2.2 Preparation of
ATN-RNA
2.2.1 In Vitro
Transcription of ATN-RNA
1. pT7/T3α18 plasmid harboring ATN-RNA sequence.
ATN-RNA was chemically synthesized form eight short
(approximately 40 nt) overlapping oligodeoxynucleotides:
four homologous to target fragment, and four complementary
to it. Complementary fragments overlap with a shift of 10 nt.
Resulting 5
0 overhangs contain sites recognized by restriction
enzymes HindIII for sense strand and EcoRI for template
strand.
2. T3 transcription kit (Thermo Fisher Scientific).
3. T7 transcription kit (Thermo Fisher Scientific).
4. Gel DNA purification kit.
5. DNA gel loading dye.
6. 10Â TBE buffer: 1 M Tris base, 1 M boric acid, 0.02 M EDTA,
in H 2 O.
7. 1% agarose gel.
8. Horizontal gel electrophoresis system.
9. Transilluminator.
2.2.2 dsRNA
Hybridization
1. Hybridization buffer: 20 mM Tris–HCl pH 7.5, 50 mM NaCl,
in H 2 O.
2. 10% PAGE with 7 M urea: 4 mL of 10Â TBE buffer, 10 mL of
20% acrylamide/bisacrylamide, 19.2 g of urea, up to 40 mL of
H 2 O.
3. RNA gel loading dye.
4. SYBR Safe.
5. Transilluminator.
2.3 MNP@PEI/dsRNA
Complex Preparation
1. ATN-RNA solution in water at a concentration of 100 ng/μL.
2. MNP@PEI solution in water at a concentration of 1000 μg
Fe/mL.
2.3.1 Gel
Retardation Assay
1. DNA gel loading dye.
2. 10Â TBE buffer: 1 M Tris base, 1 M boric acid, 0.02 M EDTA.
3. 1% agarose gel.
4. Horizontal gel electrophoresis system.
5. System for gel documentation.
6. Multidimensional images software (e.g., Digital Imaging and
Analysis System II, Serva).
2.3.2 UV–Vis
Spectrophotometry
1. Microvolume spectrophotometer (e.g. NanoDrop 2000,
Thermo Scientific).
Delivery of dsRNA to Glioblastoma Cells
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