Quantitation of Fc gamma RII mRNA in platelets and megakaryoblastic cell lines by a new method of in situ hybridization.
We have developed a highly sensitive and quantitative, non-isotopic method of in situ hybridization in which the level of probe binding to intracellular mRNA is determined using an ELISA based detection method. Highly purified cell preparations or cells from a cultured cell line are centrifuged into 96 well microtiter plates. The cells are fixed with formalin and pre-treated with Triton X-100 and Nonidet P40 before photobiotin labeled cDNA probes are applied. The biotin from the hybridization is detected using multiple applications of streptavidin and biotinylated alkaline phosphatase and then visualized by the p-NPP (p-nitrophenyl phosphate) conversion method. We have determined a number of the optimal parameters in the procedure including the effects of cell numbers per well, development times and standardization of data using ubiquitous beta-actin mRNA and poly-A+ RNA expression as controls. We have used the technique to study the level of expression of FcgR mRNA in platelets and precursors. We found that platelets and megakaryoblastic cell lines only express mRNA for Fc gamma RII. The presence of the Fc gamma RII molecules was confirmed by complementary studies using immunohistochemistry with specific monoclonal antibodies IV.3 and KB61.
Markovic
B
B
Department of Haematology, University of New South Wales, Prince of Wales Hospital, Sydney, Australia.
Wu
Z H
ZH
Chesterman
C N
CN
Chong
B H
BH
D001792:Blood Platelets / Q000737:chemistry*; D015152:Blotting, Northern; D002460:Cell Line; D018076:DNA, Complementary; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D008533:Megakaryocytes / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis* / Q000097:blood; D017452:Receptors, IgG / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D017452:Receptors, IgG