MODULE 3 – Day 1
Transfection of EGFP and p53 siRNAs into HeLa
cells
MOST STUDENTS WILL WORK IN GROUPS OF TWO
We will be studying RNAi knockdown of enhanced green fluorescent protein (EGFP)
expression in EGFP-expressing HeLa cells. We will transfect EGFP-HeLa cells with
duplexed siRNA using a modified oligofectamine transfection protocol.
You can look over the Oligofectamine Transfection protocol provided by Invitrogen
at a later date to understand how the conditions are optimized (see below).
Each group will perform three oligofectamine transfections:
(1) with EGFP siRNA
(2) with p53 siRNA
(3) without siRNA.
The p53 siRNA controls for nonspecific siRNA effects, and the experiment without
siRNA controls for oligfectamine effects.
The day prior to transfection, cells will be prepared for you at a density of 0.5 to 1 X 105
cells/well in each well of a 6 well plate.
Required Materials:
One 6-well plate of HeLa cells (per 2 students)
30 ml Phosphate Buffered Saline (PBS)
10 ml RPMI media, serum free, antibiotics free
6 eppendorf tubes
On ice in eppendorf tubes:
30 l oligofectamine
10 l EGFP siRNA, 33 M
10 l p53 siRNA, 33 M
Transfection Procedure (use sterile technique + read each task entirely
before starting).
**NOTE: read through the entire protocol first. You may want to divide the tasks by
having one partner start on step 4 while the other partner prepares the reagents in steps 2
and 3.**
1. Label your plates
You will use 2 wells for EGFP knockdown, 2 wells for p53 knockdown, and 2 wells for
oligofectamine-only controls. Label your plate accordingly. See example below:
EGFP
p53
ctrl
EGFP
p53
ctrl
2. Label 6 eppendorfs as follows: AGFP, BGFP, Ap53, Bp53, Actrl and Bctrl.
3. Prepare reagents in labeled eppendorfs according to the chart below.
 FIRST pipette the RPMI media (which has no serum or antibiotics) into all
tubes.
 THEN pipette the siRNA (EGFP for the EGFP tubes, p53 for the p53 tubes)
and oligofectamine needed for each tube. Make sure that you pipette directly
into the media and fully depress the pipette.
 Mix each tube by gently tapping on the side.
Tube A
Media
EGFP 94 l
p53
94 l
Ctrl
100 l
siRNA
6 l
6 l
-------
Tube B
Media
94 l
94 l
94 l
Oligofectamine
6 l
6 l
6 l
Transfer entire contents (l) of TUBE A into TUBE B for both siRNA transfections,
and for the Oligofectamine control. Mix by gently tapping. Incubate at room
temperature for 20 min.
4. Wash and prepare the cells while the transfection reagents are incubating:
a. Aspirate off the media.
b.
c.
d.
e.
Add 2ml of PBS to each well (add slowly, do NOT forcefully pipette in)
Rock plate gently and then aspirate off the PBS.
Repeat steps b and c.
Add 900ul media to each well
5. After the 20 min incubation, add 95 l of transfection reagent from the appropriate
eppendorf tube (GFP, p53, or ctrl) to each well. Add reagents DROPWISE, close to
the surface of the media, and spreading them evenly over each well (see diagram
below for even-distribution method). Mix gently by rocking your plate back and
forth.
6. Put your plate in the 37°C incubator.
After 6 hours of transfection, the following steps will be performed for you to complete
the procedure:
 Add 1ml of RPMI media + serum to each well.
 Incubate overnight @ 37°C, 5% CO2.
 Aspirate media and transfection reagents off. Replace with 1ml RPMI media +
serum.
 Return to incubator.
48 hours post-transfection, you will assay the plates by flow cytometry.