A specific and quick gene expression study in mouse ES cells

A specific and quick gene expression study in mouse ES cells

Author Tavares, Rubens L. C. Google Scholar
Xu, Kangpu Google Scholar
Zhang, Chenyang Google Scholar
De Freitas, Vilmon Google Scholar
Institution Cornell Univ
Universidade Federal de São Paulo (UNIFESP)
Abstract Purpose To study gene expression at single embryonic stem cell colony levels with a new RT-PCR protocol.Methods Forty-five mouse ES cell colonies were retrieved at the 5th, 10th, 15th, 20th and 25th passages. the pluripotent state was analyzed for OCT-4 and Nanog, and beta-actin as a control for the presence of templates. RT-PCR was done using the SuperScript (TM) III CellsDirect cDNA Synthesis System. Every 2 or 3 days just before passage, a single colony was loaded into a 0.5 ml PCR tube containing 10 mu l of resuspension buffer using a pulled glass pipette.Results the RT-PCR protocol was completed in less then 150 min. All colonies were positive for OCT-4 and beta-actin and 42 out of 45 were positive for Nanog.Conclusions This protocol requires as little as 10 pg of total RNA starting material and is therefore useful for low cell number tissues, such as single stem cell colonies or preimplantation embryonic materials.
Keywords ES cells
gene expression
Language English
Date 2007-08-01
Published in Journal of Assisted Reproduction and Genetics. New York: Springer/plenum Publishers, v. 24, n. 8, p. 366-372, 2007.
ISSN 1058-0468 (Sherpa/Romeo, impact factor)
Publisher Springer
Extent 366-372
Origin http://dx.doi.org/10.1007/s10815-007-9141-x
Access rights Closed access
Type Article
Web of Science ID WOS:000249205700012
URI http://repositorio.unifesp.br/handle/11600/29902

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