title: Selection of accurate reference genes in mouse trophoblast stem cells for reverse transcription-quantitative polymerase chain reaction.
abstract: Mouse trophoblast stem cells (TSCs) form colonies of different sizes and morphologies, which might reflect their degrees of differentiation. Therefore, each colony type can have a characteristic gene expression profile; however, the expression levels of internal reference genes may also change, causing fluctuations in their estimated gene expression levels. In this study, we validated seven housekeeping genes by using a geometric averaging method and identified Gapdh as the most stable gene across different colony types. Indeed, when Gapdh was used as the reference, expression levels of Elf5, a TSC marker gene, stringently classified TSC colonies into two groups: a high expression groups consisting of type 1 and 2 colonies, and a lower expression group consisting of type 3 and 4 colonies. This clustering was consistent with our putative classification of undifferentiated/differentiated colonies based on their time-dependent colony transitions. By contrast, use of an unstable reference gene (Rn18s) allowed no such clear classification. Cdx2, another TSC marker, did not show any significant colony type-specific expression pattern irrespective of the reference gene. Selection of stable reference genes for quantitative gene expression analysis might be critical, especially when cell lines consisting of heterogeneous cell populations are used.
authors: RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan.
authors: Kaori
authors: K
authors: Motomura
authors: Kimiko
authors: K
authors: Inoue
authors: Atsuo
authors: A
authors: Ogura
mesh_terms: D000818:Animals; D000071616:CDX2 Transcription Factor / Q000235:genetics; D002454:Cell Differentiation; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000235:genetics; D051519:Fibroblast Growth Factor 4 / Q000378:metabolism; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D005819:Genetic Markers; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D010936:Plant Extracts; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology*; D014157:Transcription Factors / Q000235:genetics; D014327:Trophoblasts / Q000166:cytology*
chemical_list: D000071616:CDX2 Transcription Factor; C487514:Cdx2 protein, mouse; D004268:DNA-Binding Proteins; C500759:Elf5 protein, mouse; C496445:FGF4 protein, human; D051519:Fibroblast Growth Factor 4; D005819:Genetic Markers; D010936:Plant Extracts; C510402:RN 180; D011994:Recombinant Proteins; D014157:Transcription Factors; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)