Using the Multiporator® for effective gene silencing in HeLa cells
Nina Schaffert, Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany; Natascha Weiß,
Eppendorf AG, Hamburg, Germany
RNA interference (RNAi) describes the specific post-transcriptional
inhibition of the gene expression (gene silencing).
This process is triggered by double-strand RNA
molecules (dsRNA) homologous to a target mRNA. The
highly conserved mechanism is known in many lower
and higher eukaryotic organisms and was first described
in 1998 by Fire et al.  for Caenorhabditis elegans.
The decisive molecules of the RNAi are siRNAs (small
interfering RNAs). These are the result of the RNase
activity of the protein dicer, which cuts long dsRNA into
fragments of 21-28 nucleotides. [2-4]. In a subsequent
step, the double-strand siRNA is integrated into the
multi-protein complex RISC (RNA-induced silencing
complex) and separated into single strands. The complementary
target mRNA is recognized and bonded with the
antisense strand and is ultimately degraded by nucleases
of the RISC complex [3, 5].
The use of RNAi technology for specific gene silencing
was initially impossible in most mammalian cells, as the
usage of long dsRNA molecules (> 30 bp) caused a general
defense reaction of the cells . Only in 2001 did the
group around T. Tuschl achieve a sequence-specific inhibition
of the gene expression without an unspecific
immune response by directly introducing synthetic 21-23
bp siRNA duplexes into cell lines [7-9].
The targeted and effective inhibition of the gene expression
makes RNA interference an important technique for
gene function analyses. Depending upon the objective of
the experiment, various strategies are deployed. A stable
gene silencing can be achieved with vector systems that
express shRNA (short hairpin RNA). As a result of the
cellular activity of the dicer, these are processed into
active siRNA molecules. Transfection with
synthetic siRNA, on the other hand, results in a transient
inhibition.The common transfection techniques are generally
suitable for the last method, but the efficiency can
differ depending upon the technique selected , and
non-specific changes in the gene expression profile have
been observed when using lipid-mediated transfection .
This application describes the use of the Eppendorf
Multiporator to transfect HeLa cells with siRNA molecules.
The goal was thereby to inhibit the expression of
an essential protein. In an initial experiment, various
electroporation parameters are tested. The determined
values are subsequently used for a second experiment
in which siRNA duplexes targeted against different
mRNA sequences of the same protein are used.
Materials and methods
● Cell line: HeLa SS6
● siRNA: siRNA duplex against an essential
protein, control siRNA duplex
(20 μM; in 100 mM KOAc,
2 mM MgOAc, 60 mM Hepes KOH;
● Cell preparation Trypsin, Opti-MEM® (Invitrogen)
● Washing solution: PBS (Phosphate Buffered Saline),
● Electroporator: Multiporator®
● Electroporation buffer: Eppendorf hypoosmolar
● Culture medium: DMEM/10 % FCS/100 μg/ml
● Cuvettes: Eppendorf, 4 mm, 800 μl
1. Split the cells no later than 40 hours prior to electroporation in such a way that they are in the exponential growth phase at the time of harvesting.
2. Detach the cells with trypsin. Subsequently dilute the trypsin with Opti-MEM® and pellet the cells for 5 min at 210 x g and 4°C.
3. Resuspend the cells in PBS, determine the number of cells and centrifuge again (5 min at 210 x g and 4°C). Remove the supernatant. Note: The total incubation time in the Eppendorf electroporation buffer may not exceed 30 min if successful electroporation is to be ensured.
4. Resuspend the cells in hypoosmolar electroporation buffer. Thereby set the cell concentration to 6 x 106 cells/ml.
5. Mix 20 μl of the 20-μM siRNA solution with 300 μl of hypoosmolar electroporation buffer, place in a 4 mm cuvette and incubate on ice.
6. Add 500 μl of cell suspension (corresponds to 3 x 106 cells) to the siRNA/electroporation buffer mixture in the cuvette, mix and incubate on ice for 10 min.
7. The electroporation is carried out with the Eppendorf Multiporator® using one pulse in each case. The parameters to be used are listed in the "Experiments" section.
8. Following the pulse, leave the cell suspension in the cuvette for 10 min at room temperature.
9. Add 1 ml of pre-warmed culture medium to the cuvette and transfer the suspension carefully into a cell culture dish (Ø 14.5 cm) with 11 ml of medium.
10. Incubate at 37°C for 48 hours.
11. Optional: change the medium after 6-8 hours of incubation in order to remove dead cells.
1) Determination of suitable electroporation parameters
The cells were split approx. 16 hours prior to electroporation,
so that they were in the exponential growth phase at
the time of harvesting. The cell preparation was otherwise
carried out as described in the transfection protocol (see
The following parameter combinations (voltage, time constant, temperature) were tested:
A) 1200 V /100 μs, RT
B) 1200 V/40 μs, 4°C
C) 800 V /100 μs, RT
D) 800 V/40 μs, 4°C
Each electroporation preparation (A-D) was carried out with two different cell numbers:
a) 1 x 106 cells
b) 3 x 106 cells
2) Electroporation with various siRNA duplexes
For this experiment the cells were treated according to
the transfection protocol and the electroporation was
carried out with the previously defined suitable parameters
(1200 V, 40 µsec and 4°C). Various siRNA molecules
were used. These are complementary to different
sequences of the target mRNA.
After 48 hours the cells were harvested with a Rubber
Policeman and the total cell number was determined
through a cell count with a CASYCounter TT (Schärfe
The efficiency of the transfection experiments were
determined by immunofluorescence studies and Western
blot analyses with specific antibodies, as described elsewhere
1) Determination of suitable electroporation parameters
The survival rate of the cells was calculated from the
total cell number following electroporation in relation to
the non-electroporated control. The B, C and D preparations
demonstrate the highest survival rate when using 1
x 106 cells. For 3 x 106 cells, the parameter combinations
C and B show the best results (Table 1). The highest
transfection efficiencies are achieved with the setting
of 1200 Volt (A+B) (Table 2).
2) Electroporation with various siRNA duplexes
The survival rates and efficiencies using the various
siRNA duplexes are shown in Table 3. The parameters
selected here (B: 1200 V/40 µsec/4 °C) had the best
results in the first experiment for the combination of survival
rate and efficiency. 3 x 106 cells were used in order
to obtain as much cells as possible for further analyses.
A survival rate of 100 % can be achieved as a result of the
early splitting of the cells (40 hours prior to harvesting) in
connection with the optimized electroporation conditions
(Table 3). A maximum efficiency of 90-95 % is obtained
by using a strong siRNA. It has been proven that all three
siRNA molecules perform well by using transfection
reagents in advance (not shown). The efficiency therefore
also depends on the transfection method used.
The effect of the siRNA on the expression of the protein
in question is shown in the Western blot in Fig. 1. The
amount of essential protein produced in cells transfected
with effector siRNA is reduced drastically compared with
the control samples.
In the previous experiments we described how a successful
RNAi system could be established with few optimization
steps. Based on the parameters determined in
this way, survival rates of 100 % and efficiencies of up
to 95 % were obtained.
In addition to the electroporation parameters, two factors
could be identified that had an important influence
on the success of the experiment. The survival rate of
the cells was strongly influenced by the pretreatment.
They are in an obviously better condition when they are
split significantly prior to electroporation. The selection of
the siRNA sequences is also decisive for successful inhibition
of the gene expression. Testing out of various
duplexes is therefore necessary for a successful experiment.
On the whole, we were able to show that the
Eppendorf Multiporator system, consisting of the device
and the hypoosmolar electroporation buffer, is exceptional
for the insertion of siRNA molecules into HeLa cells.
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