In situ localization of c-myc mRNA in HL-60 cells using non-radioactive synthetic oligodeoxynucleotide probes.

To analyze specific mRNA at individual cell level, non-radioactive in situ hybridization has been a powerful technique. In situ hybridization is most commonly done using double-stranded cDNA probes. We have already reported the usefulness of thymine-thymine (T-T) dimerized double-stranded cDNA as a non-radioactive probe. In this study, we extended this approach to use synthetic oligodeoxynucleotide (oligo-DNA). Two oligo-DNAs (I; 65-mer, II; 57-mer) complementary to the different regions of the human c-myc mRNA were synthesized and ultraviolet (UV)-irradiated (7, 000J/m2). When the T-T dimerized oligo-DNAs were dot-blotted to nitrocellulose filters, at least 40pg of both oligo-DNAs was detected immunohistochemically using anti-T-T antibody. By dot-blot hybridization, about 80pg of sense strand of c-myc DNA was specifically detected with T-T dimerized c-myc oligo-DNA(I). When the oligo-DNA (II) was used as a probe, however, the sensitivity of signal detection was significantly lower. Furthermore, the c-myc antisense oligo-DNAs reacted with the total RNA from HL-60 cells, but not from normal rat liver. Finally, using these oligo-DNAs, c-myc mRNA was detected in HL-60 cells.