392 | VOL.2 NO.2 | 2007 | NATURE PROTOCOLS
Reverse transfection on cell arrays for high content
Holger Erfle1, Beate Neumann1, Urban Liebel1, Phill Rogers1, Michael Held1, Thomas Walter1, Jan Ellenberg2 and
1MitoCheck Project Group, 2Gene Expression and 3Cell Biology/Biophysics Programmes, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Correspondence
should be addressed to H.E. (firstname.lastname@example.org)
Published online 1 March 2007; doi: 10.1038/nprot.2006.483
Here, we describe a robust protocol for the reverse transfection of cells on small interfering (siRNA) arrays, which, in combination
with multi-channel immunofluorescence or time-lapse microscopy, is suitable for genome-wide RNA interference (RNAi) screens in
intact human cells. The automatic production of 48 ‘transfection ready’ siRNA arrays, each containing 384 samples, takes in total 7
h. Pre-fabricated siRNA arrays can be used without loss of transfection efficiency at least up to 15 months after printing. Different
human cell lines that have been successfully transfected using the protocol are presented here. The present protocol has been
applied to two genome-wide siRNA screens addressing mitosis and constitutive protein secretion.
High-throughput parallel transfection techniques are a prereq-
uisite to perform large-scale experiments, such as genome-wide
RNAi screens, in mammalian tissue culture cells1−4. In such stud-
ies, transfections are typically performed in 96- or 384-well plates
using liquid-handling robotics. Reverse transfection of cells on
plasmid or siRNA arrays5−10 is a powerful alternative method
to perform high-throughput transfections for phenotypic data
acquisition by light microscopy.
Reverse transfection has several advantages compared with liq-
uid phase transfections in 96- or 384-well plates. First, the high
density of samples on the arrays allows increased data acqui-
sition speed by microscopy. Second, the cost of each experi-
ment is reduced owing to the small amount of siRNA needed
per spot. Third, parallel cell seeding in a single chamber for 384
knock-down experiments with no physical separation between
experiments/spots increases the screening data quality. These
Figure 1 | Workflow of the protocol for reverse cell transfection on small
interfering (siRNA) arrays. The workflow of the protocol described here is
segmented in its main factors and starts from an siRNA library. The samples
are automatically or manually prepared for the spotting process in 96- or
384-well formats (automatic preparation lasts 1 h) and are then spotted
in LabTeks (lasts 6 h for 384 samples and printing of 48 LabTeks). After
drying (minimum of 12 h) and storage (up to 15 months tested) of the
LabTeks, mammalian cells are seeded on the LabTeks. Then, typically after
20 h, live cell imaging of the samples is started. For assays involving fixed
and immunostained cells, incubation periods after cell seeding are typically
between 48 and 60 h before fixation and immunostaining.
Figure 2 | The printing process and layout of the siRNA arrays. The DNA
contact printer prints from the 384-well low volume plate (*) with eight
solid pins (arrow) in LabTeks as shown in (a). A printed LabTek chamber
with 384 spots is shown in (b) and a magnification of 84 dried spots (spot
diameter: 400 µm and spot-to-spot distance: 1,125 µm) is shown in (c).
Automatic or manual preparation of transfection solutions
(in 96- or 384-well formats)
Spotting of transfection solution on LabTeks with a contract printer
Drying and storage of printed LabTek chambers
Immunostaining of transfected
Live cell imaging
12 h to 15 months
NATURE PROTOCOLS | VOL.2 NO.2 | 2007 | 393
• siRNA oligonucleotides (Ambion), for sequences see ref. 6
• Lipofectamine 2000 (Invitrogen, cat. no. 11668-019)
• Sucrose (USB, cat. no. 21938)
• Gelatin (Sigma-Aldrich, cat. no. G-9391)
• Fibronectin, human (Sigma-Aldrich, cat. no. F0895)
• OptiMEM I + GlutaMAX I (GIBCO, cat. no. 51985-026)
• Drying pearls, orange – heavy metal free (Fluka, cat. no. 94098)
• Hoechst dye solution 33342 (Sigma, cat. no. B2261)
• Silicon grease (NEOLAB, cat. no. 1-2072)
• CO2-independent media (Gibco, cat. no. 18045-054)
• Mowiol 4-88 (Sigma-Aldrich, cat. no. 81381)
• Sodium azide (Appli Chem, cat. no. A1430)
• Paraformaldehyde (PFA) (Electron Microscopy Sciences, cat. no. 15710)
• BSA (Sigma-Aldrich, cat. no. A9056)
• Triton solution (Sigma-Aldrich, cat. no. 234729)
• Cell lines:
• Osteosarcoma, U2OS (ATCC, cat. no. HTB-96)
• Retinal pigment epithelial cells, hTERT-RPE1 (ATCC, cat. no. CRL-4000)
• Lung carcinoma cell line, A549 (ATCC, cat. no. CCL-185)
• Human umbilical vein endothelial cells, HUVEC (PromoCell, cat. no. C-12200)
• Cervix carcinoma cells, HeLa ‘Kyoto’ (Prof. Shuh Narumiya, Kyoto University
and Toru Hirota, IMP, Vienna)
well-to-well variations occur in experiments in multiwell dishes.
