Optimized Protocol sirna Test Kit for Cell Lines and Adherent Primary Cells

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› Optimized Protocol

› siRNA Test Kit for Cell Lines and Adherent Primary Cells

DSC-1001 Vs. 06-2004

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Data to come Data to come

siRNA Test Kit

for Cell Lines and Adherent Primary Cells

Kit principle Co-transfection of pmaxGFP, encoding the green fluorescent protein (GFP) from Pon-tellina p. with an siRNA directed against maxGFP in your cells of interest. Successful gene silencing is monitored as decrease of green fluorescence compared to control sample using fluorescence microscopy.

Note This kit has been designed to enable an easy set-up of siRNA transfections in your

cells of interest. In subsequent siRNA experiments, it can be used as a positive con-trol. The kit is not suitable for use with primary blood cells for which we recommend a separate protocol.

Example of siRNA mediated gene silencing of maxGFP

pmaxGFP 0.5 µg 1 µg 2 µg

no siRNA

+1.5µg siRNA

Chapter Contents

1 Procedure outline & important advice

2 Product description

3 Additional information

3.1

›

General remarks

3.2

›

Initial experimental set-up

3.3

›

Cell culture

3.4

›

Information about pmaxGFP

4 Protocol for suspension cells

NIH3T3 cells (ATTC) were nucleofected with 0.5, 1 or 2 µg of pmaxGFP only or 1.5 µg siRNA directed against max GFP, in additon. Gene silencing of maxGFP expression was monitored by fluorescence microscopy, 24 hours post-nucleofection.

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Choose the appropriate Nucleofector™ Kit and Optimized Protocol for cell type of interest.

Use recommended NucleofectorTM

pro-gram for siRNA transfections.

Use siRNA Test Kit to establish siRNA in your cells of interest.

Perform gene silencing with your gene of interest and include siRNA Test Kit as a positive control.

Use Cell Line Optimization Nucleofector™ Kit.

Optimize nucleofection conditions with pmaxGFP plasmid according to the opti-mization strategy:

Use optimized nucleofection conditions

(i.e. NucleofectorTM _{Solution and}

pro-gram) to establish siRNA experiments.

Use siRNA Test Kit to establish siRNA in your cells of interest.

Perform gene silencing with your gene of interest and include siRNA Test Kit as a positive control.

DSC-1001 Vs. 06-2004 page 2 of 8 cell lines + Optimized Protocol available / yes Optimized Protocol available / no primary cells maxGFP (3486 bp)

A simple strategy to set up siRNA in your cells of interest

Solution R T V program 1 A - 2 3 A - 2 3 A - 2 3 program 2 A - 2 7 A - 2 7 A - 2 7 program 3 T - 2 0 T - 2 0 T - 2 0 program 4 T - 2 7 T - 2 7 T - 2 7 program 5 T - 1 6 T - 1 6 T - 1 6 program 6 T - 0 1 T - 0 1 T - 0 1 program 7 G - 1 6 G - 1 6 G - 1 6 program 8 O - 1 7 O - 1 7 O - 1 7

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sample 1 sample 2 sample 3 sample 4 sample 5 sample 6 sample 7 sample 8 sample 9

pmaxGFP 0.5 µg 0.5 µg 0.5 µg 1 µg 1 µg 1 µg 2 µg 2 µg 2 µg

siRNA - 1.5 µg 1.5 µg* - 1.5 µg 1.5 µg* - 1.5 µg 1.5µg*

2

Product description

Cat. No. VSC-1001

Kit components 75 µg siRNA against maxGFP#

9 ml siRNA Suspension Buffer*

100 µg pmaxGFP (0.5 µg/µl in 10mM Tris pH 8.0)

Size 25-50 reactions

Storage and stability Store all reagents at -20°C. Repeated freeze-thaw cycles will not interfere with the siRNA

sample (dissolved or lyophilized) as long as RNAse-free conditions are strictly maintained. After thawing, spin the tube briefly, to bring contents to the bottom of the tube.

The expiry date is printed on the Kit Label.

3

Protocol

3.1

›

General remarks

›

Using the NucleofectorTM_{technology, siRNA and DNA are transfected using the}

same conditions, i.e. NucleofectorTM_{Solution and program. If the optimal}

nucleo-fection conditions are not known, e.g. for a specific cell line, we recommend using our Cell Line Optimization Kit (Cat.No. VCO-1001) to determine those.

›

Generally, it is not advisable to optimize nucleofection conditions using

fluor-escently labeled siRNA duplexes. Unless analyzed by confocal fluorescence microscopy, exact transfection efficiencies are difficult to determine.

›

The easiest way to monitor gene silencing is by fluorescence microscopy.

Flow cytometry can be used for quantitative analysis. In this case, gene silen-cing is often easier to detect by monitoring the decrease in the mean fluore-scence intensity instead of maxGFP expressing cells .

3.2

›

Initial experimental set-up

As the RNAi gene silencing mechanism varies with every cell type, we recommend testing three different amounts of DNA together with a fixed amount of siRNA duplex in an initial experiment. For details, see the table below.

