Flow cytometric quantitation of sequence-specific mRNA in hemopoietic cell suspensions by primer-induced in situ (PRINS) fluorescent nucleotide labeling.
We modified a technique of RNA detection based upon primed in situ labeling of RNA (PRINS) to quantitate poly(A) and histone mRNA in HL60 cell suspensions by flow cytometry. The direct PRINS technique was used to label poly(A) mRNA, based upon the incorporation of fluoresceinated nucleotides into cDNA chains generated by reverse transcription from the site of oligo(dT) primer-specific annealing on the poly(A) template. By this method, poly(A) fluorescent signals of up to 20-fold above background were obtained when primer concentrations were saturating. Nonspecific binding of the fluorochrome-conjugated nucleotide proved to be the greatest source of background fluorescence in cell suspensions. This was considerably reduced by preincubation of the cells in lysine and an excess of cold nucleotides prior to the PRINS reaction. The application of indirectly fluoresceinated antibodies to the FITC moiety of the conjugated nucleotide (indirect PRINS) enhanced detection of a fluorescent signal generated from cells by the direct PRINS technique after stringent histone priming. Cell cycle analysis, based upon DNA histograms obtained after PRINS labeling, showed the histone fluorescence to be primarily in S-phase cells of an exponentially growing population. Confocal laser scanning microscopy revealed good retention of cellular architecture, with cytoplasmic localization of poly(A) and histone mRNA fluorescence and heterogeneous mRNA expression among cells. Our modified PRINS labeling of RNA represents a useful advance in the precise quantitation of RNA species in cell suspension by flow cytometry.
Bains
M A
MA
Department of Haematology, University of Leicester, Leicester Royal Infirmary, United Kingdom.
Agarwal
R
R
Pringle
J H
JH
Hutchinson
R M
RM
Lauder
I
I
D005434:Flow Cytometry / Q000379:methods*; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006657:Histones / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D011061:Poly A / Q000032:analysis*; D012313:RNA / Q000032:analysis*; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D006657:Histones; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D012194:RNA-Directed DNA Polymerase