Furthermore, sample preparation steps, such as immunostaining,
do not require further automation. Another major advantage of
the reverse transfection protocol described here compared with
liquid-phase transfections in multiwell formats is the possibil-
ity to produce exact replicate arrays, derived from the same sam-
ple source plate, and once dried the printed microarrays can be
stored, as it is shown here, for at least 15 months without apparent
loss of transfection efficiency. Such replicas can be the basis for
several genome-wide screens, either for multiplexing of different
biological assays or different cell lines, yielding directly compara-
ble results because the microarrays are produced from the same
One of the limitations of reverse transfection is that applica-
tions with different cell lines have so far required variations in
the protocols to manufacture siRNA or plasmid arrays, which
involves a considerable amount of development and testing. A
further crucial aspect of the technology is the possibility of cross-
contamination of the array spots when spot densities increase;
therefore, optimization of the array layout is important. Also,
only small-scale projects have been described with the technology
and it remains to be shown that it is also applicable to large scale,
genome-wide siRNA screens.
Here, we describe a protocol (see Fig. 1 and Fig. 2 for an over-
view) that has been used for siRNA array production and appli-
cation to two genome-wide siRNA screens in HeLa cells (cervix
carcinoma cell line), and that works with similar efficiencies in
several other cell types, including human primary fibroblasts
The preparation of the samples for spotting, including the mix-
ing of transfection cocktails and siRNAs, has been automated in
a 384-well plate format using an automated liquid handler. This
process requires less than 1 h for the preparation of one 384-well
plate. The printing of these 384 samples onto 48 replicates of
chambered coverglass tissue culture dishes (LabTeks) is performed
with a contact printer (automated microarrayer) and requires 6
h. After the spots, containing siRNAs and transfection solution,
are dried on the coverglass for at least 12 h, mammalian cells are
seeded onto the LabTek dishes for transfection. Typically, cells can
be processed and analyzed by high-content-screening microscopy
20 h after cell seeding.
• Primary human skin fibroblasts (Dr. Heiko Runz, University of Heidelberg)
• 384-well low volume plates (Nalge Nunc International, cat. no. 264360)
• 96-well standard plates (Kisker, cat. no. G060)
• Sterile filter (0.45 µm) (MILLIPORE, cat. no. SCHVU01RE)
• Reservoir (Nalge Nunc International, cat. no. 370905)
• Automated liquid handling robot, ‘MICROLAB STAR’ (Hamilton); equipped
with:96-channel head and coolable carrier blocks for multiwell plates
• Standard volume tips without filter − 300 µl (Hamilton, cat. no. 235900)
• Low volume tips without filter – 10 µl (Hamilton, cat. no. 235902)
• Heraeus multifuge 3S (Kendro, cat. no. 75004361)
• Contact printers, ChipWriter ‘Compact’ and ‘Pro’ (Bio-Rad Laboratories)
• Temperature controlled plate, order no. 26-1-0 (EMBL)
• Solid pins (Point Technologies, cat. no. PTS 600)
• LabTek chambered coverglass (Nalge Nunc International, cat. no. 155361)
• Gel drying box for storage of printed LabTeks (The Stewart Company)
• Scanning microscope (scan^R, Olympus Biosystems)
• 10× objective (Olympus, cat. no. UPSLAPO 10×)
Growth medium DMEM supplemented with 10% (v/v) heat-inactivated fetal
calf serum, 2 mM glutamine, 100 U ml-1 penicillin and 100 µg ml-1 streptomycin
96-well transfection stock solution for liquid handler 18 µl OptiMEM
containing 0.4 M sucrose + 21 µl Lipofectamine 2000 per well
96-well gelatin/fibronectin stock solution for liquid handler 0.2 % (w/v)
gelatin solution containing 1 × 10-2 % (v/v) fibronectin
Preparation of spotting solution
1 | Prepare the spotting solution either manually (option A) or using a pipetting robot (option B).
