products: A15870, A15876, A15872, or A15871. The analysis software is compatible only with data obtained with those specific panels. If you see an error message that identifies that the experiment was not analyzed by the Ct method, follow the steps outlined below.
Click “Create a Project” to identify its Name, Instrument Type, Block, and Description, click “OK”, and then click “Upload Data” to start using the analysis software.
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The link for the software user guide can be found on this page.
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Retrieve your username or password using the appropriate links on the sign-in page. If you don't have a username and password, create a new account.
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Sign in using your current Thermo Fisher Scientific account information. New users to the site can quickly register by clicking the “Register for an Account” button under “Help and Support” option. Following sign in, you will be instantly redirected to the hPSC Scorecard analysis software.
The hPSC Scorecard Analysis Software is accessible through Thermo Fisher Connect and is compatible with the following web browsers:
The hPSC Scorecard Analysis Software currently only accepts data collected with the “Standard Curve” experiment type. To change your experiment type, open your data in the instrument software. In the “Set-up” menu, change your experiment type to “Standard Curve.” You may receive a warning message, but that is okay. After you change the experiment type, return to the Analysis menu and reanalyze the data by pressing the green “Analyze” button. After you save your data, you can import the file into the hPSC Scorecard Analysis Software.
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93 genes are tested per sample. There are 9 self-renewal genes, 74 lineage specific genes, and 10 housekeeping and control genes.
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Please visit the website link.
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We highly recommend that you use the template file because it contains all of the necessary experimental details for your qPCR run. If the template file is not working for you, please send an email to our stem cell technical support specialists at: stemcell-help@thermofisher.com.
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The template files contain all of the necessary experimental details for you to run your instrument. There is no need to modify any of the experimental details if you are using the template file (.edt). Step-by-step guides on how to run the qPCR instruments are available here.
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Template files are found here. Click on Instrument Compatibility to access these files.
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The hPSC Scorecard Panel analysis is also available as a service. You can send Thermo Fisher Scientific isolated RNA samples, cDNA samples, or even cells in TRIzol reagent. See Cellmodel Services for more information.
ViiA 7, QuantStudio 12K Flex, QuantStudio 7 Flex, QuantStudio 6 Flex, StepOnePlus, 7500 Fast, and 7900HT Fast Real-Time PCR systems are compatible with the hPSC Scorecard Panel. Apart from these, QuantStudio 3, QuantStudio 5, QuantStudio 6 Pro and QuantStudio 7 Pro are all able to run the panel, but require manually made templates and data exports to perform analysis. For more information, please contact Technical Support.
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The tips of most 16-channel pipettes will align with every well in each column of the plate. However, if your cDNA reactions were set up in 8 wells of a 96-well plate or in 8-well PCR strips, additional sample will be required to compensate for the dead volume. When you insert 2 tips of the 16-channel pipette into 1 well, the tips can't reach the bottom of the well, resulting in a need for additional dead volume.
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The tips of most 8-channel pipettes will align with every other well so you will need to pipette twice to load every well in each column of the plate. The first set of 8 samples can be loaded in rows A, C, E, G, I, K, M, and O. The second set of 8 samples can then be loaded into the alternate rows B, D, F, H, J, L, N, and P.
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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.
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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.
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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