Download FACS Procedure

MODULE 3 – Day 4
Transfection of ATM, ATR, EXO1 and AAG siRNAs into
HeLa cells
STUDENTS WILL CONTINUE TO WORK IN GROUPS OF TWO
We will be studying RNAi knockdown of ATM, ATR, Exo1, AAG, EGFP, and p53 expression in
EGFP-expressing HeLa cells. We will transfect EGFP-HeLa cells with duplexed siRNA using a
modified oligofectamine transfection protocol. This time, we will assay the cells for mRNA
expression using DNA microarrays.
Each group will perform two types of oligofectamine transfections:
(1) with siRNA (each group will knockdown 1 of the 6 genes)
(2) without siRNA.
Again, 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
4 eppendorf tubes
On ice in eppendorf tubes:
30 l oligofectamine
10 l siRNA, 33 M (each group will have a different gene)
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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 5 while the other partner prepares the reagents in steps 3 and 4.**
1. Label your plates
You will use 2 wells for siRNA knockdown of your gene and 2 wells for oligofectamine-only controls.
Label your plate accordingly. See example below given for the group doing ATM:
ATM
ctrl
ATM
ctrl
2. Label 4 eppendorfs as follows: AATM, BATM, Actrl and Bctrl. (Label with the gene that your group is
doing)
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 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.
ATM
Ctrl
Tube A
Media siRNA
94 l
6 l
100 l -------
Tube B
Media
94 l
94 l
Oligofectamine
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 transfection reagents are incubating.
a.
b.
c.
d.
e.
f.
Aspirate off the media
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
Incubate for 20 min
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5.
After the 20 minute incubation, add 95 l of transfection reagent from the appropriate
eppendorf tube (siRNA 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 evendistribution 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, the cells will be harvested, the RNA isolated (this will be done for
you) and this RNA will used for the DNA microarray analysis on Days 5 & 6.
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