Detection of messenger RNAs within single hemopoietic cells by in situ hybridization on small slide areas.
In situ hybridization provides a powerful tool to detect specific mRNA sequences at the cellular level. We have applied a modified in situ hybridization technique using specifically prepared regular glass microscope slides to evaluate mRNA levels in cells of small samples. Cells were derived from in vitro colonies or isolated by fluorescence-activated cell sorting and deposited on the slides. These slides were coated with polysiloxane, sparing small circular areas where adherent cells attach and can be grown directly; after preincubation of the collection areas with fibronectin, the slides can also be used to deposit and to grow nonadherent cells. In situ hybridization was performed with 35S-labeled probes. Acetylation of the slides and the cells prior to hybridization, the addition of vanadyl-ribonucleoside complexes, and a prehybridization step were found to be necessary to optimize signal-to-noise ratios, as shown by evaluation of c-myc-specific mRNA in phytohemagglutinin-stimulated T4-lymphocytes. This technique might be very useful to study mRNA expression in small samples of hemopoietic cells.
Kanz
L
L
Medizinische Universitätsklinik, Freiburg, Federal Republic of Germany.
Mielke
R
R
Löhr
G W
GW
Fauser
A A
AA
D002469:Cell Separation; D006412:Hematopoietic Stem Cells / Q000032:analysis*; D006801:Humans; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis*
D012333:RNA, Messenger