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TransMessenger™ Transfection Reagent Handbook

For transfection of eukaryotic cells with RNA and siRNA

October 2002

© 2002 QIAGEN, all rights reserved.

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siRNA. and Transmessenger Transfection Reagent Optimizing siRNA Transfection Guidelines for Transfection of siRNA Duplexes Using TransMessenger Transfection Reagent Troubleshooting Guide References Appendix Ordering Information QIAGEN International Sales and Distributors 4 4 4 4 5 5 5 6 6 6 6 6 7 7 7 8 8 9 9 10 10 10 10 12 14 15 16 18 21 21 22 27 TransMessenger Transfection Reagent Handbook 10/2002 3 .Contents Kit Contents Storage and Stability Quality Control Technical Assistance Product Use Limitations Product Warranty and Satisfaction Guarantee Safety Information Introduction General Considerations for RNA Transfection Cell culture Serum RNA structure RNA quality Handling RNA General handling Disposable plasticware DNA contamination Optimizing RNA Transfection Cell density at transfection Amount of RNA Amount of Enhancer R Ratio of TransMessenger Transfection Reagent to RNA–Enhancer R mixture Incubation period with TransMessenger–RNA complexes Protocol for Transfection of Adherent Cells with RNA RNA interference (RNAi).

QIAGEN customers are a major source of information regarding advanced or specialized uses of our products. For technical assistance and more information please call one of the QIAGEN Technical Service Departments or local distributors (see inside front cover). Inc. TransMessenger Transfection Reagent is not sensitive to oxygen. Additionally. Technical Assistance At QIAGEN we pride ourselves on the quality and availability of our technical support. TransMessenger Transfection Reagent does not need to be stored on ice during the transfection procedure. please do not hesitate to contact us. The solutions are stable for 1 year at 2–8°C. 4 TransMessenger Transfection Reagent Handbook 10/2002 . or 80 transfections in 12-well plates. and Buffer EC-R are supplied as readyto-use solutions and are shipped at ambient temperature without loss in stability. Quality Control Endotoxin levels are <10 EU/ml as determined using a Kinetic-QCL test (BioWhittaker. This information is helpful to other scientists as well as to the researchers at QIAGEN.Kit Contents 0. In contrast to many liposome-based reagents. Kit components are tested for the absence of RNase activity.). If you have any questions or experience any difficulties regarding TransMessenger Transfection Reagent or QIAGEN products in general. Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN® products. Enhancer R. TransMessenger Transfection Reagent is tested by transfection of a CAT-encoding RNA construct into NIH/3T3 cells to ensure lot-to-lot consistency. 0. and so does not require storage under an inert gas. Storage and Stability TransMessenger Transfection Reagent. they should be stored at 2–8°C upon arrival. However. 1 x 15 ml Buffer EC-R. sufficient for 60 transfections in 6-well plates.5 ml TransMessenger™ Transfection Reagent (1 mg/ml). Microbial limit tests guarantee the absence of any contaminating bacteria or fungi. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques.5 ml Enhancer R (1 mg/ml).

If a QIAGEN product does not meet your expectations. QIAGEN will replace it free of charge or refund the purchase price. disposable gloves. The purchaser must determine the suitability of the product for its particular use.asp. designed. All due care and attention should be exercised in the handling of many of the materials described in this text. please call QIAGEN Technical Services or your local distributor (see inside front cover).qiagen. where you can find. Should any product fail to perform satisfactorily due to any reason other than misuse. For more information. and print the MSDS for each QIAGEN kit and kit component.Product Use Limitations TransMessenger Transfection Reagent is developed. alter. please consult the appropriate material safety data sheets (MSDSs). 24-hour emergency information Emergency medical information in English. always wear a suitable lab coat. or modify any product to enhance its performance and design.com/ts/msds. view. A copy of QIAGEN terms and conditions can be obtained on request. It is not to be used for human diagnostic or drug purposes or to be administered to humans unless expressly cleared for that purpose by the Food and Drug Administration in the USA or the appropriate regulatory authorities in the country of use. simply call your local Technical Service Department or distributor. and German can be obtained 24 hours a day from: Poison Information Center Mainz. Product Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature. French. and sold for research purposes only. Safety Information When working with chemicals. We reserve the right to change. and protective goggles. If you have questions about product specifications or performance. We will credit your account or exchange the product — as you wish. and is also provided on the back of our invoices. These are available online in convenient and compact PDF format at www. Germany Tel: +49-6131-19240 TransMessenger Transfection Reagent Handbook 10/2002 5 .

