Taqman hpsc scorecard panel

Manufactured by Thermo Fisher Scientific

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The TaqMan hPSC Scorecard panel is a molecular biology tool designed for the comprehensive assessment of human pluripotent stem cell (hPSC) identity and potency. The panel utilizes real-time PCR technology to evaluate the expression levels of a set of marker genes that are indicative of the pluripotent state and differentiation potential of hPSCs.

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15 protocols using taqman hpsc scorecard panel

Pluripotency and Differentiation Assessment of iPSC Lines

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Applied Biosystems TaqMan®hPSC Scorecard™ Panel (Thermo Fisher Scientific) was used as an additional technique to assess pluripotency and tri-lineage differentiation potential of iPSC lines using real-time qPCR assays (Fig. 1E, Supplementary Figs. 2–24E). Total RNA from undifferentiated and EB differentiated iPSC lines was isolated using MagMAX™ mirVana™ Total RNA Isolation Kit (A27828), and 1 μg of RNA was used to make cDNA using the High Capacity cDNA Reverse Transcription Kit (4368813), both from Applied Biosystems. TaqMan qRT-PCR was carried out using the hPSC Scorecard 384w Fast plate (Life technologies, A15870) and QuantStudio 12 k Flex, following manufacturer protocol. We analysed the gene expression data from the TaqMan®hPSC Scorecard™ Panel using the web-based hPSC Scorecard™ Analysis Software (Thermo Fisher Scientific).

Toombs J., Panther L., Ornelas L., Liu C., Gomez E., Martín-Ibáñez R., Cox S.R., Ritchie S.J., Harris S.E., Taylor A., Redmond P., Russ T.C., Murphy L., Cooper J.D., Burr K., Selvaraj B.T., Browne C., Svendsen C.N., Cowley S.A., Deary I.J., Chandran S., Spires-Jones T.L, & Sareen D. (2020). Generation of twenty four induced pluripotent stem cell lines from twenty four members of the Lothian Birth Cohort 1936. Stem Cell Research, 46, 101851.

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Pluripotency Evaluation of iPSC Lines

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The Scorecard analysis was performed as outlined in Figure 4:

CJ01 and CJ02 iPSC lines and the H9 hESC line were cultured confluent in six-well plates and the colonies were collected using cell scrapper and cultured in EB media (DMEM/F12, 20% KnockOut Serum Replacement, 1 mM Non-Essential Amino Acids Solution, 55 μM β-Mercaptoethanol).

The cell suspensions were plated on non-adhesive six-well plates for seven days. Media was changed every two days.

On the seventh day, the EB’s suspension was collected and RNA was extracted using RNeasy-Plus Mini-kit (QIAGEN).

Synthesize of the cDNAs was performed using the SuperScript IV First-Strand Synthesis system (Invitrogen).

The cDNAs were used in the TaqMan hPSC Scorecard Panel (ThermoFisher Scientific) with TaqMan Fast Advanced Master Mix (ThermoFisher Scientific) as per the manufacturer’s protocol.

Data showing the relative levels of self-renewal genes, mesodermal genes, ectoderm, and endodermal genes are analyzed using the cloud-based software provided with the Scorecard.

Analyzed data shown a pass or fail results of the pluripotency test, indicating whether the cell line is pluripotent or biased to any of the germ layers.

Yang G., Hong H., Torres A., Malloy K.E., Choudhury G.R., Kim J, & Daadi M.M. (2018). Standards for Deriving Nonhuman Primate-Induced Pluripotent Stem Cells, Neural Stem Cells and Dopaminergic Lineage. International Journal of Molecular Sciences, 19(9), 2788.

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Evaluating Differentiation Potential of hPSCs

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The differentiation potential of subcultured hPSCs was estimated by forming embryoid bodies. To form embryoid bodies, hPSCs were first treated with CTK solution consisting of 1 mg/ml collagenase IV (Life Technologies), 0.25% trypsin (Life Technologies), 1 mM CaCl₂, and 20% knockout serum replacement. The cells were detached as clumps using a cell scraper and then cultured as a suspension in petri dishes containing hESC medium without fibroblast growth factor-2. The medium was changed every 2 days. After 14 days, total RNA was extracted using an RNeasy Micro Kit (Qiagen), and the expression levels of differentiation markers were estimated by quantitative PCR. The quantitative PCR was performed according to the instructions of the TaqMan hPSC Scorecard Panel (ThermoFisher Scientific). Expression of differentiation marker genes in embryoid bodies was evaluated by comparing the single data set of the TaqMan hPSC Scorecard Panel against that in undifferentiated hPSCs.

