Induced Apoptosis Investigation in Wild-type and FLT3-ITD Acute Myeloid Leukemia Cells by Nanochannel Electroporation and Single-cell qRT-PCR

摘要

Nanochannel electroporation (NEP) was applied to deliver precise dosages of myeloid cell leukemia-1 (Mcl-1)-specific siRNA and molecular beacons to two types of acute myeloid leukemia (AML) cells, FMS-like tyrosine kinase-3 wild-type (WT) and internal tandem duplications (ITD) type at the single-cell level. NEP, together with single-cell quantitative reverse transcription PCR, led to an observation showing nearly 20-folds more Mcl-1 siRNA than MCL1 mRNA were required to induce cell death for both cell lines and patient blasts, i.e., similar to 8,800 siRNAs for similar to 500 +/- 50 mRNAs in ITD cells and similar to 6,000 siRNAs for similar to 300 +/- 50 mRNAs in WT cells. A time-lapse study revealed that >75% MCL1 mRNA was down-regulated within 1 hour after delivery of a small amount of siRNA. However, additional siRNA was required to inhibit the newly transcribed mRNA for >12 hours until the cell lost its ability of self-protection recovery. A multidelivery strategy of low doses and short delivery interval, which require 77% less siRNA and has the potential of lower side effects and clinical cost, was as effective as a single high-dose siRNA delivery. Our method provides a viable analytical tool to investigate gene silencing at the single-cell level for oligonucleotide-based therapy.