罗氏siRNA转染试剂英文说明书

For life science research only. Not for use

in diagnostic procedures.

X-tremeGENE siRNA Transfection Reagent

For the transfection of siRNA into animal cells

Cat. No. 04 476 093 001 1 ml

Cat. No. 04 476 115 001 Multi-pack 5 ? 1 ml (2000 transfection)y Version 05

Content version: February 2011

Store at ?2 to ?8°C

1.What this Product Does

Number of Transfection Experiments

Using standard experimental conditions, one milliliter of X-tremeGENE siRNA Transfection Reagent transfects HeLa, NIH 3T3, HEK-293 cells, or other mammalian cells in over four-hundred wells of a 24-well plate. This is equivalent to over 1,600 transfections in 96-well plates.

Formulation

X-tremeGENE siRNA Transfection Reagent is a proprietary blend of lipids and other components supplied in aqueous solution, filtered through a 0.2 ?m pore-size membrane, and packaged in polypropy-lene tubes. The reagent is free of any animal-derived components.

Storage and Stability

X-tremeGENE siRNA Transfection Reagent is shipped at +15 to +25°C and is stabilized for extended storage at +2 to +8°C until the expiration date printed on the label when tightly closed.

N Always mix X-tremeGENE siRNA Transfection Reagent prior to use (vortex for one second or invert). Do not freeze!

Additional Equipment and Reagents Required

Refer to the following list for additional reagents and equipment required for transfection experiments.

?Prewarmed sterile, serum-free culture medium (SFM) without addi-tives or supplements. Opti-MEM I Medium is recommended for dilution of the siRNA and the X-tremeGENE siRNA Transfection reagent.

?Mammalian cell line of interest.

?siRNA solution between 0.2 ?g/?l (15 pmoles/?l, [15 ?M]) and 2 ?g/?l (150 pmoles/?l, [150 ?M]) in sterile buffer.

Application

X-tremeGENE siRNA Transfection Reagent is a multicomponent reagent that forms a complex with siRNA, and then efficiently delivers it into animal cells.

Features of X-tremeGENE siRNA Transfection Reagent include:?High transfection efficiency in many common cell types, including HeLa, NIH 3T3, HEK-293, PC-3, and COS-7.

? A single reagent for siRNA- and cotransfection-based RNA experi-ments.

?Low cytotoxicity, and the media change after the addition of trans-fection complex is not required.

?Functions exceptionally well in the presence or absence of serum; eliminates the need to change media.2.How To Use this Product

2.1Before You Begin

siRNA Purity, Fluorescently Labeled siRNAs

Determine the siRNA purity using a OD 260/280 nm ratio. The ratio should be 1.9 or higher. When using siRNA preparations from in vitro transcription reactions, a size of less than 30 base pairs of the siRNA needs to be carefully controlled, as non-embryonic animal cells show an interferon response resulting in a general translation block with larger dsRNA species (1). A recent publication (1) describes that the 5′ triphosphate residues of in vitro transcribed siRNA must be removed to avoid an interferon response. Chemically synthesized siRNAs from commercial suppliers are usually sufficiently pure for transfection.

N Use fluorescently labeled siRNAs: The uptake or non uptake of flu-orescently labeled siRNAs does not necessarily correlate with the knockdown. Even with no visible uptake of labeled siRNAs, suc-cessful knockdown was demonstrated.

Cell Culture Conditions

Minimize both intra- and inter-experimental variance in transfection efficiency by using cells that are regularly passaged, proliferating well (best when in a log-phase growth), and plated at a consistent density.

Effect of Media and Media Additives, Including Sera

?In some cell types, antimicrobial agents (e.g., antibiotics and fungi-cides) that are commonly included in cell culture media may adversely affect the transfection efficiency of X-tremeGENE siRNA Transfection Reagent. If possible, exclude additives for initial experiments. Once high-efficiency conditions have been estab-lished, these components can be added back while monitoring your transfection results.

?Different media and media components may influence the level of transfection efficiency and subsequent growth of the transfected cells, as well as knockdown of the gene of interest.

