12.2.08 -- siRNA Screen – Anja and Dr. Foley.
Things to think about before starting:
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Kind of library you want to screen. Is it compatible with your cells, for example: mouse cells,
mouse library, human cells, human library. This is very important. Each library is supplied with 34 siRNA per gene. Smaller libraries may have 300 genes while larger libraries may have 2000.
This will significantly affect the amount of work involved. You should not pick and choose what
siRNA to do, it has to be random. You can pool the sets of siRNA for each gene though. Usually
when you pool siRNA, you will have knockdown that is the average of all of the individual siRNA
for that gene.
Transfection efficiency of your cells – needs to be optimized for siRNA. Several different
methods to check. Lipofectamine works for me so far for DNA, but I don’t know how it will work
with siRNA, N-Ter (Sigma) is another transfection reagent that is based on peptide delivery
system which may be something that you can check. Need to get very good efficiency for siRNA
to make sure that you are able to deplete the genes that you are looking at. A good way to
optimize this is to use siRNA for GAPDH and an activity assay for GAPDH (colour change) to
determine if it is reduced. This can be bought as a kit.
Optimize cell number for 96 well plates so there is optimal signal for reporter assay as well as
cells not dying because they are overgrown.
Make stable cell line of the reporter assay, or can also transfect reporter assay at the same time
as the siRNA if need be.
siRNA needs about 72 hours to work properly, growth of cells has to be appropriate for this and
that the cells will not die if overgrown. A special problem to ES cells is that if they are confluent,
they differentiate – need to optimize this. Also, ES cells need LIF to prevent differentiation and
LIF turns on the STAT3 pathway, which works partially with WNT and NOTCH pathways, the
pathways that we are interested in looking at. Will have to take this into consideration when
doing the reporter assay.
Readout assay – luciferase, colour change. Has to be more than 2 fold difference between the
two cell types that you are testing, preferably much higher. For example: for the CGR8 and G45,
I use the b-catenin luciferase reporter assay which is basically on in the wildtype and off in G45,
hopefully will be an easy assay to detect. Also need to optimize luciferase signal from a 96 well
format. The plate reader has very high sensitivity which hopefully allows this to work.
Controls that you need: negative control for wildtype (doesn’t reduce luciferase signal), positive
control for wildtype (siRNA that reduces something that is known – deplete GAPDH and do
activity assay for GAPDH (colour change)), negative control for G45 (doesn’t reduce luciferase
signal) and positive control for G45 (something that increases signal – this is not known and
therefore cannot be included – this is what I am looking for). You will also need scrambled siRNA
controls to account for overlap in recognition of the siRNA. Each plate will need cells incubated
without siRNA and transfection reagent, cells with non-silencing siRNA (scrambled), cells with
functional siRNA #1, cells with functional siRNA #2, cells with functional siRNA #3 (set of three
for one gene). Plan two wells for each, or four wells for each if stimulating with a drug such as
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SB415286 (which may be a good control, as it inhibits GSK3B). Also have a row all around with
water or media for humidity.
Do a literature search to find out if others have already optimized conditions for your cells with
siRNA screens. This will be a good starting point for your experiments.
Start with a smaller library if possible and get that working first and then do larger library when
the conditions have been optimized
The data mining is also a very large task. There are programs that can help with this. You need to
be careful to keep an objective view of the results to make sure that you do not “make” genes
fit into what you expect.
Cells are plated onto the transfection rather than plating cells first and then doing the
transfection. This makes for less work.
Once you identify genes that are affected in your reporter assay, you will need to do followup
biochemistry to verify that those genes are affected under your conditions. This may include
immunoprecipitation to identify protein-protein interactions, western blot analysis under your
conditions to determine that protein levels are affected, immunocytochemistry to identify
whether the protein is localized properly, and other methods as well. This is just a start.
Transfection protocols
General remarks from Anja:
These are the two transfection protocols I have used, the first one uses Dharmafect transfection
reagent and has been optimized by me (and was subsequently used to do my screen), the
second one uses the N-TER peptide transfection reagent and is not optimized yet – I only
followed the recommendations from SIGMA and downscaled it to 96 well format (that’s why
there are some weird volume numbers in it)
- each siRNA experiment should assay the following conditions
-cells incubated without any siRNA and transfection reagent
-cells with a non-silencing siRNA
-cells with a functional siRNA#1
-cells with a functional siRNA#2
-cells with a functional siRNA#3
and so on ...