including siRNA (see page 14). This ensures normal cell metabolism and increases the likelihood of RNA uptake.Introduction TransMessenger Transfection Reagent has been designed to efficiently transfect eukaryotic cells with RNA. In addition. may increase or decrease the efficiency of translation in a given cell type. The TransMessenger–RNA complexes are then mixed with serum-free medium and added directly to cells. TransMessenger Transfection Reagent is added to the condensed RNA to produce TransMessenger–RNA complexes. the RNA is condensed by interaction with Enhancer R in a defined buffer system. particularly when transfecting cells with RNA. General Considerations for RNA Transfection Transfection efficiencies are affected by a variety of parameters.. A cap can be incorporated into an in vitro-transcribed 6 TransMessenger Transfection Reagent Handbook 10/2002 . However. Messenger RNA (mRNA) species in eukaryotic cells usually terminate at the 5'-end with a cap and at the 3'-end with a poly-A tail. poly-A tail. The following factors should be considered carefully. In the first step of TransMessenger–RNA complex formation. and supplement requirements. A cap is a modified nucleotide incorporated at the 5'-end of RNA transcripts that enhances mRNA stability and is necessary in some cell types to ensure optimal translation. In the second step. Serum In general. serum. This handbook contains guidelines and protocols for the transfection of single-stranded RNA (pages 9–13) and double-stranded siRNA (pages 14–17). We strongly recommend subculturing cells for a minimum of 24 hours before transfection. we do not recommend using serum during transfection of RNA. such as a cap. mycoplasma) and fungi should be avoided. We recommend using cells with a low passage number (<50 splitting cycles) to ensure that the cell genotype does not become altered. Different cells or cell types have very specific medium. The TransMessenger Principle TransMessenger Transfection Reagent is based on a lipid formulation and is used in conjunction with a specific RNA-condensing reagent (Enhancer R) and an RNA-condensing buffer (Buffer EC-R). the presence of specific RNA characteristics.g. Contamination with bacteria (e. Cell culture A healthy cell culture lays the foundation for successful transfection. RNA structure The structure of the transfected RNA molecule can influence the efficiency of transfection by influencing the formation of the TransMessenger Reagent–RNA complex. to avoid contamination with ribonucleases (RNases). since this can drastically alter transfection results. Best results are obtained using the confluence levels indicated in the appropriate protocol sections. or internal ribosomal entry site (IRES). the use of serum enhances transfection.

also stabilizes mRNA. and solutions (including water and culture medium) are RNase-free. Hands and dust particles may carry bacteria and molds. Ensure that all glassware. Change gloves frequently and keep tubes closed. General handling Proper microbiological. Therefore only RNA of the highest quality. Handling RNA Ribonucleases (RNases) are very stable and active enzymes that do not generally require cofactors to function. should be used. An absorbance of 1 unit at 260 nm corresponds to ~40 µg RNA per milliliter. thereby enhancing translation. do not use any plasticware. it is likely that the RNA has suffered major degradation during its preparation. If the band is not sharp but appears as a smear containing smaller sized RNAs. glassware. RNA purified with RNeasy® Kits and Oligotex® mRNA Kits is highly recommended for transfection. a stretch of adenylate residues added 3' to the mRNA coding sequence. The RNA should appear as a sharp band on the stained gel. A poly-A tail.RNA by adding an RNA cap-structure analog to the transcription reaction. An IRES is a specific sequence which helps the ribosome to access the mRNA. Since RNases are difficult to inactivate and minute amounts are sufficient to destroy RNA. However in some cell types. A poly-A tail can be incorporated into an in vitro-transcribed RNA by using a relevant DNA template or added post-transcriptionally using a poly-A polymerase. aseptic technique should always be used when working with RNA. RNA integrity and size can be checked by denaturing agarose-gel electrophoresis and ethidium bromide staining. In order to create and maintain an RNase-free environment when working with RNA. Great care should be taken to avoid inadvertently introducing RNases during the transfection procedure. or solutions without first eliminating possible RNase contamination. RNA concentration and purity can be determined by measuring the absorbance at 260 nm (A260) and 280 nm (A280) using a spectrophotometer. RNA species containing an IRES will not be efficiently translated due to a lack of factors necessary for translation initiation at an IRES in these cells. plasticware. which is free of contaminating DNA and proteins. The functionality of the transcript can be determined by in vitro translation. TransMessenger Transfection Reagent Handbook 10/2002 7 . Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin. We strongly recommend checking RNA quality before starting the transfection. RNA quality Optimal transfection results are achieved when RNA of the highest purity is used for transfection. the precautions listed below must be followed throughout the procedure. and are the most common sources of RNase contamination.

disposable. For RNA transfection. digestion of the purified RNA with RNase-free DNase is recommended. even when no DNA is visible on an agarose gel. DNA contamination No currently available purification method can guarantee that RNA is completely free of DNA. The RNA can then be repurified using the RNeasy Kit. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases.Disposable plasticware The use of sterile. plastic tubes throughout the procedure is recommended. 8 TransMessenger Transfection Reagent Handbook 10/2002 .

Sufficient cells should be seeded such that the culture is 80–90% confluent on the day of transfection. they should be kept constant in every experiment using a particular cell type/RNA combination. Use a volume suitable for the cell culture format. This is achieved by counting cells prior to seeding and by keeping the time period between seeding and transfection (minimum 24 hours) constant. Cell density at transfection Table 1 lists the recommended number of adherent cells to seed per culture well the day prior to transfection of single-stranded RNA. The optimal confluency for transfection of adherent cells with single-stranded RNA is 80–90%.Optimizing RNA Transfection To achieve optimal transfection efficiency for every cell type–RNA combination. RNA. while the optimal confluency for transfection of adherent cells with siRNA is 50–80%. and TransMessenger–RNA complexes The cell number/confluency The length of exposure of cells to TransMessenger–RNA complexes Once the parameters yielding maximum transfection efficiency have been determined. The optimal confluency should be determined for every new cell type to be transfected and kept constant in future experiments. of adherent cells to seed* 2–3 x 104 4–8 x 104 8–10 x 104 2–3 x 105 4–6 x 105 * Be sure that cells are seeded a minimum of 24 hours before transfection of RNA. Recommended number of cells to seed the day before transfection of single-stranded RNA Culture format 96-well plate 48-well plate 24-well plate 12-well plate 6-well plate Suggested no. we recommend optimizing a number of parameters: • • • The amounts of TransMessenger Transfection Reagent. TransMessenger Transfection Reagent Handbook 10/2002 9 . Table 1. This will ensure that the cell density is not too high and that the cells are in optimal physiological condition on the day of transfection. Actual cell number used depends on cell type and size. The volume of medium used is not critical.