L Any medium can be used for cell cultivation and during trans-fection. H owever, Opti-MEM I Medium is recommended for dilution of the siRNA and the X-tremeGENE siRNA Transfection reagent (see section 2.2).

? Test different media and optimize the level of each medium compo-nent for these effects. Although it is not usually necessary to remove the transfection reagent:siRNA complex following the transfection step, it is necessary to feed your cells with fresh media for extended growth periods. This is particularly important when the transfected cells are allowed to grow for 3–7 days for maximal knockdown with very stable proteins.

Verification of Transfection Efficiency

As the knockdown efficiency is dependent on the gene as well as on the cell line, determination of the transfection efficiency is recom-mended when using a new cell line. The knockdown of the endoge-nous human HPRT housekeeping gene can be measured with qPCR. The knockdown of the endogenous lamin A/C gene with anti-lamin A/C antibodies (BD Biosciences) was demonstrated in western blots.

Incubation Time

Incubate the cells for 24–72 hours. The length of incubation depends upon the siRNA, cell type being transfected, stability of the mRNA, or the protein being targeted. After this incubation period, measure the knockdown using an assay that is appropriate for your system.

2.2Procedure

Preparation of Cells for Transfection

One day prior to the transfection experiment, trypsinize, adjust the cell concentration, and plate the cells in the chosen cell culture vessel. For most cell types, plating 0.1 – 0.8 x 105 cells in a 24-well plate in 0.45 ml of medium overnight will achieve the desired density of 30–50% con-fluency. If using culture plates of a different size, adjust the starting volume of X-tremeGENE siRNA Transfection Reagent and the starting mass of siRNA in proportion to the relative surface area (see Table 1).

Ratio Overview

Preparation of a complex that is sufficient for a single well of a 24-well plate at three different concentrations.

Preparation of X-tremeGENE siRNA Transfection Reagent: siRNA Complex and Transfection of Cells in a 24-well Plate

For initial optimization, use 10:2, 2.5:0.5, and 1:0.2 ratios of X-trem-eGENE siRNA Transfection Reagent (μl) to siRNA (μg), respectively. The preparation of the complex for a single well of a 24-well plate is described below. These ratios will function very well for commonly used adherent cells.

N The X-tremeGENE siRNA Transfection Reagent:siRNA complex must be prepared in medium that does not contain serum, even if the cells are transfected in the presence of serum.

L For additional optimization tips, visit

https://www.sodocs.net/doc/8814811262.html,/

1Dilute X-tremeGENE siRNA Transfection Reagent with serum-free Opti-MEM I Medium (without antibiotics or

fungicides).

N Serum-free medium must be pipetted first. The order and manner of addition is critical.

?Label three small sterile tubes: 10, 2.5, and 1. Pipet 40 ?l of

serum-free medium into the first, 47.5 ?l into the second, and

49 μl into the last tube.

?Pipet the X-tremeGENE siRNA Transfection Reagent directly

into the medium without allowing contact with the walls of the

plastic tube: 10 ?l X-tremeGENE siRNA Transfection Reagent

into the first tube, 2.5 ?l of the reagent into the tube labeled

2.5, and 1 ?l of the reagent into the tube labeled 1.

Tube label SFM X-tremeGENE siRNA

Transfection Reagent (?l) 104010

2.547.5 2.5

1491

?Mix cautiously by pipetting up and down.

N Do not vortex!

N To avoid adversely affecting transfection efficiency, do not allow undiluted X tremeGENE siRNA Transfection Reagent

to come into contact with plastic surfaces (such as the walls

of the tube containing the serum-free medium) other than

pipette tips. Diluted siRNA should be combined with trans-

fection reagent (step 3) within 5 minutes.2Dilute siRNA with serum-free Opti-MEM I Medium (without antibiotics or fungicides).

N Serum-free medium must be pipetted first. The order and manner of addition is critical.

Label three small sterile tubes:

50 + 2, 50 + 0.5, and 50 + 0.2.

Pipet serum-free medium into all tubes to yield a final volume of

50 ?l.