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Kanamycin

pUC ori

DSC-1001 Vs. 06-2004 page 4 of 8 Esp 3l (2667) BspTl (1891) Eco31l (1896) Esp3l (1909) Bglll (1676) Xhol (1680) Sacl (1687) Kpnl (980) Nhel (988) Eco47lll (993) Agel (997) Esp 3l (7) Eco 31l (18) Nsil (27) 3.3

›

Cell culture

For detailed information on cell culture conditions and cell numbers please refer to the cell-type specific Optimized Protocol or contact amaxa's Scientific Support Team for further assistance.

3.4

›

Information about pmaxGFP

pmaxGFP encodes the green fluorescent protein (GFP) from copepod Potellina p. The fluorescence intensity of pmaxGFP is comparable to, or slightly exceeds, that of eGFP.

4 Protocol for suspension cell lines

4.1 ›

Preparation of siRNA

Preparation of Always work in an RNase-free environment.

siRNA sample

›

Add 250 µl of the siRNA Suspension Buffer to the lyophilized siRNA to obtain a

20 µM solution (0.30 µg/µl).

›

Heat the tube to 90o_{C for 1 min.}

›

Incubate at 37o_{C for 60 min.}

This procedure will disrupt higher aggregates which may have formed during the lyo-philization process. It is necessary to maximize siRNA silencing potential. You can

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4.2

›

Nucleofection protocol

One nucleofection

›

2x105_{- 5x10}6_cells sample contains

›

0.5, 1 or 2 _µg pmax GFP

›

1.5 _µg siRNA (i.e. 5 _µl)

›

100 _µl NucleofectorTM_Solution

Preparation of 1. Cultivate the required number of cells (2x105_{to 5x10}6_{cells). For details see the} cell-samples type specific Optimized Protocol (www.amaxa.com/protocols).

2. Prepare the required amount of siRNA and pmaxGFP.

3. Pre-warm the cell-type specific supplemented Nucleofector™ Solution to room tem-perature. Pre-warm an aliquot of culture medium containing serum and

supple-ments at 37°C in a 50 ml tube (500 _µl per sample).

4. Prepare 12-well plates by filling the appropriate number of wells with 1 ml of culture medium containing serum and supplements and pre-incubate plates in a humidified 37°C/5% CO

2incubator.

5. Take an aliquot of cell culture and count the cells to determine the cell density.

6. Centrifuge the required number of cells (2x105_{- 5x10}6 _{cells per sample) at}

90-200xg for 10 min. Discard supernatant completely so that no residual medium covers the cell pellet.

7. Resuspend the pellet in appropriate room temperature Nucleofector™ Solution to a

final concentration of 2x105 _{- 5x10}6_cells/100_µ_{l. Avoid storing the cell suspension}

longer than 15 min in NucleofectorTM_{Solution, as this reduces cell viability and gene}

transfer efficiency.

8. Mix 100 _µl of cell suspension with 1.5 _µg siRNA (5 _µl) and 0.5 - 2 _µg pmaxGFP as

recommended in table under 3.2

IImmppoorrttaanntt:: SStteeppss 99--1122 sshhoouulldd bbee ppeerrffoorrmmeedd ffoorr eeaacchh ssaammppllee sseeppaarraatteellyy..

Nucleofection 9. Transfer the sample into an amaxa certified cuvette. Make sure that the sample covers the bottom of the cuvette, avoid air bubbles while pipetting. Close the cuvette with the blue cap.

10. Select the appropriate NucleofectorTM _{program (see Nucleofector}TM_{Manual for}

details). Insert the cuvette into the cuvette holder (NucleofectorTM _{I : rotate carousel}

to final position) and press the “X” button to start the program.

11. TToo aavvooiidd ddaammaaggee ttoo tthhee cceellllss rreemmoovvee tthhee ssaammppllee ffrroomm tthhee ccuuvveettttee iimmmmeeddiiaatteellyy

a

afftteerr tthhee pprrooggrraamm hhaass ffiinniisshheedd (display showing "OK"). Take the cuvette out of the

holder. To transfer the cells from the cuvettes, we strongly recommend using the

plastic pipettes provided in the kit to prevent damage and loss of cells. Add 500 _µl

of the pre-warmed culture medium containing serum and supplements to the

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DSC-1001 Vs. 06-2004

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te and transfer the sample into the prepared 12-well plates. Alternatively, transfer the sample into a 1.5 ml microcentrifuge tube and place it in a 37°C heat block.

12.Press the “X” button to reset the NucleofectorTM_.

13.Repeat steps 9-12 for the remaining samples.

Cultivation 14.If you have incubated the samples in 1.5 ml microcentrifuge tubes transfer them into

post nucleofection the prepared 12-well plates.

15.Incubate cells in a humidified 37°/5% CO

2incubator. Analyze gene silencing after

24 hours by fluorescence microscopy.

