3.2 Preparation of
ATN-RNA
3.2.1 In Vitro
Transcription of ATN-RNA
1. Digest 50 μg of plasmid harboring ATN-RNA construct separately with restriction enzymes EcoRI and HindIII.
2. Carry out the digestion process by minimum 8 h or overnight.
3. Separate the whole reaction on the 1% agarose gel and examine
it on the transilluminator.
4. From agarose gel cut out the digested form of plasmid and
purify the plasmid from the gel using gel DNA purification kit.
5. Independently transcribe the two strands of RNA with T3 and
T7 polymerases for 1 μg of EcoRI and HindIII digested plasmids, respectively. Incubate the reaction overnight.
6. Purify the reaction loading whole mixture onto spin column
plugged into sterile 1.5 mL collection tubes, then centrifuge at
750 Â g, 2 min.
7. Apply 20 μL of nuclease-free H 2 O on spin tubes and repeat
centrifugation to new collection tubes.
8. Store transcripts in À20
C.
3.2.2 dsRNA
Hybridization
1. Mix equal quantities of T3 and T7 transcripts. Add 20 μL of 5Â
hybridization buffer and fill up with H 2 O to 100 μL.
2. Incubate: 95
C, 3 min, then follow the incubation for 75
C,
30 min.
3. Slowly cool down the reaction for minimum 4 h or overnight
to 25
C.
4. Check the quality of dsRNA by separating with accompanying
T3 and T7 non-hybridized transcripts on 10% polyacrylamide
denaturing gel.
5. Rinse the gel for half an hour in SYBR Safe in water solution to
visualize the bands.
3.3 MNP@PEI/dsRNA
Complexes
Preparation
1. Thaw dsRNA on ice and stir it gently by pipetting.
2. Vortex vigorously nanoparticles to break up the sediment and
scatter nanoparticles in ultrasonic disperser for 15 min.
3. Mix dsRNA with MNP@PEI in a series of Fe to RNA weight
ratios (1, 2, 3, 4, 5, 8, 10 Mag@PEI to ATN-RNA wt:wt
ratios). Manipulate only with the amount of nanoparticles, as
each complex must have an identical amount of dsRNA.
4. Adjust the volume of the complexes up to 50 μL with nucleasefree water.
5. Incubate: room temperature, 30 min.
6. Determine the rate of binding by gel retardation assay (Subheading 3.3.1) or UV-VIS spectrophotometry (Subheading
3.3.2). Use the free RNA as a control.
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Małgorzata Grabowska et al.
ATN-RNA
3.2.1 In Vitro
Transcription of ATN-RNA
1. Digest 50 μg of plasmid harboring ATN-RNA construct separately with restriction enzymes EcoRI and HindIII.
2. Carry out the digestion process by minimum 8 h or overnight.
3. Separate the whole reaction on the 1% agarose gel and examine
it on the transilluminator.
4. From agarose gel cut out the digested form of plasmid and
purify the plasmid from the gel using gel DNA purification kit.
5. Independently transcribe the two strands of RNA with T3 and
T7 polymerases for 1 μg of EcoRI and HindIII digested plasmids, respectively. Incubate the reaction overnight.
6. Purify the reaction loading whole mixture onto spin column
plugged into sterile 1.5 mL collection tubes, then centrifuge at
750 Â g, 2 min.
7. Apply 20 μL of nuclease-free H 2 O on spin tubes and repeat
centrifugation to new collection tubes.
8. Store transcripts in À20
C.
3.2.2 dsRNA
Hybridization
1. Mix equal quantities of T3 and T7 transcripts. Add 20 μL of 5Â
hybridization buffer and fill up with H 2 O to 100 μL.
2. Incubate: 95
C, 3 min, then follow the incubation for 75
C,
30 min.
3. Slowly cool down the reaction for minimum 4 h or overnight
to 25
C.
4. Check the quality of dsRNA by separating with accompanying
T3 and T7 non-hybridized transcripts on 10% polyacrylamide
denaturing gel.
5. Rinse the gel for half an hour in SYBR Safe in water solution to
visualize the bands.
3.3 MNP@PEI/dsRNA
Complexes
Preparation
1. Thaw dsRNA on ice and stir it gently by pipetting.
2. Vortex vigorously nanoparticles to break up the sediment and
scatter nanoparticles in ultrasonic disperser for 15 min.
3. Mix dsRNA with MNP@PEI in a series of Fe to RNA weight
ratios (1, 2, 3, 4, 5, 8, 10 Mag@PEI to ATN-RNA wt:wt
ratios). Manipulate only with the amount of nanoparticles, as
each complex must have an identical amount of dsRNA.
4. Adjust the volume of the complexes up to 50 μL with nucleasefree water.
5. Incubate: room temperature, 30 min.
6. Determine the rate of binding by gel retardation assay (Subheading 3.3.1) or UV-VIS spectrophotometry (Subheading
3.3.2). Use the free RNA as a control.
74
Małgorzata Grabowska et al.