while conversely. page 13 Roughly half the listed ratios and quantities Double the listed ratios and quantities. Efficient condensation of RNA with Enhancer R is determined by the mass quantity of RNA. Toxic effects may arise if too much RNA is used. For transfection using other culture formats.. page 11.Amount of RNA The amount of RNA used is a critical factor for efficient transfection. The recommended amount of RNA for transfection using 6-well plates is 2 µg. wash the cells with PBS.g. sialylated glycoproteins) on the cell surface requires a slightly positive net charge. Therefore. Incubation period with TransMessenger–RNA complexes Remove TransMessenger–RNA complexes 3 hours after their addition to the cells. page 13. the amount of RNA transfected should be optimized for every new RNA and/or new cell type used. The ratio of TransMessenger Transfection Reagent (µl) to RNA (µg) is an important factor to optimize for every new cell type and RNA used. Optimal binding of TransMessenger–RNA complexes to negatively charged groups (e. If no cytotoxic effects are observed. A pipetting scheme for optimizing the transfection of adherent cells in 6-well plates is provided in Table 2. 10 TransMessenger Transfection Reagent Handbook 10/2002 . Ratio of TransMessenger Transfection Reagent to RNA–Enhancer R mixture The overall charge of the TransMessenger–RNA complex is determined by the ratio of TransMessenger Transfection Reagent to RNA–Enhancer R mixture. and add fresh medium. expression levels may be too low if insufficient RNA is used. Amount of Enhancer R The RNA (µg) to Enhancer R (µl) ratio of 1:2 given in the pipetting scheme on page 11 should not be changed. please refer to Table 3. A pipetting scheme for optimizing the transfection of adherent cells in 6-well plates is provided in Table 2. To optimize transfection in other culture formats. As a starting point for optimization we recommend using an RNA:TransMessenger Transfection Reagent ratio of 2 µg RNA to 8 µl TransMessenger Reagent when using 6-well plates. the incubation time can be increased up to 4 hours. prepare separate transfection mixtures using: • • • The starting point RNA and TransMessenger Transfection Reagent quantities listed in Table 3.

Table 2. Pipetting scheme for optimizing the transfection of adherent cells in 6-well plates Amount of RNA 1:2 1 µg 1 µg RNA 2 µl Enhancer R 2 µl TransMessenger Reagent 2 µg 2 µg RNA 4 µl Enhancer R 4 µl TransMessenger Reagent 4 µg 4 µg RNA 8 µl Enhancer R Ratio of RNA to TransMessenger Transfection Reagent 1:4 1 µg RNA 2 µl Enhancer R 4 µl TransMessenger Reagent 2 µg RNA 4 µl Enhancer R 8 µl TransMessenger Reagent 4 µg RNA 8 µl Enhancer R 1:8 1 µg RNA 2 µl Enhancer R 8 µl TransMessenger Reagent 2 µg RNA 4 µl Enhancer R 16 µl TransMessenger Reagent 4 µg RNA 8 µl Enhancer R 32 µl TransMessenger Reagent 8 µl TransMessenger 16 µl TransMessenger Reagent Reagent TransMessenger Transfection Reagent Handbook 10/2002 11 .

Important notes before starting • • • • We strongly recommend reading this handbook carefully before starting. Incubate cells under their normal growth conditions (generally 37°C and 5% CO2). On the day of transfection. Check the integrity and functionality of the RNA (see “RNA quality”. 2. • Always keep the ratio of RNA to Enhancer R constant. dilute 4 µl Enhancer R in 92 µl Buffer EC-R. See Table 1 on page 9 for the recommended number of cells to seed. The day before transfection.g. The RNA should be capable of being efficiently translated in the cell type used for transfection (e. or by vortexing for 10 s. Subculture the cells a minimum of 24 hours before transfection. 5. Starting points for optimizing transfection in other formats are listed in Table 3 on page 13. Optimal transfection conditions should be determined for every cell type–RNA combination to obtain the highest transfection efficiency with TransMessenger Transfection Reagent.5 µg/µl. if the RNA concentration is 0. 3.. The final volume should be 100 µl. Note: Make sure that cells are in good condition and are seeded at least 24 h before transfection. Cells should be 80–90% confluent on the day of transfection. Add 8 µl TransMessenger Transfection Reagent to the RNA–Enhancer R mixture. then add 4 µl RNA solution. For example. dilute 4 µl Enhancer R in Buffer EC-R. IMPORTANT: • Always mix Enhancer R with Buffer EC-R before adding RNA. use 2 µg RNA for transfection in 6-well plates. seed 4–6 x 105 cells (depending on the cell type) per well of a 6-well plate in 2 ml appropriate growth medium containing serum and antibiotics. page 6). TransMessenger Transfection Reagent Handbook 10/2002 Protocol Procedure 1. see page 14.Protocol for Transfection of Adherent Cells with RNA The following protocol is for transfection of adherent cells using single-stranded RNA in one well of a 6-well plate. Incubate at room temperature (15–25°C) for 5 min. see “RNA structure”. IRES compatibility. page 7).1 µg/µl) and mix by vortexing for 10 s. RNA purified with RNeasy Kits and Oligotex mRNA Kits is highly recommended. The cells should be in optimal physiological condition on the day of transfection. then spin down the mixture for a few seconds to collect drops from the top of the tube. The optimal confluency for transfection is 80–90%. Note: The best results are achieved when RNA of the highest purity is used for transfection. 4. Add 2 µg RNA (minimum RNA concentration 0. Mix by pipetting up and down 5 times. Please refer to the optimization guidelines on pages 9–10. As a starting point. For transfection of siRNA. 12 .

