Assay in vitro for oligodeoxynucleotide activity with E. coli RNase H against c-myc mRNA in total RNA isolated from KYO1 cells. Reaction mixtures containing 1 μg RNA and 0.5 U RNase H in 18 μl were incubated at 37°C for 30 min in the presence, where indicated, of 22 μM oligodeoxynucleotide. Following RT-PCR with c-myc-specific primers straddling the antisense target site, products were separated by 1.5% agarose / ethidium bromide gel electrophoresis and viewed under UV light. M, markers. The expected product size from uncleaved mRNA is 899 bp. Lane 11 was loaded with a no oligodeoxynucleotide, no reverse transcriptase control. Only lanes 3 and 4 loaded with 20-mer antisense replicates showed reductions in signal intensity consistent with significant RNase H-mediated cleavage of c-myc mRNA.
Tidd et al. BMC Molecular Biology 2001 2:13 doi:10.1186/1471-2199-2-13