Technical Tip

The artificial uptake of new genetic material by mammalian cells is called transfection, or DNA transfer, to distinguish it from the natural process, transformation. Vaheri and Pagano first described in 1965, the transfection of cells using DEAE-dextran. The second method, coprecipitation with calcium phosphate, established by Graham and Van der Eb in 1973, is still widely used. A new generation of transfection reagents, multicomponent transfection reagents (e.g., the Roche Applied Science X-tremeGENE DNA Transfection Reagents), has shown outstanding performance. These newer transfection reagents can transfect cells efficiently and are biodegradable, resulting in significantly reduced cytotoxicity.

Successful Transfection

Published byRoche Diagnostics GmbHSandhofer Straße 11668305 MannheimGermany

© 2011 Roche Diagnostics. All rights reserved.

06341314001  0211

More recently, transfection is not only used to deliver DNA into the cells, but also RNA, proteins and biologically active macromolecules. The transfection process is now an integral part of many application fields focused on under-standing gene function and regulation, gene silencing, as well as protein expression and function.

1. Maximizing transfection efficiency

No single transfection protocol can be applied to all types of cells and experiments. For each condition, an optimization step is required to obtain the desired level of transfection. Depending on the assay, you may want to reach a very high level of transfection efficiency (e.g., for the production of protein in large scale) or a lower transfection efficiency (e.g., for morphological studies).

The critical variables are: Condition of cell culture��Quality of the material to be transfected �� (e.g., plasmid DNA)Transfection method/technique��

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2. Key Cell Parameters

Three parameters are very important for the success of your transfection:

Poorly mixed cells to be transfected are pipetted��Pipette is not calibrated�� For multichannel pipettes, different volumes in each ��channelIncorrect volume or cell number calculations��Limited experience with new cell types ��

(with special needs for cell titration)

Tip: The Roche Applied Science Cedex and CASY Cell Coun-ters and Analyzers standardize cell counting for accurate and reliable calculations.Tip: The Roche Applied Science xCELLigence System of Real-Time Cell Analyzer (RTCA) Instruments continually measure the growth characteristics of your cells in real-time, allowing rapid:

Pinpointing of the growth phase verifying cell status�� Determining when cell up- and downscaling are appropriate��Identifying the presence of cell clumps ��

a) Main situations which can lead to the wrong number of cells used

b) Status of the cells

Subculturing

Cell density

Mycoplasma contamination-free

Tip: Use normally proliferating cells with a low passage number. Accurately evaluate the influence of passage number on cell behavior using the xCELLigence System from Roche Applied Science.

Tip: Use a monolayer of cells that is 70 to 90 % confluent to quickly optimize transfection using Roche’s X-tremeGENE 9 or HP DNA Transfection Reagents.

Tip: Use the Roche Applied Science Mycoplasma PCR ELISA to verify that your cell cultures are free of Myco-plasms.

c) Cell viability

Knowing the number of healthy cells is essential for deter-mining the number of cells to be used for a given assay. Vital dyes, such as trypan blue are still widely used. It is also possible to measure metabolic activity by incubating cells with tetrazolium salts using the Roche Cell Proliferation Reagent WST-1.

Important Note: Apply trypsin only until cells begin to detach, usually 1-2 minutes at +37°C. Too long a trypsin treatment negatively affects cell viability. Too short a treatment can produce cell clumps or aggregates.

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3. Monitoring Changes in Transfection Effficiency

Reagents: Compare the effect of old and new lots of all reagents ��used for cell culture Verify reagent expiration dates and appropriate storage ��conditions

Cells: Signs of contamination (�� e.g., color of media, change of morphology, growth speed)Density of cell culture ��Passage number (too old, too recently thawed)��Grade of confluence��Splitting protocol (�� e.g., inactivation of trypsin incomplete)

Vessel:

New scale (upscale/downscale)��New plastic��

Plasmid:

Quality of the purification��Control vector��New vector�� Functionality of the vector (�� e.g., structural integrity, degradation after long storage)

Transfection complex:Reagent/DNA ratio��Ionic strength��Buffer/pH��Temperature��Time��Volumes��

Application of the transfection complex:Dropwise, or complexed with media��

Transfection media:With and without serum ��

Media exchange:

When media exchange is required, all solutions should ��have the appropriate temperature when used.

Functional Analysis:Relevance and functionality of downstream assay ��

(e.g., for microscopy, FACs, biochemistry)

Tip: The Genopure Plasmid Midi Kit and Genopure Plas-mid Maxi Kit from Roche Applied Science are ideal for the efficient purification of plasmid DNA. Resulting plasmid DNA should be stored short-term at +2 to + 8°C or, for long-term storage, in aliquots at -15 to -25°C. Note that freezing and thawing can lead to DNA strand breaks and decreased trans-fection efficiencies.

Tip: X-tremeGENE 9 and HP DNA Transfection Reagents can be used with media with or without serum.

Tip: The Roche Applied Science Cellavista System allows you to microscopically and quantitatively evaluate cells in culture.

Published byRoche Diagnostics GmbHSandhofer Straße 11668305 MannheimGermany

© 2011 Roche Diagnostics. All rights reserved.

06341314001  0211

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

TrademarksXCELLIGENCE, X-TREMEGENE, GENOPURE, CASY, CEDEX and CELLAVISTA are a trademark 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.

References

1. R. Ian Freshney (2005). Culture of animals cells. A manual of basic techniques, 5th edition (ISBN: 0-471-45329-3).

2. R. A. Dixon and R. A. Gonzales (1994). Plant Cell Culture. A Practical Approach, 2nd edition., Oxford University Press.Publication (ISBN: 0-19-963402-5).

4. What is the best way to transfect hard-to-transfect cell types using X-tremeGene Transfection Reagent?

X-tremeGENE DNA Transfection Reagents are designed for effective DNA delivery into even hard-to-transfect cell types.

For detailed cell-type specific protocols, please visit: www.x-tremegene.roche.com

Product Cat. No. Pack Size

X-tremeGENE HP DNA Transfection Reagent 06 366 244 001 06 366 236 001 06 366 546 001

0.4 ml 1.0 ml 5 x 1 ml

X-tremeGENE 9 DNA Transfection Reagent 06 365 779 001 06 365 787 001 06 365 809 001

0.4 ml 1.0 ml 5 x 1 ml

X-tremeGENE siRNA Transfection Reagent 04 476 093 001 04 476 115 001

1 ml 5 x 1 ml

Ordering Information