Abstract
Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo.
Highlights
During mammalian pre-implantation development a series of asynchronous divisions result in the formation of the blastocyst
Our results suggest that normalization of Quantitative reverse transcription-polymerase chain reaction (qPCR) data using the geometric means of the transcripts listed above will yield the most accurate quantification of gene expression between these three unique stem cell types
Our results indicate normalizing quantitative RTPCR measurements using the geometric mean CT values obtained for the Pgk1, Sdha and Tbp mRNAs, offers the most reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo
Summary
During mammalian pre-implantation development a series of asynchronous divisions result in the formation of the blastocyst. A novel statistical algorithm, (geNORM) Vandesompele and colleagues demonstrated that the geometric mean of three reference genes provided the most accurate and reliable means of normalizing qPCR expression data [13]. This experimental strategy has been validated and additional algorithms written and utilized to identify the most suitable reference genes for a variety of experimental conditions [14,15,16,17,18,19,20,21,22,23,24,25]
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