title: Selection of stable reference genes for quantitative rt-PCR comparisons of mouse embryonic and extra-embryonic stem cells.
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.
authors.affiliation: College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States of America.
authors.forename: Kylee J
authors.initials: KJ
authors.lastname: Veazey
authors.forename: Michael C
authors.initials: MC
authors.lastname: Golding
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism*; D001755:Blastocyst / Q000166:cytology; D002454:Cell Differentiation; D019070:Cell Lineage; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology; D004707:Endoderm / Q000166:cytology; D018507:Gene Expression Regulation, Developmental; D005796:Genes / Q000502:physiology*; D051379:Mice; D012333:RNA, Messenger / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology
chemical_list: D015415:Biomarkers; D012333:RNA, Messenger
references.citation: J Biol Chem. 2004 Mar 5;279(10):9424-31
references.pmid: 14672929
references.citation: Genes Dev. 2011 Mar 15;25(6):594-607
references.pmid: 21357675
references.citation: Proc Natl Acad Sci U S A. 2007 Aug 7;104(32):12955-61
references.pmid: 17670940
references.citation: Biochem Biophys Res Commun. 2008 Sep 12;374(1):106-10
references.pmid: 18602371
references.citation: J Clin Invest. 2010 Sep;120(9):3340-9
references.pmid: 20679726
references.citation: Methods. 2001 Dec;25(4):402-8
references.pmid: 11846609
references.citation: Development. 2005 Apr;132(7):1649-61
references.pmid: 15753215
references.citation: RNA. 2010 Feb;16(2):450-61
references.pmid: 20040593
references.citation: BMC Dev Biol. 2005 Dec 03;5:27
references.pmid: 16324220
references.citation: Cancer Res. 2004 Aug 1;64(15):5245-50
references.pmid: 15289330
references.citation: Clin Chem. 2009 Apr;55(4):611-22
references.pmid: 19246619
references.citation: Genome Biol. 2002 Jun 18;3(7):RESEARCH0034
references.pmid: 12184808
references.citation: Development. 2004 Aug;131(15):3727-35
references.pmid: 15240554
references.citation: Mol Cell. 2004 Nov 19;16(4):655-61
references.pmid: 15546624
references.citation: Genes Dev. 1998 Jul 1;12(13):2048-60
references.pmid: 9649508
references.citation: Development. 2011 Sep;138(17):3667-78
references.pmid: 21775415
references.citation: Stem Cells. 2010 Jun;28(6):1030-8
references.pmid: 20506138
references.citation: Proc Natl Acad Sci U S A. 2007 Jan 9;104(2):525-30
references.pmid: 17194760
references.citation: Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8424-8
references.pmid: 8378314
references.citation: Science. 2007 Jan 5;315(5808):97-100
references.pmid: 17204650
references.citation: J Mol Neurosci. 2009 Mar;37(3):238-53
references.pmid: 18607772
references.citation: Development. 2009 Mar;136(5):701-13
references.pmid: 19201946
references.citation: Dev Cell. 2004 Aug;7(2):155-64
references.pmid: 15296713
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13344-9
references.pmid: 14581617
references.citation: Nat Protoc. 2008;3(6):1101-8
references.pmid: 18546601
references.citation: Stem Cells. 2009 Jan;27(1):49-58
references.pmid: 18832594
references.citation: Science. 1998 Dec 11;282(5396):2072-5
references.pmid: 9851926
references.citation: Nat Struct Mol Biol. 2004 Sep;11(9):822-9
references.pmid: 15300239
references.citation: BMC Dev Biol. 2007 Mar 06;7:14
references.pmid: 17341302
references.citation: Hum Mol Genet. 2008 Jun 1;17(11):1653-65
references.pmid: 18287259
references.citation: Plant Biotechnol J. 2008 Aug;6(6):609-18
references.pmid: 18433420
references.citation: Nat Struct Mol Biol. 2004 Jul;11(7):650-5
references.pmid: 15195148
references.citation: Nature. 1988 Dec 15;336(6200):684-7
references.pmid: 3143916
references.citation: Int J Dev Biol. 2006;50(7):627-35
references.pmid: 16892176
references.citation: Biotechnol Lett. 2004 Mar;26(6):509-15
references.pmid: 15127793
references.citation: Biol Reprod. 2009 Aug;81(2):362-70
references.pmid: 19403927
references.citation: Hum Mol Genet. 2010 Jan 1;19(1):36-51
references.pmid: 19805400
references.citation: Biomed Biochim Acta. 1983;42(11-12):S263-7
references.pmid: 6689547
references.citation: Biotechniques. 2006 Feb;40(2):173-7
references.pmid: 16526406
references.citation: Proc Natl Acad Sci U S A. 1981 Dec;78(12):7634-8
references.pmid: 6950406
references.citation: Mol Cell Biol. 2000 Oct;20(20):7419-26
references.pmid: 11003639
references.citation: J Cell Biol. 1995 Jul;130(2):393-405
references.pmid: 7615639
references.citation: Nat Struct Mol Biol. 2004 Sep;11(9):816-21
references.pmid: 15300240
references.citation: J Embryol Exp Morphol. 1975 Jul;33(4):991-1001
references.pmid: 1176885
references.citation: Cell Stem Cell. 2010 May 7;6(5):457-67
references.pmid: 20452320
references.citation: Development. 2005 May;132(9):2093-102
references.pmid: 15788452
references.citation: Development. 1990 Nov;110(3):815-21
references.pmid: 2088722