(A) Manual preparation of the spotting solution:
(i) Prepare an siRNA stock solution by dissolving lyophilized siRNAs with milliQ water to a final concentration of 30 µM.
(ii) Transfer 3 µl of OptiMEM, containing 0.4 M sucrose, to each well of a 384-well low volume plate.
▲ CRITICAL STEP Prepare the OptiMEM (containing 0.4 M sucrose) freshly.
(iii) Add 3.5 µl Lipofectamine 2000 to each well of the 384-well low volume plate and mix thoroughly.
▲ CRITICAL STEP The same batch of the transfection reagent should be used for the whole experiment. We observed
a high batch-to-batch variation in performance of the Lipofectamine 2000 transfection reagent. Therefore, we
394 | VOL.2 NO.2 | 2007 | NATURE PROTOCOLS
recommend testing different batch numbers of the transfection reagent and then using only one batch for a whole
genome-wide project using reverse transfection of siRNAs.
(iv) Add 5 µl of the respective siRNA stock solution to each well of the 384-well low volume plate and mix thoroughly.
(v) Incubate for 20 min at room temperature (20–23 °C).
(vi) Add 7.25 µl of a 0.2 % (w/v) gelatin solution containing 1 × 10-2 % (v/v) fibronectin to each well of the 384-well
low volume plate and mix thoroughly.
▲ CRITICAL STEP Prepare the gelatin solution freshly. Dissolve the gelatin powder at 56 °C for 20 min in milliQ
water. Cool it down to room temperature and filter the solution with a sterile filter (0.45 µm).
(B) Automatic preparation of the spotting solution for 384 samples on a Hamilton ‘MICROLAB STAR’ pipetting robot:
(i) Prepare a 96-well transfection stock solution plate manually, and place it in the pipetting robot. Keep it at room
▲ CRITICAL STEP Prepare the OptiMEM (containing 0.4 M sucrose) freshly. The same batch number of the
transfection reagent should be used for the whole project. We observed a high batch-to-batch variation in
performance of the Lipofectamine 2000 transfection reagent. Therefore, we recommend testing different batch
numbers of the transfection reagent and then use of only one batch for a whole genome-wide project using reverse
transfection of siRNAs.
(ii) Prepare a 96-well gelatin/fibronectin stock solution plate manually, with 48 µl gelatin/fibronectin stock solution and
place it in the pipetting robot. Keep it at room temperature.
▲ CRITICAL STEP Prepare the gelatin solution freshly. Dissolve the gelatin powder at 56 °C for 20 min in milliQ
water. Cool it down to room temperature and filter the solution with a sterile filter (0.45 µm).
(iii) Prepare the siRNA stock solution by dissolving lyophilized siRNAs (4 nmols, delivered by manufacturer in 96-well
plates) in four standard 96-well plates with 130 µl milliQ water taken from reservoir, filled with 400 ml of milliQ
water (final concentration: 30 µM) per well on cooled carrier blocks (14 °C) using standard 300 µl volume tips
without filters. The liquid handler is thereby adjusted to a mix volume of 100 µl and eight cycles of mixing using the
(iv) Transfer 6.5 µl of the transfection stock solution (from Step 1B(i)) into each well of a 384-well low volume plate
using 10 µl low volume tips without filters (using the 96-channel head) at room temperature.
(v) Transfer 5 µl of the siRNA stock solution (from Step 1B(iii)) into the 384-well low volume plate. The liquid handler is
thereby adjusted to a mix volume of 7 µl and eight cycles of mixing (using 96-channel head) at room temperature.
▲ CRITICAL STEP This step is crucial because the number of cycles influences the transfection efficiency.
(vi) Incubate for 20 min at room temperature.
(vii) Add 7.25 µl of the gelatin/fibronectin stock solution into each well of the 384-well low volume plate. The liquid
handler is thereby adjusted to a mix volume of 10 µl and eight cycles of mixing (using the 96-channel head).
2 | Using the multifuge 3S, centrifuge the 384-well low volume plate for 15 s at 54g at room temperature to straighten the
surface of the samples and place immediately in the contact printer.