Incubation time is determined by the assay and RNA used. IMPORTANT: Use medium without serum or antibiotics to avoid RNase contamination. 11.or 48-well plates. Incubate cells with the transfection complexes for 3 h under their normal growth conditions.25 0. optimal expression levels may be obtained earlier following transfection with RNA.6 2.0 1. 9.5–2 times the volume of medium used for cell seeding. Mix by pipetting up and down twice. 7. gently aspirate the growth medium from the plate.5† 4 6 8 † Culture format Protocol step 96-well plate 48-well plate 24-well plate 12-well plate 6-well plate RNA (µg) 3 0. Note: If no cytotoxic effects are observed. † If transfections are performed in 96.2 4. IMPORTANT: Do not allow the cells to become dry. TransMessenger Transfection Reagent Handbook 10/2002 13 . 10. Gently swirl the plate to ensure uniform distribution of the transfection complexes.5 0.6. the incubation time can be increased up to 4 h.6 3. then add 2 ml fresh medium containing serum and antibiotics to the cells. before addition to the RNA–Enhancer R mixture prepared in step 3. Remove the complexes from the cells.5 1. Incubate cells under their normal growth conditions to allow for protein expression.5 2.8 1. While complex formation takes place. Add 900 µl cell growth medium without serum or antibiotics to the tube containing the transfection complexes.0 Enhancer R (µl) 3 0. Starting points for optimizing the transfection of adherent cells in different formats Final volume of TransMessenger RNA–Enhancer R Transfection Reagent in Buffer EC-R (µl) (µl) 3 15 30 100 100 100 5 1. Note: Compared to DNA transfection. and carefully wash cells once with sterile PBS using 1. dilute TransMessenger Transfection Reagent with Buffer EC-R to a total volume of 10 µl or 20 µl. wash cells once with PBS. then immediately add the transfection complexes drop-wise onto the cells.0 Volume of medium* added to complexes (µl) 8 25 50 100 300 900 * We recommend using medium without serum or antibiotics to avoid possible RNase contamination. Table 3. Incubate the samples for 10 min at room temperature (15–25°C) to allow transfectioncomplex formation. Cells transfected with a chloramphenicol acetyltransferase (CAT) reporter RNA are typically incubated for approximately 24 h post-transfection to obtain maximal levels of protein expression. Minimize the time they are without medium. respectively. Protocol 8.

Designing an siRNA The design of an siRNA is a critical factor in its ability to mediate gene-specific silencing. QIAGEN provides an siRNA oligonucleotide synthesis service that offers expert advice on the design of an siRNA to achieve optimal gene-silencing effects.qiagen. and confluency of the cells at the time of transfection. cell type. If you would like to discuss optimization of your conditions please contact our Technical Services Department (see inside front cover). passage number.com/transfectiontools/. Library siRNAs that are directed against human gene products and have been shown to deliver gene-specific silencing in RNAi experiments are available from the QIAGEN siRNA Oligonucleotide Synthesis Service (see page 24 for ordering information). gene functional analysis.RNA interference (RNAi).qiagen. Controls for RNAi experiments Controls should always be included in RNAi experiments. or FACS®. and our own experiences. 14 TransMessenger Transfection Reagent Handbook 10/2002 . An siRNA with a scrambled oligonucleotide sequence can be used as a negative control. and the efficiency of transfection can be dependent on many different parameters. immunofluorescence. Visit the Transfection Tools site for answers to your RNAi questions — www. and TransMessenger Transfection Reagent Scientists at QIAGEN have successfully performed RNAi experiments using TransMessenger Transfection Reagent and a gene-specific siRNA (1).com/sirna. For more information visit www. Please keep in mind that these are only guidelines. General recommendations The guidelines and recommendations below are based on RNAi experiments performed in 24-well plates. siRNA. QIAGEN offers the QuantiTect™ SYBR® Green RT-PCR Kit and the QuantiTect Probe RT-PCR Kit for highly sensitive and specific two-step and one-step real-time RT-PCR analysis. Fluorescently labeled siRNA allows transfection efficiency to be easily followed. Silencing can also be monitored at the mRNA level by real-time RT-PCR. Variations of these parameters should be considered if varying the amounts and ratios of siRNA and TransMessenger Reagent used does not lead to the desired results. Measuring the gene-silencing effect The gene-silencing effect can be monitored at the protein level by western blotting. All expression data should be compared to levels of a “housekeeping” gene to exclude the possibility of non-specific effects. RNAi information online The QIAGEN Transfection Tools web site contains siRNA transfection protocols and the most up-to-date information and literature on QIAGEN reagents for RNAi. such as siRNA quality. Below are some guidelines based on information from current literature (2–5).

