Target Discovery and Validation Reviews and Protocols

158 Erfle and Pepperkok
cells away from the spot region. To retrieve the spot regions and to highlight
successfully transfected cells of siRNA experiments, a Cy3-labeled DNA
oligonucleotide is used as cotransfection marker. In this case, 0.5 µL of a
40 µM marker solution is included in step 1 of the protocol, resulting in a total
oligonucleotide volume of 1.5 µL (1 µL of siRNA plus 0.5 µL of Cy3-labeled
oligonucleotide). With this protocol, it is possible to cotransfect plasmid cDNA
and siRNA. In this case, 1 µL of siRNA plus 1 µL of plasmid cDNA are added
in step 1, resulting in a total volume of 2 µL.
1. Add 1 µL of the respective siRNA stock solution (20 µM in RNA dilution buffer
as supplied by the manufacturer) to each well. For plasmid transfections, 1 µL of
plasmid DNA at a stock concentration of 500 ng/µL is added.
2. Add 7.5 µL EC buffer (part of the Effectene transfection kit, QIAGEN) containing
0.2 M sucrose and mix thoroughly by pipeting three times up and down. The EC
buffer can be replaced by water, also containing 0.2 M sucrose for LipofectAMINE
2000 (Invitrogen).
3. Incubate the mixture for 10 min at room temperature.
4. Add 4.5 µL of Effectene transfection reagent (QIAGEN) or 3.5 µL of
LipofectAMINE 2000 (Invitrogen).
5. Incubate for 10 min at room temperature.
6. Add 7.25 µL of 0.08% gelatin (G-9391, Sigma) containing 3.5 × 10 –4 % fibronectin
(Sigma).
7. Spin down the 384-well plate for 1 min at 216g. The solution is now ready for the
spotting process.
3.2. Spotting the Transfection Solution on Uncoated Lab-Tek Dishes
As a substrate, we use untreated chambered coverglass dishes, allowing
live cell imaging and easy-to-perform immunostaining. We use a ChipWriter
Compact robot (Bio-Rad, Hercules, CA) equipped with PTS 600 (Point
Technologies) solid pins. The distance between the spots is 1125 µm, resulting
in spot diameters of approx 450 µm. The spotted solutions on the Lab-Tek
chamber are dried at room temperature for at least 12 h after printing before
cells are plated. The solutions of one 384-well plate (see Subheading 3.1.)
are sufficient to spot at least 50 identical Lab-Tek chambers, which can be
stored for later use in a dry environment for several months without obvious
loss in transfection efficiencies. The outer dimensions of a Lab-Tek chamber
are 57.2 × 25.6 mm (length × width). Of this area we use, 34.875 mm in
length and 12.375 mm in width to spot 384 samples. Spotting 384 samples on
48 Lab-Tek chambers, by using eight solid pins in the x-direction mode typically
lasts 6 h.
1. We adjust the temperature of the 384-well plate with an in-house–built watercooled
plate to 12°C.