3 | Adjust the number of pins in the contact printer. We routinely use eight solid pins PTS 600. The spot diameter of the
solid pins PTS 600 pins is about 400 µm. Smaller pin sizes create a smaller spot diameter (e.g., for PTS 400 pins the spot
size is about 270 µm) resulting in less cells per spot.
4 | Adjust the temperature of the 384-well plate on an in-house built temperature controlled plate to 12 °C to avoid
evaporation of the sample.
5 | Set the spot-to-spot distance in the contact printer menu with respect to the application in mind. We routinely use
either 900 µm, 1,125 µm, 1,500 µm or 2,250 µm. This correlates to the following number of sample spots per LabTek: 900
µm distance = 600 samples per LabTek; 1,125 µm distance = 384 samples per LabTek; 1,500 µm distance = 216 samples per
LabTek; and 2,250 µm distance = 96 samples per LabTek. For HeLa cells we routinely use 1,125 µm. Increase the spot-to-
spot distance if cross-contamination is seen for different cell lines.
6 | Set the dwell time of the pins in the 384-well low volume plate (time pins stay in the spotting solution) in the contact
printer menu to 0.5 s.
7 | Set the LabTek dwell time (time pins stay on LabTek chambered coverglass) in the contact printer menu to 0.3 s.
NATURE PROTOCOLS | VOL.2 NO.2 | 2007 | 395
8 | Carry out solid pin washing between individual samples.
The procedure is setup in the following way: Pins remain in
the washing container for 10 s at room temperature. The
washing container is an inbuilt basin filled with milliQ water.
The insertion depth of the pins in the basin is adjusted to ~3
mm. Pins remain in the sonication container for 10 s at room
temperature. The sonication container is an inbuilt sonication
basin filled with milliQ water. The insertion depth of the pins
in the basin is adjusted to ~3 mm. Move the pins above the
holes of the vacuum drying array of the contact printer and
vacuum dry the pins for 10 s at room temperature.
9 | After printing, immediately place the printed LabTeks in
a gel drying box, add 50 g of drying pearls and securely and
carefully close the box. LabTek chambers need to be dried
for at least 12 h in this way before they can be used for
■ PAUSE POINT Printed LabTeks can be stored for more
than 15 months without any apparent loss of transfection
efficiencies (see Fig. 3).
Cell seeding on siRNA spotted LabTeks
10 | To aid identification of the siRNA spot-matrix on the
microscope, the first siRNA spot of the array is marked with
a permanent marker pen on the opposite side of the cell
growth area prior to cell plating.
▲ CRITICAL STEP The microscope acquires images in defined
distances related to the first spot (see Steps 5, 25 and 26).
11 | Split confluent stock cell cultures of HeLa cells (1:3)
24 h prior to their seeding on the dried siRNA LabTek arrays.
The split ratio might vary from cell type to cell type and
needs to be determined individually for alternative cell
▲ CRITICAL STEP This step helps to avoid cell clumping on
the LabTeks and further ascertains that an actively growing
cell population is plated.
12 | Following trypsinization and cell counting, plate 1.5
ml of 7.5 × 104 cells ml-1 actively growing HeLa cells in a
suspension of growth medium upon each LabTek by carefully
dispensing the cell suspension to the centre of the LabTek
using a 5 ml pipette. While dispensing, contact should be
maintained between the pipette tip and the dispensed cell
suspension to ensure spread of the cell suspension upon the
LabTek. Bubbles and any contact between the pipette tip
and the glass of the LabTek itself should be avoided as this
could disrupt the array. For other cell lines we also plate 1.5
ml of cell suspension with the following number of actively
growing cells per ml: A549, 8 × 104; U2OS, 10 × 104; hTERT–
RPE1, 10 × 104; primary human skin fibroblasts, 5 × 104;
HUVEC, 8 × 104.
Figure 3 | The penetrance for the three positive controls is plotted against
the number of days between spotting and cell seeding. For each control,
the penetrance value shown in the diagram was calculated as the average of
the penetrance values obtained for the single control experiments on each
LabTek dish. For each control experiment and time-point, the percentage of
cell nuclei showing the control-specific phenotype (see examples in Fig. 5)
in the spot of interest was determined. The penetrance value of the single
control experiment was then defined as the maximal percentage obtained.