This is achieved by counting cells prior to seeding and by keeping the time period between seeding and transfection (minimum 24 hours) constant.Optimizing siRNA Transfection To achieve the best results in siRNA transfection we recommend optimizing the following parameters.8 µg. prepare separate transfection mixtures using: • • • The starting point siRNA and TransMessenger Reagent quantities that are given in the protocol on page 16 Half the listed quantities and ratios Double the listed ratios and quantities. Always use a 1:2 ratio of nucleic acid (µg) to Enhancer R (µl). The recommended starting amount for transfection of siRNA in 24-well plates is 0. TransMessenger Transfection Reagent Handbook 10/2002 15 . Ratio of TransMessenger Transfection Reagent to siRNA The ratio of TransMessenger Transfection Reagent to siRNA should be optimized for every new cell type and siRNA combination used. This will ensure that the cell density is not too high and that the cells are in optimal physiological condition on the day of transfection. To optimize siRNA transfection in 24-well format. Amount of siRNA and Enhancer R The amount of siRNA used is a critical factor for efficient transfection and gene silencing. As a starting point for optimization we recommend using an siRNA to TransMessenger Transfection Reagent ratio (µg:µl) of 1:5 when using 24-well plates. A pipetting scheme for optimizing the transfection of siRNA in adherent cells in a 24-well format is provided in Table 4 on page 16. Cell density at transfection The optimal confluency for transfection of adherent cells with siRNA is 50–80%. The optimal confluency should be determined for every new cell type to be transfected and kept constant in future experiments.

000 to 100. (2). 1. seed 50.6 µl Enhancer R 2 µl TransMessenger Reagent 1.2 µl Enhancer R 4 µl TransMessenger Reagent 1:5 0. Make sure that cells are in good condition and are seeded 24 h before transfection.6 µg siRNA 3.6 µg 1.Table 4. This procedure is provided as a starting point for optimization of siRNA transfection in mammalian cells using TransMessenger Transfection Reagent. For specific cell type and targets. optimal conditions could be different from those described here.8 µg 0.5 0.2 µl Enhancer R 8 µl TransMessenger Reagent 1:10 0.8 µl Enhancer R 1 µl TransMessenger Reagent 0.000 cells (depending on cell type and the time point of analysis) per well of a 24-well plate in 0.2 µl Enhancer R 16 µl TransMessenger Reagent Guidelines for Transfection of siRNA Duplexes Using TransMessenger Transfection Reagent The procedure below is based on the TransMessenger Reagent standard procedure and RNAi studies using targets described by Elbashir et al. Pipetting scheme for optimization of siRNA transfection in 24-well plates Amount of siRNA Ratio of siRNA to TransMessenger Transfection Reagent (µg:µl) 1:2.8 µl Enhancer R 2 µl TransMessenger Reagent 0. Please be sure to read the background information and the protocol notes on pages 9 and 10 of this handbook before starting. 16 TransMessenger Transfection Reagent Handbook 10/2002 .6 µg siRNA 3.6 µl Enhancer R 8 µl TransMessenger Reagent 1.4 µg siRNA 0.4 µg 0.5 ml appropriate growth medium containing serum and antibiotics. The day before transfection. Cells should be 50–80% confluent on the day of transfection.8 µg siRNA 1.4 µg siRNA 0.8 µl Enhancer R 4 µl TransMessenger Reagent 0.8 µg siRNA 1.6 µl Enhancer R 4 µl TransMessenger Reagent 1.4 µg siRNA 0.6 µg siRNA 3.8 µg siRNA 1.

if RNase contamination is not a concern. and method of analysis. or if your serum is validated to have no detectable RNase activity. On the day of transfection. 7. Add 300 µl cell growth medium (without serum or antibiotics) to the tube containing the siRNA–TransMessenger Reagent complexes.8 µg siRNA (minimum siRNA concentration. Add 4 µl TransMessenger Transfection Reagent to the siRNA–Enhancer R mixture. dilute 1. Mix by pipetting up and down twice.1 µg/µl) and mix by vortexing. Remove complexes from the cells. 3. Change medium as required. Double-stranded siRNA is more resistant to degradation by RNAses than singlestranded RNA and therefore. Incubate cells under their normal growth conditions and monitor gene silencing after an appropriate incubation time. While complex formation is taking place. Note: The optimal time point for gene silencing analysis is dependent on cell type. Incubate cells with the transfection complexes for 3 h under their normal growth conditions. 11. medium containing serum can be used.2. or vortexing for 10 s. 6. and then add 500 µl fresh medium containing serum and antibiotics to the cells. 0. TransMessenger Transfection Reagent Handbook 10/2002 17 . 10. Add 0. the incubation time can be increased up to 4 h. Incubate cells under their normal growth conditions (typically 37°C and 5% CO2). Always keep the ratio (µg:µl) of siRNA to Enhancer R constant (1:2). then immediately add the transfection complexes drop-wise onto the cells. Note: Use of media without serum or antibiotics avoids the potential introduction of RNases. gently aspirate the growth medium from the plate.5–2 times the volume of media used for cell seeding. IMPORTANT: Always mix Enhancer R with Buffer EC-R before adding RNA. IMPORTANT: Do not allow the cells to become dry. and carefully wash cells once with sterile PBS using 1. A time course experiment should be performed to determine the appropriate incubation time. Gently swirl the plate to ensure uniform distribution of the transfection complexes. wash cells once with PBS. and then centrifuge the mixture for a few seconds to collect drops from the top of the tube.6 µl Enhancer R in Buffer EC-R. 9. It is possible that transfection in the presence of serum may give improved results. the gene targeted. Incubate the samples for 10 min at room temperature (15–25°C) to allow transfectioncomplex formation. Minimize the time they are without medium. Mix by pipetting up and down 5 times. Note: If no cytotoxic effects are observed. 5. The final volume should be 100 µl. 8. 4. Incubate at room temperature (15–25°C) for 5 min.