Tube label SFM (μl) + siRNA (?g)Final volume (?l)

50 + 250 + 2

(~150 pmoles)

50

50 + 0.550 + 0.5

(~40 pmoles)

50

50 + 0.250 + 0.2

(~15 pmoles)

50

?Pipet the siRNA directly into the medium without allowing con-

tact with the walls of the plastic tube: 2 ?g siRNA into the first

tube, 0.5 ?g of the siRNA into the tube labeled 50 + 0.5, and

0.2 ?g of the siRNA into the tube labeled 50 + 0.2.

?Mix cautiously by pipetting up and down.

N Do not vortex!

N To avoid adversely affecting transfection efficiency, do not allow undiluted siRNA to come into contact with plastic sur-

faces (such as the walls of the tube containing the

serum-free medium) other than pipette tips.

Diluted siRNA should be combined with transfection reagent

(step 3) within 5 minutes!

3Mix and incubate the complex.

?Mix the contents of the tube from Step 1 with that of Step 2 in

the following order:

- Tube labeled 10 with 50 + 2

- Tube labeled 2.5 with 50 + 0.5

- Tube labeled 1 with 50 + 0.2

?Mix cautiously by pipetting up and down.

N Do not vortex!

?Incubate the transfection reagent: siRNA complex for 15 -

20 minutes at +15 to +25°C.

?Add the entire volume to each well of a 24-well plate.

4Add complex to the cells.

?Remove culture vessel from the incubator.

?Removal of growth medium is not necessary.

?Add the transfection reagent: siRNA complex dropwise to the

cells, and swirl the wells or flasks cautiously to ensure distribu-

tion over the entire plate/flask surface.

5Return the cells to the incubator until the assay for gene knock-down is performed. Once the X-tremeGENE siRNA Transfection Reagent:siRNA complex has been added to the cells, there is no need to remove and replace with fresh medium (as is necessary with some other transfection reagents).

L The exposure of most common laboratory cell types (COS-7, NIH 3T3, HEK-293, HeLa) to the reagent:siRNA complex until

measurement of the gene-knockdown (24–72 hours later)

does not affect the results. When using serum-free medium

during the transfection procedure (step 4), replace the

medium with serum-containing medium 3–8 hours after

transfection.

Optimizing Ratio of X-tremeGENE siRNA Transfection Reagent, siRNA, and Plasmid DNA in Various Plate Formats Refer to Table 1 when setting up your transfection reactions in other plate formats. The starting volume and mass is based on a

X-tremeGENE siRNA Transfection Reagent:siRNA ratio of 5:1. The ranges cover mutiple ratios. When varying the siRNA concentration, keep the X-tremeGENE siRNA Transfection Reagent in a ratio of 2-10:1 to the siRNA mass.

Culture plate diameter (mm)96-well

plate

24-well

plate

6-well

plate

Surface area cm20.4 1.99

Plated cells [?105] Starting point 0.05-0.2

0.1

0.1-0.8

0.4

1-4

2

Medium volume (ml)0.150.45 2.0

siRNA (?g) range Starting point (?g) Dilution volume (?l)0.05-0.3

0.15

15

0.1-1.0

0.5 (~40 pmoles)

50

0.4-5.0

2.0

100

X-tremeGENE siRNA Transfection Reagent (?l) Starting point (?l) Dilution volume (?l)0.1-4.0

0.8

15

0.5-10

2.5

50

2-35

10

100

Total volume (ml) 0.180.55 2.2

Table 1.

Co-Transfection Experiments

The X-tremeGENE siRNA Transfection Reagent has the potential for co-transfection of short inhibitory RNAs and plasmid DNA. Usually transfecting plasmids requires a higher cell density at the point of transfection compared to siRNA. Note the following suggestions when performing co-transfection experiments:

?Plate cells such that they reach 90% confluency at the time of trans-fection.

?Maintain the same total reagent:total nucleic acid ratio as that used for siRNA alone in your system. If you need to increase the total amount of nucleic acid, then increase the amount of transfection reagent in proportion to the total amount of nucleic acid (?g).?Use Table 2 for optimizing X-tremeGENE siRNA Transfection Reagent, siRNA, and plasmid DNA in the various plate formats.