The typical phenotypes obtained by the control siRNAs are shown in Fig. 5c–
e. Results obtained with the siRNA targeting KIF11 (kinesin family member
11) (a); results obtained with the siRNA targeting INCENP (inner centromere
protein) (b); results obtained with the siRNA targeting COPB (coat protein,
subunit beta 1) (c).
396 | VOL.2 NO.2 | 2007 | NATURE PROTOCOLS
13 | Once the cell suspension has been dispensed, gently
tilt the LabTek to each of the four corners to ensure
thorough coverage of cells across the chamber surface.
14 | Incubate cells plated on LabTeks in 1.5 ml growth
medium at 37 °C with 5% CO2.For time-lapse microscopy
we typically incubate them for 20 h. For time-lapse
microscopy replace the medium 20 h after cell seeding
with 5 ml CO2-independent growth medium just prior to
imaging and seal the LabTeks at the lid with silicon grease
to avoid drying.
Immunostaining of cell arrays
15 | Depending on the primary antibody used for
immunostaining, and the cellular structures to be labeled,
fix cells with 4% (w/v) PFA in PBS11 for 10 min at room
temperature or in 100% methanol for 2 min at –20 °C.
When fixation with methanol is used, cells already become
permeabilized such that antibodies can cross the plasma
membrane and gain access to intracellular antigen. In this
case Step 16 can be omitted. This kind of fixation is very
good for staining the cytoskleleton, the Golgi complex and
the endoplasmic reticulum. In the case of PFA fixation, the
plasma membrane stays intact and only antigene domains
accessible from the outer side of the plasma membrane can
be stained. To stain intracellular structures in PFA fixed
cells, they need to be permeabilized as described in Step 16. Which antibodies give better results by which fixation method
needs to be tested using separate experiments before applying it to the staining of cell arrays.
16 | Wash twice for 2 min at room temperature with 1 ml of 1 × PBS/0.1% (v/v) Triton solution.
17 | Incubate for 30 min at room temperature with a 3% BSA/1 × PBS (w/v) solution.
18 | Apply 250−800 µl of the first antibody solution by carefully and gently distributing it over the spotted area. Incubate
for 10 min (time may vary depending on the primary antibody used) at room temperature.
19 | Wash twice for 5 min at room temperature with 1 ml PBS.
20 | Incubate with secondary antibody as recommended by the supplier.
■ PAUSE POINT Store stained dishes either by embedding in Mowiol (ref. 11) or by adding 1 ml PBS containing 0.01% (w/v)
azide. In the latter case, cell stainings are preserved for long-term storage by a further incubation of stained cells for 2 min
at room temperature with 4% (w/v) PFA in PBS (post-staining fixation) followed by two washes with PBS.
21 | Stain cell nuclei with 1.5 ml Hoechst dye solution (1 µg ml-1 final concentration, in PBS) for 10 min at room
temperature, followed by an additional washing step with PBS for 5 min. Because Hoechst staining is a strong and robust
way to highlight cell nuclei, we routinely use this staining during image acquisition for automated identification of the focal
plane12. It also serves for the identification of all cell nuclei during automated image analysis (see for example refs 12,13).
22 | In our laboratory we image cell arrays using a scan^R fluorescence microscope (Fig. 4) equipped as described in ref. 6.
Place four LabTeks in an in-house built holder (see Fig. 4) and secure them tightly with springs.
23 | Choose objective (e.g., a 10×/0.4 air objective).
24 | Choose exposure time and filter set according to dye.
25 | Assign the first spot on each of the four LabTeks. For this, observe LabTeks in bright-field illumination, which allows
Figure 4 | The automatic microscope and substrate (LabTek) holder. The
automatic scan^R microscope with incubation chamber is shown in (a). A
drawing of the holder for four LabTek chambers is shown in (b). An image of
the holder with four LabTeks in position is shown in (c).
NATURE PROTOCOLS | VOL.2 NO.2 | 2007 | 397
clear identification of each first spot owing to its labeling
with indelible ink (see Step 10). Store each of the four
positions in the computer using the scan^R software.
▲ CRITICAL STEP The microscope acquires images in
defined distances related to the first spot (see Step 5).
26 | Assign the spot-to-spot distance with respect to the
values used for array production (see section on ‘Contact
27 | Start automated data acquisition.
