Human ESC Germ Layer PCR Kit

Human ESC Germ Layer PCR Kit

Chemicon (Millipore)

100 reactions

Millipore’s Human ESC Germ Layer PCR Kit enables researchers to quantitatively monitor the health of the starting undifferentiated human ESC population and to analyze the capacity of human ESC to differentiate into cell derivatives of the three germ layers.

Culturing human embryonic stem cells requires specialized training and meticulous and diligent care. Even in the most optimal in vitro conditions, some degree of spontaneous differentiation is evident in the human ES colonies. It has become a routine and essential part of culturing human ES cells to visually inspect and remove differentiated cell colonies to ensure the overall health and undifferentiated status of the ES cell population. Consequences for neglect can be dire and include decreased cell proliferation, low cell viability upon freeze-thawing and also decreased capacity of cells to differentiate into the three embryonic germ layers (endoderm, mesoderm and ectoderm). Although important, the use of visual inspection alone can be subjective and qualitative.

Stem Cell Culture

Millipore’s Human ESC Germ Layer PCR Kit enables researchers to quantitatively monitor the health of the starting undifferentiated human ESC population and to analyze the capacity of human ESC to differentiate into cell derivatives of the three germ layers. Provided in the kit are optimized and validated primer sets for two pluripotent markers (Nanog and Oct4), an endoderm marker (AFP), a mesoderm marker (-MHC), an ectoderm marker (Pax6) and a constitutive marker (GAPDH) along with control cDNAs from undifferentiated human ESC and from human ESC that have been differentiated as embryoid bodies (EB). Nanog and Oct4 are essential transcriptional factors that help maintain the pluripotency of ESCs (2). The down-regulation of Nanog and Oct4 expressions are closely associated with progression of differentiation. Expressions of AFP, cardiac muscle specific myosin heavy chain 6 (-MHC), and the neuronal marker, Pax6, indicate the presence of cells that have differentiated into endodermal, mesodermal and ectodermal lineages, respectively. AFP and Pax6 transcripts can be detected in differentiated embryoid bodies (EB) by RT-PCR by 7 days of differentiation (3, 4). The two cDNA controls are provided as useful bench-marks that can be used to measure the relative health and undifferentiated status of human ESC (ES cell cDNA control) and their capacity to give rise to cell derivatives of the three embryonic germ layers (EB cDNA control).

PCR conditions have been optimized and are provided. Millipore’s Human ESC Germ Layer PCR kit is compatible for use with cDNAs that have been generated from commercially available kits for RNA isolation and reverse transcription. Control cDNAs from undifferentiated human ESC and differentiated EBs were generated from reverse transcription of high-quality total RNA using oligo-dT as the primer. DNA primers have been validated to be specific to human ES cells and do not recognize and amplify nucleic acids from murine sources. Because the primers do not recognize murine nucleic acids, the kit can be used on cDNAs generated from human ESC that have been cultured on murine embryonic fibroblasts (MEF).

For Research Use Only; Not for use in diagnostic procedures

Mammals

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

• Human Embryonic Stem Cells
• Induced Pluripotent Stem Cells

Characterization

1. GAPDH primer set: (Part No. CS201372) 10 µM mixed primers, 50 µL, sufficient for 100 reactions.
2. Nanog primer set: (Part No CS201373) 10 µM mixed primers, 50 µL, sufficient for 100 reactions.
3. Oct4 primer set: (Part No CS201374) 10 µM mixed primers, 50 µL, sufficient for 100 reactions.
4. AFP primer set: (Part No CS201375) 10 µM mixed primers, 50 µL, sufficient for 100 reactions.
5. -MHC primer set: (Part No CS201376) 10 µM mixed primers, 50 µL, sufficient for 100 reactions.
6. PAX-6 primer set: (Part No CS201377) 10 µM mixed primers, 50 µL, sufficient for 100 reactions.
7. Embryonic Stem Cell (ES) cDNA Control: (Part No CS201378) From total RNA at 10 ng/µL, 90 µL
8. Embryoid Body (EB) cDNA Control: (Part No CS201379) From total RNA at 10 ng/µL, 90 µL
9. Sterile distilled water : (Part No 90442) 1.3 mL
10. 5x loading dye: (Part No CS201380) 500 µL

Chemicon

This kit is stable for 6 months upon date of receipt under proper storage (-20C). All of the reagents in the kit are provided in a ready-to-use format. Primers are supplied at optimal, validated concentrations. Primers and cDNA controls should be stored at -20C until use. Aliquot and freeze primers and cDNA controls to avoid repeated freeze-thaw cycles.

Human ESC Germ Layer PCR Kit

1.RNA isolation kit
2.Reverse transcriptase
3.Hot-start DNA polymerase.
4.Thin-wall PCR tubes
5.Agarose gel and electrophoresis apparatus and reagents