N Always use a volume of X-tremeGENE siRNA Transfection Reagent that is at least 3-fold in excess of the total final mass of nucleic acid.

Culture plate diameter (mm)96-well

plate

24-well

plate

6-well

plate

Surface area cm20.4 1.99

Plated cells [?105] Starting point 0.05 - 0.2

0.1

0.1 - 0.8

0.4

1 - 4

2

Medium volume (ml)0.150.45 2.0

siRNA (?g) range Starting point (?g)0.05 - 0.3

0.08

0.1 - 1.0

0.25

0.4 - 5.0

1.0

DNA (?g)0.150.5 2.0 Dilution Volume1550100

X-tremeGENE siRNA Transfection Reagent (?l) Starting point (?l) Dilution volume (?l)0.1 - 4.0

0.8

15

0.5 - 10

2.5

50

2 - 35

10

100

Total volume (ml)0.180.55 2.2 Table 2.3.Troubleshooting

Observations Possible Cause Recommendation

Low/No

knockdown

levels

observed

Low transfection efficiency.

Cells were grown

confluent at the time

of transfection.

Seed cells at a lower density so

they reach 30-50% confluency at

the time of transfection.

Cells passaged too

often or used from

stationary phase.

Use cells with low passage num-

ber and only cell cultures that

were regularly passaged at log

phase.

Not enough siRNA

used.

Increase the amount of siRNA.

Not enough

X-tremeGENE siRNA

Transfection

Reagent used.

Optimize transfection conditions

for each cell line by varying the

amount of siRNA:X-tremeGENE

transfection complex and the

ratio of siRNA:X-tremeGENE

siRNA Transfection Reagent.

Antibiotics added

during transfection.

Do not add antibiotics during

transfection.

Serum present

during siRNA:

X-tremeGENE

complex formation.

Use serum-free medium during

complex formation.

siRNA not active.

Target sequence not

suitable.

Select another target region.

d-siRNA degraded.Check integrity of siRNA on poly-

acrylamide or agarose gels.

Do not store siRNA in water.

Use a sterile RNAse-free buffer

containing 10 mM Tris, pH 8.0, 20

mM NaCl, 1 mM EDTA for stor-

age.

Store siRNA aliquoted at

-15 to -25°C and avoid repeated

freeze/thaw cycles.

Large cellular

amounts or high sta-

bility of the targeted

mRNA or protein.

Perform a time course experiment

and determine the time when the

highest degree of knockdown is

obtained.

Perform qPCR analysis (e.g.,

using the LightCycler? Instru-

ment) to measure mRNA levels

when only low knockdown on

protein level is observed.

Repeat the addition of siRNA:

X-tremeGENE transfection com-

plex and refresh medium for

every long-lived target protein

species.

Cytotoxic

effects after

transfection

observed

Too much

X-tremeGENE

siRNA Transfection

Reagent used.

Reduce/optimize amounts of

X-tremeGENE siRNA Transfec-

tion Reagent for each cell line.

Cells are very

sensitive to

transfection.

Remove transfection medium and

add new prewarmed serum con-

taining medium after 4-6 hours.

This will not reduce transfection

efficiency.

Unpurified d-siRNA

used for transfection.

To avoid a general translation

block and initiation of apoptosis,

carefully purified siRNA of less

than 30 nucleotides is necessary

for most somatic mammalian cell

lines (7, 8).

Nonspecific

off-target

gene

knockdown

observed

Target sequence

siRNA sense or anti-

sense strand con-

tains strong

homology to other

genes.

Select a new target sequence.

Lower the concentration of the

siRNA.

Limit the size of the target

sequence to <1.0 kb when using

d-siRNA.

