Development of flow cytometric method to detect antisense oligonucleotides delivery into human platelets: the significance in thrombocytopenia]

Antisense oligonucleotides act to hybridize with RNA in a sequence-related manner and to modulate the selective translation in living cells. Antisense oligonucleotides are expected to be potential therapeutic agents toward desired molecular targets. However, little efforts have been made to realize the use of antisense oligonucleotides as powerful laboratory markers for detecting selective transcriptional levels. We describe here that FITC-labeled antisense for 18S ribosomal RNA were delivered into human platelets which were countable with flow cytometry. In healthy volunteers, the FITC positive platelets were 2% of PECD42b positive platelets. The rate was not affected by reaction time of 20 to 360 min and by reaction temperature at 4, 37 degrees C, or room temperature. The FITC positive rate was unchanged in final concentrations of both 10(-6) and 10(-5) M of FITC-labeled antisense. However, the rate was three times higher in patients with thrombocytopenia. There was a significant negative relationship between platelet count and FITC positive rate. FITC-labeled antisense for c-Mpl mRNA were more sensitive than that for 18S ribosomal RNA. These results suggest that fluorescence-labeled antisense oligonucleotides might be potential laboratory tool to reveal the distribution of RNA containing cells in the population.