Miyazaki T., Isobe T., Nakatsuji N, & Suemori H. (2017). Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner. Scientific Reports, 7, 41165.

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Assessing Pluripotency of hPSC Lines

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Functional pluripotency of each hPSC line used in this study was assessed using the Applied Biosystems TaqMan hPSC Scorecard Panel (Thermo Fisher Scientific). Total RNA from undifferentiated hPSCs and EBs differentiated for 14 days was isolated using the Qiagen RNeasy Plus Mini Kit (Qiagen), and 1 μg RNA was reverse transcribed to cDNA using the Super-Script VILO cDNA Synthesis Kit (Thermo Fisher Scientific). TaqMan qRT-PCR was carried out using the hPSC Scorecard 384w Fast Plate (Thermo Fisher Scientific, A15870) on a QuantStudio 7 qPCR device following the manufacturer’s recommended protocol. Gene expression data from the Scorecard Panel were analyzed using the web-based hPSC Scorecard Analysis Software (Thermo Fisher Scientific).

Pitstick A.L., Poling H.M., Sundaram N., Lewis P.L., Kechele D.O., Sanchez J.G., Scott M.A., Broda T.R., Helmrath M.A., Wells J.M, & Mayhew C.N. (2022). Aggregation of cryopreserved mid-hindgut endoderm for more reliable and reproducible hPSC-derived small intestinal organoid generation. Stem Cell Reports, 17(8), 1889-1902.

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Quantitative RT-PCR for Gene Expression Analysis

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Total RNA was isolated using the RNeasy Plus Mini Kit (QIAGEN GmbH). Single-stranded cDNA was synthesized from 0.1 to 2 μg of total RNA in 20 μL reactions containing random primers using a Superscript III or IV First Strand cDNA Synthesis System (Thermo Fisher Scientific). For qRT–PCR, we used SYBR Green–based assays (Luna Universal qPCR Master Mix; NEB). Transcript levels were determined using a QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific). All qRT–PCR reactions using SYBR Green were conducted in triplicate or quadruplicate, and relative quantification was performed using GAPDH as a reference gene. In addition, we ran multiple gene expression assays with EBs derived from wild-type and hsa-miR-302/367 heterozygous knockout iPSCs by qRT–PCR, using TaqMan™ hPSC Scorecard™ Panel (Thermo Fisher Scientific).

Sugawara T., Kawamoto Y., Kawasaki T., Umezawa A, & Akutsu H. (2022). A single allele of the hsa-miR-302/367 cluster maintains human pluripotent stem cells. Regenerative Therapy, 21, 37-45.

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Comprehensive Transcriptomics and Genomics Analysis

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RNA was isolated using an RNeasy kit (Qiagen) and reverse transcription was performed using SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Quantitative RT-PCR was performed using SYBR® Green PCR Master Mix (Applied Biosystems™) on the 7500 Step OnePlus™ Real-Time PCR System (Applied Biosystems^TM). All samples were run in triplicate and relative quantification was done using the ΔΔCt method with normalization to GAPDH. A list of primers can be found in Supplementary Table 2. Genomic DNA was isolated using a DNeasy kit (Qiagen) and sequencing was performed by LGC Genomics (Teddington, UK). The TaqMan® hPSC Scorecard™ Panel (Life Technologies—A15870—HPSC scorecard panel 384) kit was used to determine the expression of markers from the three germ layers.

Guo W., Naujock M., Fumagalli L., Vandoorne T., Baatsen P., Boon R., Ordovás L., Patel A., Welters M., Vanwelden T., Geens N., Tricot T., Benoy V., Steyaert J., Lefebvre-Omar C., Boesmans W., Jarpe M., Sterneckert J., Wegner F., Petri S., Bohl D., Vanden Berghe P., Robberecht W., Van Damme P., Verfaillie C, & Van Den Bosch L. (2017). HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients. Nature Communications, 8, 861.

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Quantitative Analysis of Pluripotency Markers

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Total RNA was extracted using an RNeasy micro kit (74004, QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. One μg of total RNA was used to synthesize cDNA with the QuantiTect Reverse Transcription Kit (205311, QIAGEN). Quantitative PCR (qPCR) was performed with TaqMan hPSC Scorecard Panel (A15870, Life Technologies)[19 (link)], using a qRT-PCR device (QuantStudio 12K Flex, Life Technologies).