Image processing and data analysis
28 | Analysis of images acquired automatically depends
strongly on the assay applied and therefore requires custom
developments or adaptation of commercial software. Perform
analysis either using the analysis package provided by the
scan^R microscope system (when fluorescence intensities
are to be measured, see also refs 12,13) or using software
that has been developed for assay-specific phenotyping of
cells6,14. As a general point, we use our scrambled controls
– siRNA not targeting any gene – (Fig. 5) to normalize our
29 | In total, we distribute 16 control siRNAs on the LabTek
(Fig. 5). These comprise negative controls (scrambled
siRNA) that have been tested not to interfere with cell
growth, mitosis and secretion. Ideally, distribute the
negative controls throughout the entire LabTek. In addition,
we have also spotted three different positive controls
targeting different genes. These siRNAs cause clearly visible
phenotypes as shown in Fig. 5. As some of these positive
control siRNAs are spotted at the edges of the array (see
Fig. 5b), identification of their phenotypes by automated
image acquisition and analysis determines the correct
positioning and alignment of the arrays in use. Only when
these phenotypes can be clearly seen on the array, it can
be assumed that the array is aligned and siRNA transfection
has in principle been successful. Characterize the positive
control siRNAs by the following phenotypes: (i) siRNA
targeting INCENP (inner centromere protein): cells exhibit
butterfly like nuclei (Fig. 5c). This shape phenotype is
visible between 20 h and 64 h after cell seeding and then
cells start to undergo cell death. This INCENP phenotype
is long lasting and is therefore suitable for assays with
different time windows. (ii) siRNA targeting KIF11 (kinesin
family member 11): cells show a prometaphase arrest starting 17 h after cell seeding (Fig. 5d). Cells stay between 12 and
20 h in this prometaphase arrest and then undergo cell death. This control is more suitable for assays investigating early
time points after cell seeding. (iii) siRNA targeting COPB (coat protein, subunit beta 1): protein secretion from cells is
inhibited and cells start to undergo cell death 40 h after cell seeding (Fig. 5e). Maximum penetrance of this phenotype
occurs between 60 and 70 h after cell seeding.
Steps 1–2: Preparation of the spotting solution: 1 h (automatically)
Steps 3–8: Contact printing: 6 h (48 LabTeks)
Figure 5 | Cell proliferation and phenotypes of control genes on a cell
array. Typical results obtained for the cell density and cell proliferation from
one LabTek in a time-lapse experiment studying cell cycle progression and
mitosis (see ref. 6) are shown. The color-coded cell numbers are shown in
a heat map 20 h after cell seeding, where each square represents one spot
(a). The color-coded proliferation rate, which was calculated by dividing
the cell number at the end of the time-lapse experiment by the number at
the beginning, is shown in (b). The axes indicate the columns and rows of
the siRNA cell array and the letters the control genes (c–f). Typical images
of cell nuclei obtained in spots containing the positive control siRNAs are
shown in (c)–(f). (c) I = INCENP (inner centromere protein); (d) K = KIF11
(kinesin family member 11); (e) B = COPB (coat protein, subunit beta 1);
(f) S = cells on a scrambled control spot. Arrows point to typical phenotype
specific cells. Scale bars = 20 µm
398 | VOL.2 NO.2 | 2007 | NATURE PROTOCOLS
Step 9: Storage: 12 h – 15 months
Steps 10–14: Cell seeding: 1 h
Steps 15–21: Immunostaining of cell arrays: 2 h
Steps 22–27: Scanning microscopy: typically 4 h for fixed time point assays and 48 h for time-lapse experiments
Step 28: Image processing and data analysis: depending on assay and processing speed
Step 29: Quality control: typically 10 min automatically
See Table 1 for troubleshooting guidance.
TABLE 1 | troubleshooting table
Step 5: Cross contamination of samples after cell seeding.
Increase spot-to-spot distance to at least 1,125 µm, 1,500 µm or
2,250 µm. These distances show no intergrid spacing.
Sonicate pins after spotting process and carefully clean holes in the
pin-head with a brush.
Allow a longer drying period.
Step 8: Spots missing on LabTek owing to one or more pins getting
stuck during the spotting procedure.
Step 9: Cross contamination of samples after cell seeding.
