Design and synthesis of anti-HBV siRNA
SiRNA targeting X-gene of HBV was selected from a study by Wooddell et
al. (siHBV74)64. PCR product encoding siRNA under T7
promoter was synthesized using Q5 polymerase and following primers:
fw_AAGCTAATACGACTCACTATAGGGACCAATTTATGCCTACAGCCttcaagagaGGCTGT,
rev_AGACATAAAAAACAAAAAAAGACCAATTTATGCCTACAGCCtctctt. Mock siRNA was
synthesized with primers fw_AAGCTAATACGACTCACTATAGGTTCTCCGAAC and
rev_AGACATAAAAAACAAAAAAAAAACGTGACACGTTCGGAGAAC. T7 containing PCR
product was used as a template for in vitro transcription (IVT) reaction
using the HiScribe Quick T7 High Yield RNA synthesis kit (NEB) according
to the manufacturer’s protocol. The IVT reaction was incubated overnight
and then treated with DNAse I (NEB) for 15 min at 37°C followed by
isopropanol purification. In brief, isopropanol and 0.5 M NaCl were
added to the reaction mixture and centrifuged for 30 min. Next, the
pellet was consecutively washed twice with 70% and 95% ethanol. The
air-dried pellet was resuspended in RNase-free water and stored at
-80°C.