title: Prolonged hybridization with a cRNA probe improves the signal to noise ratio for in-tube in situ hybridization for quantification of mRNA after fluorescence-activated cell sorting.
abstract: We developed an in-tube in situ hybridization method for mRNA quantification after fluorescence-activated cell sorting (FACS-mQ). A specific RNA in a particular cell type is stained with a cRNA probe and a fluorescent dye, which allows the stained cells to be selected by FACS without excessive RNA degradation. Our previous protocol required 4 h for hybridization with a cRNA probe, which might not produce enough fluorescence signal for sorting genes with low expressions. We determined the effect of prolonged hybridization for in-tube in situ hybridization on quantitative measurement of intracellular RNAs. During the hybridization step, the quantity of ACTB mRNA decreased gradually until 4 h, but remained constant from 4 to 16 h below 63.6° C. For flow cytometry, cells hybridization with cRNA probes for TG mRNA at 60° C for 16 h showed both increased signal and decreased background fluorescence compared to those hybridized for 4 h. These results indicate that when performing in-tube in situ hybridization, hybridization temperature can be raised to 63.6° C and the hybridization step can be extended up to 16 h without excessive intracellular RNA degradation.
authors.affiliation: Department of Laboratory Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565 - 0871, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Yamada
authors.forename: N
authors.initials: N
authors.lastname: Yamakawa
authors.forename: M
authors.initials: M
authors.lastname: Watanabe
authors.forename: Y
authors.initials: Y
authors.lastname: Hidaka
authors.forename: Y
authors.initials: Y
authors.lastname: Iwatani
authors.forename: T
authors.initials: T
authors.lastname: Takano
mesh_terms: D000818:Animals; D002460:Cell Line; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry*; D017403:In Situ Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction; D018075:RNA, Complementary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D059629:Signal-To-Noise Ratio
chemical_list: D005456:Fluorescent Dyes; D018075:RNA, Complementary; D012333:RNA, Messenger