One of the main applications of the protocol described here is in genome-wide siRNA screens using high-content-screening
microscopy. In our laboratory, arrays produced with the described protocol have been used for two genome-wide siRNA
screens, one investigating mitosis and cell-cycle progression by time-lapse microscopy (data not shown; see also ref. 6 and
Fig. 5) and the other one studying constitutive protein secretion using quantitative microscopy in fixed cells (data not
shown), see also ref. 7). Evenness of cell seeding in LabTeks is demonstrated by Fig. 5a, showing cell numbers in the spots
20 h after seeding. In our experience, in these large-scale
experiments the fraction of arrays that failed to reproduce
the phenotypes of the positive control siRNAs is less
than 10% (Fig. 5). Furthermore, the arrays produced can
still be used even 15 months after printing without any
apparent loss in efficiency (Fig. 3). An example testing the
reproducibility of the method is shown in Fig. 6.
Tests have also shown that the arrays produced with the
protocol described here can be used for reverse transfecting
A549, U2OS, hTERT–RPE1, primary human skin fibroblasts,
HUVEC and HeLa cells.
Figure 6 | The reproducibility of the method for the reverse transfection
of cells on arrays is shown in this figure. Results of positive (KIF11 and
INCENP) and negative (scrambled) controls from three replicates of the same
LabTek are shown. The maximum percentages of cell nuclei per spot area and
per time-lapse movie, showing the specified phenotype, either ‘shape’ for
INCENP showing butterfly like nuclei or ‘prometaphase’ for KIF11 showing
condensed nuclei, (called penetrance) are shown.
ACKNOWLEDGMENTS The authors would like to acknowledge funding within the
MitoCheck consortium by the European Commission (FP6-503464 to J.E.), as well
as by the Federal Ministry of Education and Research (BMBF) in the framework of
the National Genome Research Network (NGFN) (NGFN-2 SMP-RNAi, FKZ01GR0403
to J.E. and NGFN-2 SMP-Cell FKZ01GR0423, NGFN-1 FKZ01GR0101, FKZ01KW0013
to R.P.). The J.E. and R.P. labs are supported by a grant of the Landesstiftung
Baden Wuerttemberg in the framework of the research programme ‘RNS/RNAi’.
COMPETING INTERESTS STATEMENT The authors declare that they have no
competing financial interests.
Published online at http://www.natureprotocols.com/
Reprints and permissions information is available online at http://npg.nature.
1. Pelkmans, L. et al. Genome-wide analysis of human kinases in clathrin- and
caveolae/raft-mediated endocytosis. Nature 436, 78−86 (2005).
2. Sonnichsen, B. et al. Full-genome RNAi profiling of early embryogenesis in
Caenorhabditis elegans. Nature 434, 462−469 (2005).
3. Mukherji, M. et al. Genome-wide functional analysis of human cell-cycle
regulators. PNAS 103, 14819−14824 (2006).
4. Bard, F. et al. Functional genomics reveals genes involved in protein
secretion and Golgi organization Nature 439, 604−607 (2006).
5. Ziauddin, J. & Sabatini, D.M. Microarrays of cells expressing defined cDNAs.
Nature 411, 107−110 (2001).
6. Neumann, B. et al. High-throughput RNAi screening by time-lapse imaging of
live human cells. Nature Methods 3, 385−390 (2006).
7. Erfle, H. et al. siRNA cell arrays for high-content screening microscopy.
Biotechniques. 37, 454−458, 460, 462 (2004).
8. Silva, J.M. et al. RNA interference microarrays: High-throughput loss-of-
Controls in LabTek LT0010
NATURE PROTOCOLS | VOL.2 NO.2 | 2007 | 399
function genetics in mammalian cells. PNAS 101, 6548−6552 (2004).
9. Mousses, S. et al. RNAi microarray analysis in cultured mammalian cells.
Genome Res. 13, 2341−2347 (2003).
10. Kumar, R. et al. High-throughput selection of effective RNAi probes for gene
silencing. Genome Res. 13, 2333−2340 (2003).
11. Pepperkok, R. et al. Imunofluorescence Microscopy. Monoclonal Antibodies: A
Practical Approach 355–370 (Oxford University Press, New York, 2000).
12. Liebel, U. et al. A microscope-based screening platform for large-scale
functional protein analysis in intact cells. FEBS Lett. 554, 394−398 (2003).
13. Starkuviene, V. et al. High-content screening microscopy identifies novel
proteins with a putative role in secretory membrane traffic. Genome Res. 14,
14. Conrad, C. et al. Automatic identification of subcellular phenotypes on
human cell arrays. Genome Res. 14, 1130−1136 (2004).