4.Additional Information on this Product

Background Information

The RNAi Pathway

RNA interference (RNAi) is the process of sequence specific, post-transcriptional gene silencing in animals and plants, induced by double-stranded RNA (dsRNA) that triggers the degradation of com-plementary mRNA. In eukaryotic organisms, in vivo or in vitro gener-ated long double-stranded RNAs are cleaved into 21-23 nucleotide long short interfering RNAs (siRNA) by a RNAse III like enzyme activity called DICER (2). The siRNA is then taken up by the RNA-induced silencing complex (RISC), which anneals one of the siRNA strands to the complementary region of the mRNA and finally cleaves the mRNA

(3).

References

1Kim, D-H et al (2004) Nat. Biotech.22, 321-325.

2Bernstein, E. et al (2001) Nature409, 363-366.

3Hammond, S.M. et al (2000) Nature404, 293-296.

4Bass, B.L. (2001) Nature411, 428-429.

5Hannon, G.J (2002) Nature418, 244-251.

6Kaufmann, R.J. (1999) Proc. Natl. Acad. Sci. USA96, 11693-11695.

7Stark, G.R. et al (1998) Annu. Rev. Biochem.67, 227-264.

Quality Control

Activity assay: 1-2.5 μl of X-tremeGENE siRNA Transfection Reagent is combined with 0.1-0.35 μg of siRNA against the HPRT housekeep-ing gene, and used to transfect COS-7 cells (in a monolayer [30–50% confluent]) in the presence of 10% fetal bovine serum (FBS). Following transfection, the decrease of HPRT mRNA cells is analyzed with the LightCycler? Instrument. Typically, a knockdown between 80% - 95 % is observed.

Cytotoxicity analysis: X-tremeGENE siRNA Transfection Reagent is routinely controlled for cytotoxicity with the WST-1 Cell Proliferation Reagent on COS-7 cells that are exposed to siRNA/X-tremeGENE complexes for 72 hours in the presence of serum and without a change of the medium.

Absence of microbial contamination: The absence of microbial contamination is verified by a 1 week incubation of 25 μl of X-tremeGENE siRNA Transfection Reagent.Conventions

Text Conventions

To make information consistent and memorable, the following text conventions are used in this Package Insert:

Text Convention Usage

Numbered stages

labeled ?, ? etc.

Stages in a process that usually occur in

the order listed.

Numbered instructions

labeled ?, ? etc.

Steps in a procedure that must be per-

formed in the order listed.

Asterisk *Denotes a product available from Roche

Applied Science.

Symbols

In this Instruction Manual, the following symbols are used to highlight important information:

Symbol Description

L

Information Note:

Additional information about the current topic or proce-

dure.

N

Important Note:

Information critical to the success of the procedure or use

of the product.

Changes to Previous Version

?New Ordering Information

?Editorial changes

Ordering Information

Roche Applied Science provides a large selection of reagents and sys-tems for life science research. For a complete overview of related products and manuals, visit and bookmark our homepage, https://www.sodocs.net/doc/8814811262.html,, and our Special Interest Site on transfection, https://www.sodocs.net/doc/8814811262.html,.

Product Pack Size Cat. No. Apoptosis and Cell Death

Cell Proliferation

Reagent WST-1

8 ml (800 tests)

25 ml (2,500 tests)

05 015 944 001

11 644 807 001 Cytotoxicity Detection

Kit PLUS (LDH)

1 kit (400 tests in

96 wells)

1 kit (2,000 tests in

96 wells)

04 744 926 001

04 744 934 001

Mycoplasma Detection

Mycoplasma Detection

Kit

1 kit (25 tests)11 296 744 001 Mycoplasma PCR ELISA1 kit (96 reactions)11 663 925 910 Plasmid Isolation

Genopure Plasmid Midi

Kit

1 kit (for up to 20 prepara-

tions)

03 143 414 001

Genopure Plasmid Maxi

Kit

1 kit (for up to 10 prepara-

tions)

03 143 422 001 Protease Inhibitor T ablets and Lysis Reagents

cOmplete20 tablets in glass vials

3 x 20 tablets in glass vials

20 tablets in EASYpacks

11 697 498 001

11 836 145 001

04 693 116 001 cOmplete, EDTA-free20 tablets in a glass vial

3 x 20 tablets in glass vials

20 tablets in EASYpacks

11 873 580 001

05 056 489 001

04 693 132 001 cOmplete Lysis-M (for

mammalian cell lysis)