g. neutral. This can lead to insufficient uptake of complexes into the cells or inefficient processing of the RNA of interest. Different cell types achieve maximal expression levels at different times post-transfection. increase the overall amount of TransMessenger–RNA complex added to the cells. Observation Possible cause Comments and suggestions If the ratio of TransMessenger Transfection Reagent to RNA is sub-optimal. or strongly positive. or poly-A tail) can influence its stability and/or the expression rate. Be sure to seed cells a minimum of 24 h prior to transfection. Factors such as the size of the RNA and special features contained within it (e. or lower gene-silencing effects than expected are observed.. If the transfection efficiency is lower than expected and cytoxicity is acceptably low. Refer to the pipetting scheme in Table 2 on page 11. cells may not be in the optimal growth phase. TransMessenger Transfection Reagent Handbook 10/2002 Low transfection Sub-optimal efficiency TransMessenger Reagent:RNA ratio Insufficient TransMessenger– RNA complex Sub-optimal incubation time for protein expression Influence of the RNA Sub-optimal cell density 18 . If the time point of maximal expression is not known for a particular cell type–RNA combination. cap. This should be kept in mind when determining the length of incubation after transfection. which can lead to inefficient adsorption to the cell surface. Optimize the TransMessenger Transfection Reagent to RNA ratio according to the optimization guide (pages 9–10). If cell density at the time of TransMessenger –RNA complex addition is not at an optimal level. For adherent cells. higher cytotoxicities. IRES. a time course experiment may be necessary.Troubleshooting Guide The following troubleshooting guide is helpful if lower transfection efficiencies. Some RNA features may not be compatible with every cell type. the optimal confluency for transfection is 80–90% (for single-stranded RNA) or 50–80% (for siRNA). the overall charge of the complexes may be negative. Comments and suggestions are listed in the order in which they should be considered.

Poor RNA quality Reporter assay problem Serum Excessive cell death Excessive exposure of cells to TransMessenger–RNA complexes Concentration of If cell death is still excessive after reducing TransMessenger–RNA the exposure time. For adherent cells. decrease the amount of complexes is too high TransMessenger–RNA complexes added to cells.Observation Possible cause Cell type–RNA combination Comments and suggestions RNA species with features such as a cap. Serum may contain RNases. RNA should be checked for degradation before transfection. If no expression is observed change the cell type or.. Impurities present in RNA preparations can potentially lower transfection efficiency. RNA used for transfection should be of high quality. the RNA. the optimal confluency for transfection is 80–90% (for singlestranded RNA) or 50–80% (for siRNA). if possible. primary cells) with 1. wash particularly sensitive cells (e. TransMessenger Transfection Reagent Handbook 10/2002 19 . Cells are stressed Avoid stressing cells with temperature shifts and long periods without medium during washing steps. For example. Be sure to seed cells a minimum of 24 h before transfection. ensure that cell density is not too low at transfection. or QIAGEN DNA/RNA Kits. We recommend purifying RNA using RNeasy Kits. If excessive cell death is observed after 3 h incubation. or IRES will not necessarily be processed in every cell type. Oligotex mRNA Kits. In addition. We therefore recommend transfection in the absence of serum. It is particularly important for RNA transfection that the cells are in good condition. Therefore. Include positive controls to ensure that the reporter assay is working properly.5–2 volumes of medium without serum instead of PBS after removal of the TransMessenger–RNA complexes. an RNA with an IRES may not be translated in COS-7 and HeLa cells. reduce the time of cell–complex incubation to 1–2 h. Degradation of RNA leads to decreased expression levels.g. poly-A tail.

