Target Discovery and Validation Reviews and Protocols

RNAi Libraries 137
3.3.1. Puromysin Selection
1. Twenty-four hours after transfection, replate the transfecetd cells in a new 96-well
plate and add 1 µg/mL (final concentration) puromycin to each well (see Note 6).
2. Incubate the cell at 37°C for 36 h.
3. Wash the wells twice with PBS and add fresh DMEM, transfer the cells into new
96-well plate, and incubate at 37°C for additional 24 h.
3.3.2. Induction of Apoptosis by Transfection With Poly(I:C)
1. For each well, mix 25 µL of Opti-MEM I, 0.125 µg of poly(I:C), and 0.375 µL
FuGENE 6 (see Note 7).
2. After incubation for 15 min at room temperature, add the mixture to the cells.
3. Culture these cells for 24 h.
3.3.3. Assay
1. After 24-h incubation, wash the wells twice with PBS and fix with 4% paraformaldehyde
for 15 min at room temperature.
2. Incubate the fixed cells in 0.2% crystal violet liquid (prepared in PBS) for 15 min
and then wash the cells twice with PBS.
In this assay, nonapoptotic cells would be stained with crystal violet. In these
cells, genes that are involved in the induction of apoptosis are more likely inhibited.
3.4. Validation of Functional siRNA Expression Vectors
by Western Blot Analysis
After the first screening, positive clones should be confirmed with more
stringent conditions, which aims to remove the false positives. Check the levels
of protein or mRNA knockdown.
1. Plate HeLa S3 cells at 2 × 10 5 cells/well in a six-well plate and grow for 24 h.
2. For each well, mix 2 µg of siRNA expression vector and 250 µL of Opti-MEM I.
3. For each well, mix 5 µL of LipofectAMINE 2000 reagent and 250 µL of Opti-
MEM I medium.
4. Mix both solutions and incubate at room temperature for 15 min.
5. Add the mixture to the cells and incubate at 37°C for 24 h.
6. After incubation, split the cells and transfer approx 1/2 to 1/4 of the split cells to
a new six-well plate and add 2 mg/mL puromysin (final concentration) and
culture for 36 h. The 96-well plate is assayed for apoptosis as described in
Subheading 3.3.
7. Remove selection medium, wash the cells twice with PBS, harvest the cells, and
centrifuge at 1000g for 5 min.
8. Remove the supernatant, add lysis buffer to the pellet, and then sonicate the cells.
9. After sonication, centrifuge the lysates at 10,000g for 15 min and determine the
protein concentration in the supernatants by using the DC Protein Assay kit.