title: Expression analysis of rare cellular subsets: direct RT-PCR on limited cell numbers obtained by FACS or soft agar assays.
abstract: Since tissues and tumors are heterogenous populations containing different cell types, their transcriptomes are blends of multiple mRNA expression profiles. Although fluorescence-activated cell sorting (FACS) allows isolation of individual cell types, RNA isolation and quantification remain problematic from rare subsets, such as tissue stem cells. Likewise, identification of transcriptional changes relevant to the tumorigenic potential of mammalian cells while they are actively growing as colonies in soft agar is also hampered by limited amounts of starting material. Here we describe a convenient method that fills the gap between single cell and whole tissue mRNA analysis, enabling mRNA quantification for individual colonies picked from soft agar. Our method involves direct lysis, reverse transcription and quantitative PCR (RT-qPCR) on 500 sorted cells or a single soft agar colony, thus allowing evaluation of up to 20 transcripts in functionally distinct subpopulations without the need for RNA isolation or amplification.
authors.affiliation: Laboratory of Mammary Gland Biology, National Cancer Centre Singapore, Singapore.
authors.forename: Victor
authors.initials: V
authors.lastname: Ho
authors.forename: Shi Yun
authors.initials: SY
authors.lastname: Yeo
authors.forename: Kamini
authors.initials: K
authors.lastname: Kunasegaran
authors.forename: Duvini
authors.initials: D
authors.lastname: De Silva
authors.forename: Gerard A
authors.initials: GA
authors.lastname: Tarulli
authors.forename: P Mathijs
authors.initials: PM
authors.lastname: Voorhoeve
authors.forename: Alexandra M
authors.initials: AM
authors.lastname: Pietersen
mesh_terms: D000362:Agar / Q000737:chemistry; D000818:Animals; D001940:Breast / Q000166:cytology* / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics; D002452:Cell Count; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D012333:RNA, Messenger; D000362:Agar