If the gene targeted in an RNAi experiment is key to the survival of the cell.Observation Possible cause Poor RNA quality Comments and suggestions RNA used for transfection should be of high quality. Degradation of RNA leads to decreased expression levels. Cells should be seeded no later than 24 h before transfection. Variations in the growth behavior of mycoplasma-infected cells will lead to different transfection efficiencies between replicate experiments. and transfectability. decreased transfection efficiencies may be observed in later experiments. or if too much RNA is used. RNA-related effects Key gene is silenced Variable transfection efficiencies in replicate experiments Inconsistent cell confluencies in replicate experiments Possible mycoplasma Mycoplasma contamination influences transfection contamination efficiency. Toxic effects may arise if RNA encoding a toxic protein is transfected. Keep incubation times between seeding and complex addition consistent between experiments. 20 TransMessenger Transfection Reagent Handbook 10/2002 . morphology. Count cells prior to seeding to ensure that the same number of cells is seeded for each experiment. Impurities present in RNA preparations can potentially lower transfection efficiency. Cells have been passaged too many times Cells that have been passaged a large number of times tend to change their growth behavior. Optimize the amount of RNA according to the optimization guide (pages 9–10) for each new RNA and/or cell type used. silencing this gene will lead to cell death. RNA should be checked for degradation before transfection. if insufficient RNA with a low translation rate is used. or QIAGEN DNA/RNA Kits. When cells with high passage numbers are used for replicate experiments. We recommend using cells with a low passage number (<50 splitting cycles). We recommend purifying RNA using RNeasy Kits. transfection efficiency may be too low. Oligotex mRNA Kits. In contrast.

QIAGEN offers siRNA that has been functionally tested for specific gene silencing (see ordering information). The gene silencing effect observed on the protein level is dependent on a protein’s expression level and its rate of turnover within the cell.Observation Possible cause Comments and suggestions The design of an siRNA can have a large effect on its gene silencing efficiency.qiagen. For more information visit www. No or very small Design of siRNA gene silencing sub-optimal effect observed after siRNA transfection Incubation time post-transfection too short Problems with experimental design TransMessenger Transfection Reagent Handbook 10/2002 21 .com/sirna. RNAi effects may not be seen for some genes targeted with certain siRNAs in some cell types. Include where possible both positive and negative controls in your experiments. QIAGEN offers expert advice on siRNA design for maximum gene silencing effect. If possible. repeat experiments using a different cell type and/or siRNA. Perform a time course experiment to determine the optimal time point for analysis.

and Kosik. Acad. L. Appendix Buffer 1x PBS (phosphate-buffered saline) Composition 137 mM NaCl 2. Sci. either by a simple keyword search or by specifying the application. Caplen. (2002) RNAi functions in cultured mammalian neurons. 2. Tyrberg. Proc.M. trademarks. etc. 11926. Dennig. J. TransMessenger™. FACS is a registered trademark of Becton Dickinson and Company. Nature 411. 22 TransMessenger Transfection Reagent Handbook 10/2002 . up-to-date online database of scientific publications utilizing QIAGEN products. used in this document. N. For a complete list of references. title. 2002 No. Cambridge. even when not specifically marked as such. J. are not to be considered unprotected by law. K.7 mM KCl 4..References QIAGEN maintains a large. Acad. and Levine.. R. Metab. Fire. and Morgan. and Konrad. Clin. 1. Jarvis.M. Elbashir. Comprehensive search options allow you to find the articles you need. S.qiagen. Oligotex®..47 mM KH2PO4 Adjust to a final pH of 7. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.S. 4.. Krichevsky. 7. (2002) c-Myc controls proliferation versus differentiation in human pancreatic endocrine cells. Proc.3 mM Na2HPO4 1. etc.A. Registered names. research area... MA. Sci..Imani. Endocrinol.. Natl. C. visit the QIAGEN Reference Database online at www. Demeterco. 9742. R.4 Storage Room temperature Trademarks and disclaimer Patented or patent-pending and/or registered or registration-pending trademarks of QIAGEN: QIAGEN®. P. Itkin-Ansari. F. Parrish. QIAGEN News. et al. (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. USA 99. 3. 2.. siRNA technology licensed to QIAGEN is covered by various patent applications. 5.. (2002) TransMessenger Transfection Reagent enables targeted gene silencing in Hela-S3 cells through RNA interference.com/RefDB/search. 3475.asp or contact QIAGEN Technical Services or your local distributor. USA and others. owned by the Massachusetts Institute of Technology. Ford. A.. RNeasy®. 87.J. A. Natl. J. 494. USA 98. B. F. S.

Small Spin Columns. RNase-free Reagents and Buffers 74104 301525 RNeasy Midi Kits — for isolation of up to 1 mg total RNA from animal cells or tissues. Collection Tubes.5 ml and 2 ml). and bacteria RNeasy Midi Kit (10)* 10 RNeasy Midi Spin Columns.Ordering Information Product Contents Cat. Collection Tubes (50 ml). please inquire † Not available in Japan TransMessenger Transfection Reagent Handbook 10/2002 23 . and bacteria RNeasy Maxi Kit (12) 12 RNeasy Maxi Spin Columns. TransMessenger Transfection Reagent for transfection of eukaryotic cells with RNA TransMessenger Transfection For 60 transfections in 6-well plates Reagent (0. No. yeast. and bacteria RNeasy Mini Kit (50)* 50 RNeasy Mini Spin Columns. RNase-free Reagents and Buffers 75142 RNeasy Maxi Kits — for isolation of up to 6 mg total RNA from animal cells or tissues. yeast. RNase-free Reagents and Buffers 70022 * Larger kit sizes available.5 ml) or 80 transfections in 12-well plates Related products RNeasy Mini Kits — for isolation of up to 100 µg total RNA from animal cells or tissues. yeast. Collection Tubes (15 ml). Collection Tubes (1. RNase-free Reagents and Buffers 75162 Oligotex mRNA Kits† — for isolation of poly A+ mRNA from total RNA Oligotex mRNA Mini Kit* For 12 mRNA preps from up to 250 µg total RNA each: 200 µl Oligotex Suspension.

