title: Isolation and quantification of messenger RNA from tissue models by using a double-barrel carbon probe.
abstract: In this study, we introduce the double-barrel carbon probe (DBCP)-a simple, affordable microring electrode-which enables the collection and analysis of single cells independent of cellular positioning. The target cells were punctured by utilizing an electric pulse between the two electrodes in DBCP, and the cellular lysates were collected by manual aspiration using the DBCP. The mRNA in the collected lysate was evaluated quantitatively using real-time PCR. The histograms of single-cell relative gene expression normalized to GAPDH were fit to a theoretical lognormal distribution. In the tissue culture model, we focused on angiogenesis to prove that multiple gene expression analysis was available. Finally, we applied DBCP for the embryonic stem (ES) cell-derived cardiomyocytes to substantiate the capability of the probe to collect cells, even from high-volume samples such as spheroids. This method achieves high sensitivity for mRNA at the single-cell level and is applicable in the analysis of various biological samples independent of cellular positioning.
authors.affiliation: Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai, 980-8579, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nashimoto
authors.forename: Yasufumi
authors.initials: Y
authors.lastname: Takahashi
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Takano
authors.forename: Kosuke
authors.initials: K
authors.lastname: Miyashita
authors.forename: Shukuyo
authors.initials: S
authors.lastname: Yamada
authors.forename: Kosuke
authors.initials: K
authors.lastname: Ino
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Shiku
authors.forename: Tomokazu
authors.initials: T
authors.lastname: Matsue
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D004560:Electricity; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D020043:Genes, Essential; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000235:genetics / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D006801:Humans; D051379:Mice; D008839:Microelectrodes; D032383:Myocytes, Cardiac / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D015415:Biomarkers; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)