2.8.2 Removal of
Residual DNA and Reverse
Transcription
1. DNA removal kit (Ambion).
2. cDNA reverse transcription kit (Roche).
3. RNA gel loading dye.
4. 10Â TBE buffer: 1 M Tris base, 1 M boric acid, 0.02 M EDTA.
5. 1% agarose gel.
6. Vertical gel electrophoresis system.
7. Transilluminator.
8. Microvolume spectrophotometer.
2.8.3 Real-Time
Polymerase Chain Reaction
1. RT-PCR probe mix 2Â.
2. Primers for the gene of interest.
3. Primers for the endogenous gene.
4. Probe from the Universal Probe Library (UPL, Roche).
5. 96-well PCR plates.
6. Adhesive PCR plate foils.
7. Real-time PCR detection system.
2.9 Cell-Migration
Analysis
1. DMEM High Glucose.
2. PBS.
3. 12-well cell culture plate.
4. 200 μL micropipette tip.
5. Microscope with Â5 magnification.
6. Software with edge-detection algorithm to wound-healing
assays analysis (e.g., TScratch, CSElab).
3 Methods
3.1 Synthesis and
Functionalization of
Magnetic
Nanoparticles
1. Mix FeCl 3 ·6H 2 O (135 mg, 0.5 mmol) with FeCl 2 ·4H 2 O
(50 mg, 0.25 mmol) in 5 mL of water and degassed with N 2
(see Note 1).
2. Heat the mixture up to 80
C. Add 1 mL of working solution
and continue heating for 120 min. Cool down the mixture and
collect nanoparticles by an external magnet (see Note 2).
3. Wash nanoparticles with water (2 Â 150 mL) and collected by
an external magnet. Finally, redisperse nanoparticles in 10 mL
of water.
Delivery of dsRNA to Glioblastoma Cells
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