Takenaka C., Miyajima H., Yoda Y., Imazato H., Yamamoto T., Gomi S., Ohshima Y., Kagawa K., Sasaki T, & Kawamata S. (2015). Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes. PLoS ONE, 10(6), e0129855.

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Quantitative Real-Time PCR for Gene Expression

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Relative gene expression was determined using a two-step quantitative real-time PCR method. Total RNA was isolated with the RNeasy Isolation kit with on-column DNase I treatment (Qiagen) and reverse-transcribed using the cDNA Synthesis Kit (Quanta biosciences). Quantitative reverse transcription PCR (RT–PCR) was performed with the Quanta SYBR Green Supermix (Quanta biosciences) on the ABI Prism 7500 Real-Time PCR System (Applied Biosystems). Fold changes in gene expression were determined using the comparative C_T method (ΔΔCt) with normalization to the housekeeping genes GAPDH or B2M. The primer sequences used in the study are shown in Supplementary Table 2. The TaqMan hPSC Scorecard panel was used to assess pluripotency and trilineage differentiation potential according to manufacturer's instructions (Life Technologies). Data analysis was performed using the hPSC Scorecard software (Life Technologies).

Karakikes I., Stillitano F., Nonnenmacher M., Tzimas C., Sanoudou D., Termglinchan V., Kong C.W., Rushing S., Hansen J., Ceholski D., Kolokathis F., Kremastinos D., Katoulis A., Ren L., Cohen N., Gho J.M., Tsiapras D., Vink A., Wu J.C., Asselbergs F.W., Li R.A., Hulot J.S., Kranias E.G, & Hajjar R.J. (2015). Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy. Nature Communications, 6, 6955.

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Characterization of TDP-43 iPS Cell Lines

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Novel iPS cell lines were characterized in this study:
TDP-43_A315T and TDP-43_G298S. Dermal biopsies were
obtained from 2 familial ALS patients with mutations in the
TARDBP locus. From these explants, fibroblasts were
generated and expanded in KO-DMEM (Life Technologies™) supplemented with
10% FBS (Hyclone™). TDP-43 iPS cells were derived by transduction of
fibroblasts with retroviruses expressing OCT4, SOX2 and
KLF4. After 3-4 weeks, primary iPS cell colonies were
picked based on morphology and independently expanded in mTeSR™1 medium
(STEMCELL Technologies) to generate 4-6 different iPS cell lines/patient. The
presence of the mutations was confirmed by PCR amplification of a genomic region
surrounding Exon 6, followed by Sanger DNA sequencing. The
pluripotency of the iPS cell lines used was validated using the TaqMan®
hPSC Scorecard™ Panel (Life Technologies™). Expression of
transcription factors and cell surface antigens characteristic of the
pluripotent state was confirmed using immunocytochemistry. Primary antibodies
and dilution factors used for this purpose were NANOG (1:100, R&D), OCT3/4
(1:500, Santa Cruz), SSEA-4 (1:1000, Santa Cruz) and TRA-1-81 (1:1000,
Millipore).

Alami N.H., Smith R.B., Carrasco M.A., Williams L.A., Winborn C.S., Han S.S., Kiskinis E., Winborn B., Freibaum B.D., Kanagaraj A., Clare A.J., Badders N.M., Bilican B., Chaum E., Chandran S., Shaw C.E., Eggan K.C., Maniatis T, & Taylor J.P. (2014). Axonal transport of TDP-43 mRNA granules in neurons is impaired by ALS-causing mutations. Neuron, 81(3), 536-543.

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Comprehensive hPSC Transcriptome Profiling

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Total RNA was extracted from frozen cell pellets using Ambion Pure Link RNA Mini Kit (Life Technologies). Eight cDNA reactions were set-up from 1μg of total RNA per sample using High-Capacity cDNA RT Kit (Life Technologies). qPCR was performed on 384-well TaqMan hPSC Scorecard plates using Viia7 RUO software and Applied Biosystems ViiA7 instrument. The qPCR assay used in this study is commercially available as the TaqMan hPSC Scorecard panel (Life Technologies).

Tsankov A.M., Akopian V., Pop R., Chetty S., Gifford C.A., Daheron L., Melton D.A., Tsankova N.M, & Meissner A. (2015). An improved ScoreCard to assess the differentiation potential of human pluripotent stem cells. Nature biotechnology, 33(11), 1182-1192.

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