- plan 4 wells for each of the above conditions – 2 of them will be stimulated with TNF/CHX, two
of them will not
- plate layout could look like this:
1... no siRNA
1 2 3 4
1 2 3 4
1 2 3 4
1 2 3 4
No
stimulation
2… non-silencing siRNA
TNF/CHX
4… siRNA#2
1 row of water/medium
3… siRNA#1
to prevent evaporation
day 1: reverse transfection with Dharmafect/other transfection Reagent
- one well of a 96-well plate will finally contain:
10 ul of 200 nM siRNA dilution in RNase-free water (= 1:100 dilution of your 20 uM siRNA stock)
15 ul OptiMEM containing 0.2 ul transfection reagent (Dharmafect 1)
75 ul culture medium (DMEM) containing 10% FBS (but no antibiotics!) and 6500 HeLa cells (=
0.867 x 105 cells/ml)
- scale up according to the number of wells you have… (always make a little more master mix for each
siRNA, master mix of OptiMEM with Dharmafect, master mix of medium with cells)
- transfection in detail:
1) dilute your 20 uM siRNA stock in RNase-free water – for one well you need:
0.1 ul of 20 uM siRNA
9.9 ul RNase-free water
2) add 10 ul siRNA dilution to each well – prevent drying of the siRNA solution in the wells by storing
completed plates in a moist environment (for example a Styrofoam container with lid and moist paper
towels on the bottom - this is more important for screens with large amounts of plates – for one plate I
usually don’t do it…)
3) mix OptiMEM with transfection reagent – for one well you need:
0.2 ul Dharmafect 1
14.8 ul OptiMEM
- incubate for 5 minutes at room temperature
4) in the meantime: trypsinize your cells (if adherent)
5) add 15 ul OptiMEM containing transfection reagent to the wells with the siRNA solution, incubate for
20-30 min (approximately, but not too long), mix by moving the plates in a horizontal direction, do NOT
keep on a shaker for the incubation time!
6) in the meantime: count cells
7) add 75 ul of culture medium containing 6500 cells, mixing not required (mixing in my experience
often leads to clumping)
8) pipette 100 ul medium or water into the wells surrounding your sample wells – that is to avoid edge
effects and evaporation
day 1: reverse transfection with N-TER
- one well of a 96-well plate will finally contain:
3.1 ul of siRNA/peptide complexes (siRNA concentration will lead to the same final
concentration of 20 nM)
96.9 ul culture medium (DMEM) containing 10% FBS and antibiotics (that does not matter here)
and 6500 HeLa cells (0.671 x 105 cells/ml)
- transfection in detail:
1) in a tube, mix siRNA with siRNA dilution buffer – for a single well you need:
0.1 ul of your 20 uM siRNA stock
1.43 ul of siRNA dilution buffer
- make a master mix for each siRNA you have (always make a little more than you need)
2) in a tube, dilute the N-TER peptide in water – for a single well you need:
0.25 ul of N-TER peptide
1.3 ul of RNase-free water
- make a master mix for all transfections… (always make a little more)
- incubate for 5 minutes
3) mix each siRNA dilution made in 1) with 1.53 ul of the N-TER peptide dilution made in 2)
4) incubate for 20-30 minutes at room temperature to let the siRNA/peptide complexes form
5) in the meantime: trypsinize your cells (if adherent) and count them
6) spot 3.1 ul of the siRNA/peptide solution into each well (your cells should be ready to go at that
moment, because 3.1 ul of siRNA/peptide mix dries out very fast)
7) add 96.9 ul of culture medium containing 6500 cells, mixing not required (mixing in my experience
often leads to clumping)
8) pipette 100 ul medium or water into the wells surrounding your sample wells – that is to avoid edge
effects and evaporation
you can see that this protocol is not optimized for screens yet, it would be too complicated to mix each
siRNA from a 96 well plate library in a tube; on the other hand we can also not mix the siRNA and the
peptide in the well because the volumes are so small, but eventually one could use another 96 well plate
in which one could prepare the master mix? or increase the total volumes?