1 kit (200 ml lysis reagent

and 20 complete Protease

Inhibitor Cocktail Tablets)

04 719 956 001

Roche Diagnostics GmbH Roche Applied Science 68298 Mannheim Germany

Contact and Support

To ask questions, solve problems, suggest enhancements or report new applications, please visit our Online Technical Support Site at:

https://www.sodocs.net/doc/8814811262.html,/support

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Trademarks

LIGHTCYCLER, LC, COMPLETE, GENOPURE, X-TREMEGENE, CASY, CEDEX, and CELLAVISTA are trademarks of Roche.

E-PLATE and ACEA BIOSCIENCES are registered trademarks of ACEA Biosciences, Inc. in the US.

Other brands or product names are trademarks of their respective holders.

Regulatory Disclaimer

For life science research only. Not for use in diagnostic procedures.

cOmplete Lysis-M,EDTA-free (for mammalian cell lysis) 1 kit (200 ml lysis reagent and 20 complete, EDTA-free Protease Inhibi-tor Cocktail Tablets)04 719 964 001Reporter Gene Assays CAT ELISA

1 kit (19

2 tests)

11 363 727 001

?-Gal Reporter Gene Assay,

chemiluminescent 1 kit (500 assays, micro-plate format, 250 assays, tube format)11 758 241 001?-Gal ELISA 1 kit (192 tests)11 539 426 001hGH ELISA

1 kit (19

2 tests)

11 585 878 001Luciferase Reporter Gene Assay, high sensi-tivity

200 assays 1,000 assays 11 669 893 00111 814 036 001

SEAP Reporter Gene Assay,

chemiluminescent 1 kit (500 assays, micro-plate format, or 250 assays, tube format)

11 779 842 001Selection Antibiotics G-418 Solution 20 ml 100 ml 04 727 878 00104 727 894 001Hygromycin B 1 g (20 ml)

10 843 555 001Transfection X-tremeGENE 9 DNA Transfection Reagent

0.4 ml 1 ml 5 x 1 ml 06 365 779 00106 365 787 00106 365 809 001X-tremeGENE HP DNA Transfection Reagent 0.4 ml 1 ml

5 x 1 ml 0

6 366 244 00106 366 236 00106 366 546 001Western Blotting

Lumi-Light PLUS Western Blotting Kit

(Mouse/Rabbit)

1 kit

(1,000 cm 2 membrane)12 015 218 001

Lumi-Light PLUS Western Blotting Substrate

100 ml

(1,000 cm 2 membrane)12 015 196 001PVDF Western Blotting Membranes 1 roll

(30 cm × 3.00 m)03 010 040 001Western Blocking Reagent, Solution

100 ml

(10 blots, 100 cm 2 membrane)6 × 100 ml

(60 blots, 100 cm 2 membrane)

11 921 673 00111 921 681 001

Cellular Analysis RTCA Analyzer 05 228 972 001RTCA SP Station 05 229 057 001RTCA MP Station 05 331 625 001RTCA Control Unit 1.105 454 417 001E-Plate 96 6 Units 6 x 6 Units 05 232 368 001 05 232 376 001E-Plate VIEW 96 6 Units 6 x 6 Units

06 472 451 001 06 472 460 001Cellavista Basic System Magnification: 4x, 10x Illumination: Brightfield only

05 651 522 001

Cellavista Medium System

Magnification: 4x, 10x, 20x

Illumination: Brightfield and Fluorescence, UV, Blue, Green

05 651 549 001

Product Pack Size Cat. No.Cellavista High End System

Magnification: 2x, 4x, 10x, 20x, 40x

Illumination: Brightfield and Fluorescence,UV, Blue, Cyan, Green, Amber, Red

05 651 557 001

Cedex XS Analyzer with Control Unit 05 926 432 001

Cedex Smart Slides 15 x 8

measurements

05 650 801 001CASY Model TT 45, 60, 150 μm

05 651 735 001

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Pack Size Cat. No.