Biology Reference
In-Depth Information
4.1.2
RNAi library
Previous to our screen, many RNAi screens have been performed in
Drosophila
cul-
ture cells to investigate various cellular phenomena. In 2006, two reports focused on
the problem of “off-target” effects (downregulation of genes other than the intended
ones) of genome-wide RNAi screens (
Kulkarni et al., 2006; Ma, Creanga, Lum, &
Beachy, 2006
). The dsRNAs used in most
Drosophila
cell screens are usually a few
hundred base pairs long compared to the 21-23 bp siRNAs or shRNAs used in mam-
malian systems. Inside the cells the long dsRNAs are processed into smaller RNAs.
As many dsRNAs used in previous screens contain tandem trinucleotide repeats and
regions that match unintended targets, the off-target false-positive effects were high.
To minimize the off-target effects, we used the specially designed UCSF DmRNAi
libraries (
Goshima et al., 2007
) and also performed a secondary screen using dsRNAs
targeting different regions than the ones used in the primary screen. The UCSF
DmRNAi library version 2 was designed to minimize off-target effects by computa-
tionally selecting dsRNA-targeted regions that avoid low-complexity repeats and those
with significant sequence homology elsewhere in the genome. With this design, 90%
of the library avoids 21 bp identical sequence overlapping with to any unintended
genes. In addition to these cautiously designed libraries we synthesized a second
set of dsRNAs to target a different region for the 847 genes we identified from the
primary genome-wide screen to confirm the effects in the secondary screen.
4.1.3
Screening materials
UCSF DmRNAi library version 1 and version 2 (1
m
g/well predried in 96-well
tissue-culture plates)
Drosophila
S2 cells and culture medium (with 10% fetal bovine serum (FBS), 1%
penicillin-streptomycin)
96-well tissue-culture plates and glass-bottom plates
Oleate/BSA stock (7.5 mM)
BODIPY493/503, DAPI, Concanavalin-A (Con-A), formaldehyde, PBS
IC100 (Beckman) or ImageXpress Micro (Molecular Devices) automated
microscope
4.1.4
Screening procedure
1.
Aliquot 1
m
g dsRNA per well in 96-well tissue-culture plates, air dry, and store
80
C.
2.
Transfer healthy S2 cells both in medium and on the bottom of the flask by
gentle pipetting to conical tubes, wash with serum free medium (SFM) once,
add SFM, and count cell numbers.
3.
Dilute the cells to 2.7
10
6
cells/ml, and apply 35
at
l/well to dsRNA preloaded
96-well culture plates. Incubate at 25
C for 50 min, and then add 65
m
m
l 15%