Rhodamine (5nm) GAS41 siRNA (5 nm) Lamin B2 siRNA (5 nm) Emerin siRNA (5 nm) LAP2 siRNA (5 nm) 5 nmol fluorescein-labeled duplex siRNA. functionally tested for 1022159 specific gene silencing in RNAi experiments 5 nmol duplex siRNA. No. functionally tested for 1022070 specific gene silencing in RNAi experiments Luciferase GL3 siRNA (5 nm) 5 nmol duplex siRNA.Ordering Information Product Contents Cat. functionally tested for 1022073 specific gene silencing in RNAi experiments Control (non-sil.) siRNA (5 nm) 5 nmol duplex siRNA. functionally tested for 1022064 specific gene silencing in RNAi experiments 5 nmol duplex siRNA. Fluorescein (5nm) Control (non-sil. for use as a non-silencing control in RNAi experiments Control(non-sil. functionally tested for 1022055 specific gene silencing in RNAi experiments 5 nmol duplex siRNA. functionally tested for 1022153 specific gene silencing in RNAi experiments 5 nmol duplex siRNA. for use as a non-silencing control in RNAi experiments 5 nmol rhodamine-labeled duplex siRNA. functionally tested for 1022156 specific gene silencing in RNAi experiments 5 nmol duplex siRNA.) siRNA. Library siRNA oligonucleotides Lamin A/C siRNA (5 nm) Vimentin siRNA (5 nm) NuMa siRNA (5 nm) Lamin B1 siRNA (5 nm) GFP-22 siRNA (5 nm) CAT-22 siRNA (5 nm) 5 nmol duplex siRNA. functionally tested for 1022061 specific gene silencing in RNAi experiments 5 nmol duplex siRNA. functionally tested for 1022067 specific gene silencing in RNAi experiments Luciferase GL2 siRNA (5 nm) 5 nmol duplex siRNA.) siRNA. functionally tested for 1022162 specific gene silencing in RNAi experiments 24 TransMessenger Transfection Reagent Handbook 10/2002 . functionally tested for 1022050 specific gene silencing in RNAi experiments 5 nmol duplex siRNA. for use as a non-silencing control in RNAi experiments 1022076 1022079 1022083 5 nmol duplex siRNA. functionally tested for 1022058 specific gene silencing in RNAi experiments 5 nmol duplex siRNA.

available in 0. and information on how to order custom siRNA. standard purity siRNA. available in 0. r(XY) overhang. purified siRNA. available in 0. and 10 µmol synthesis scales Standard purity (80–85% pure) siRNA.qiagen. and 10 µmol synthesis scales Ultrapure (>97% pure) siRNA. available in 0. and 10 µmol synthesis scales Standard purity (80–85% pure) siRNA. and 10 µmol synthesis scales Ultrapure (>97% pure) siRNA. No. Custom siRNA oligonucleotides siRNA. r(UU) overhang. standard purity siRNA.2.2.2. purified siRNA.2. Library siRNA oligonucleotides Nucleoporin (Nup) 153 siRNA (5 nm) β-actin siRNA (5 nm) 5 nmol duplex siRNA. d(TT) overhang. functionally tested for 1022165 specific gene silencing in RNAi experiments 5 nmol duplex siRNA. functionally tested for 1022168 specific gene silencing in RNAi experiments All library siRNAs are also available in 10 and 20 nmol amounts. standard purity Ultrapure (>97% pure) siRNA. purified siRNA. purified siRNA.2. TransMessenger Transfection Reagent Handbook 10/2002 25 . d(TT) overhang. available in 0. 1. 1.2. 1. and 10 µmol synthesis scales Ultrapure (>97% pure) siRNA. For an up-to-date list. r(UU) overhang.Ordering Information Product Contents Cat. available in 0. d(XY) overhang.2. 1. please visit www.com/sirna. and 10 µmol synthesis scales Standard purity (80–85% pure) siRNA. 1. d(XY) overhang. and 10 µmol synthesis scales Standard purity (80–85% pure) siRNA. 1. available in 0. standard purity siRNA.2. r(XY) overhang. 1. available in 0. and 10 µmol synthesis scales Inquire Inquire Inquire Inquire Inquire Inquire Inquire Inquire Numerous 3' and 5' modifications are available on request. 1. please inquire.

Ordering Information Product Contents Cat. 2 x 2. QuantiTect Kits — for quantitative real-time PCR QuantiTect SYBR Green RT-PCR Kit (200) For 200 x 50 µl reactions: 3 x 1.7 ml 204243 QuantiTect SYBR Green RT-PCR Master Mix (providing a final concentration of 4 mM MgCl2). 100 µl QuantiTect RT Mix.0 ml RNase-free water For 200 x 50 µl reactions: 3 x 1.7 ml QuantiTect Probe RT-PCR Master Mix (providing a final concentration of 4 mM MgCl2). 2 x 2. No.0 ml RNase-free water 204443 QuantiTect Probe RT-PCR Kit (200) 26 TransMessenger Transfection Reagent Handbook 10/2002 . 100 µl QuantiTect RT Mix.

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