5

› Protocol for adherent cell lines

4.1 5.1

5.1

›

Preparation of siRNA

Preparation of Always work in an RNase-free environment

siRNA sample

›

Add 250 µl of the siRNA Suspension Bufferto the lyophilized siRNA to obtain a

20 µM solution (0.3 µg/µl).

›

Heat the tube to 90o_{C for 1 min.}

›

Incubate at 37o_{C for 60 min.}

Perform your experiment or store at 20o_{C This procedure will disrupt higher}

aggrega-tes, which may have formed during the lyophilization process. It is necessary to maxi-mize siRNA silencing potential. You can perform your experiment directly or store

siRNA stock at -20o_C.

5.2

›

Nucleofection Protocol One nucleofection

›

2x105_{- 5x10}6_cells.

sample contains

›

0.5, 1 or 2 µg pmax GFP.

›

1.5 µg siRNA (i.e. 5 µl).

›

100 µl NucleofectorTM_Solution.

Preparation of 1. Cultivate the required number of cells (2x105_{to 5x10}6_{cells). For details see the cell} samples type specific Optimized Protocol (www.amaxa.com/protocols).

2. Prepare the required amount of siRNA and pmaxGFP.

3. Pre-warm the cell-type specific supplemented Nucleofector™ Solution to room tem-perature. Pre-warm an aliquot of culture medium containing serum and

supple-ments at 37°C in a 50 ml tube (500 _µl per sample).

4. Prepare 6-well plates by filling the appropriate number of wells with 1.5 ml of culture medium containing serum and supplements and pre-incubate plates in a humidified

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37°C/5% CO

2incubator.

5. Remove the medium from the cultured cells. Wash cells once with PBS. Aspirate and discard PBS.

6. Harvest the cells, e.g. with trypsin/EDTA and stop the trypsinization with

supplemen-ted culture medium or PBS/0.5% BSA (see NucleofectorTM_{Manual, for details).}

7. Take an aliquot of the trypsinized cell suspension and count the cells to determine the cell density.

8. Centrifuge the required number of cells at 200xg for 10 min. Discard supernatant completely so that no residual medium covers the cell pellet.

9. Resuspend the pellet in appropriate room temperature Nucleofector™ Solution to a

final concentration of 2x105_{- 5x10}6_cells/100_µ_{l. Avoid storing the cell suspension}

longer than 15 min in NucleofectorTM_{Solution, as this reduces cell viability and gene}

transfer efficiency.

10.Mix 100 _µl of cell suspension with 1.5 _µg siRNA (5 _µl) and 0.5 - 2 _µg pmax GFP as

recommended in table under 3.2

IImmppoorrttaanntt:: SStteeppss 1111--1144 sshhoouulldd bbee ppeerrffoorrmmeedd ffoorr eeaacchh ssaammppllee sseeppaarraatteellyy..

Nucleofection 11. Transfer the sample into an amaxa certified cuvette. Make sure that the sample covers the bottom of the cuvette, avoid air bubbles while pipetting. Close the cuvette with the blue cap.

12. Select the appropriate NucleofectorTM _{program (see Nucleofector}TM_{Manual for}

details). Insert the cuvette into the cuvette holder (NucleofectorTM _{I : rotate carousel}

to final position) and press the “X” button to start the program.

13.TToo aavvooiidd ddaammaaggee ttoo tthhee cceellllss rreemmoovvee tthhee ssaammppllee ffrroomm tthhee ccuuvveettttee iimmmmeeddiiaatteellyy

a

afftteerr tthhee pprrooggrraamm hhaass ffiinniisshheedd (display showing "OK"). Take the cuvette out of the

holder. To transfer the cells from the cuvettes, we strongly recommend using the

plastic pipettes provided in the kit to prevent damage and loss of cells. Add 500 _µl of

the pre-warmed culture medium containing serum and supplements to the cuvette and transfer the sample into the prepared 6-well plates. Alternatively, transfer the sample into a 1.5 ml microcentrifuge tube and place it in a 37°C heat block.

14.Press the “X” button to reset the NucleofectorTM_.

15.Repeat steps 11-14 for the remaining samples.

Cultivation 16.If you have incubated the samples in 1.5 ml microcentrifuge tubes transfer them into

post nucleofection the prepared 6-well plates.

17. Incubate cells in a humidified 37°/5% CO

2incubator. Analyze gene silencing after

24 hours by fluorescence microscopy.

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DSC-1001 Vs. 06-2004

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This kit contains a proprietary nucleic acid coding for a proprietary copepod fluorescent protein intended to be used as a positive control with this amaxa product only. Any use of the proprietary nucleic acid or protein other than as a positive control with this amaxa product is strictly prohibited. USE IN ANY OTHER APPLICATION REQUIRES A LICENSE FROM EVROGEN. To obtain such a license, please contact Evrogen at [email protected]

The CMV promoter is covered under U.S. Patents 5,168,062 and 5,385,839 and its use is permitted for research purposes only. Any other use of the CMV promoter requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA.

#

# Manufactured by QIAGEN® for distribution by amaxa.