For transfection of eukaryotic cells with RNA and siRNA 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 RNAEnhancer R mixture
Incubation period with TransMessengerRNA complexes
Protocol for Transfection of Adherent Cells with RNA RNA interference (RNAi), siRNA, and Transmessenger Transfection Reagent
Optimizing siRNA Transfection
Guidelines for Transfection of siRNA Duplexes Using TransMessenger Transfection Reagent
Troubleshooting Guide
References
Appendix

Kit Contents

0.5 ml TransMessenger Transfection Reagent (1 mg/ml), 0.5 ml Enhancer R (1 mg/ml), 1 x 15 ml Buffer EC-R, sufficient for 60 transfections in 6-well plates, or 80 transfections in 12-well plates.

Storage and Stability

TransMessenger Transfection Reagent, Enhancer R, and Buffer EC-R are supplied as readyto-use solutions and are shipped at ambient temperature without loss in stability. However, they should be stored at 28C upon arrival. The solutions are stable for 1 year at 28C. In contrast to many liposome-based reagents, TransMessenger Transfection Reagent is not sensitive to oxygen, and so does not require storage under an inert gas. Additionally, TransMessenger Transfection Reagent does not need to be stored on ice during the transfection procedure.

Quality Control

Endotoxin levels are <10 EU/ml as determined using a Kinetic-QCL test (BioWhittaker, Inc.). TransMessenger Transfection Reagent is tested by transfection of a CAT-encoding RNA construct into NIH/3T3 cells to ensure lot-to-lot consistency. Microbial limit tests guarantee the absence of any contaminating bacteria or fungi. Kit components are tested for the absence of RNase activity.

Technical Assistance

At QIAGEN we pride ourselves on the quality and availability of our technical support. Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in molecular biology and the use of QIAGEN products. If you have any questions or experience any difficulties regarding TransMessenger Transfection Reagent or QIAGEN products in general, please do not hesitate to contact us. QIAGEN customers are a major source of information regarding advanced or specialized uses of our products. This information is helpful to other scientists as well as to the
researchers at QIAGEN. We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques. For technical assistance and more information please call one of the QIAGEN Technical Service Departments or local distributors (see inside front cover).

Product Use Limitations

TransMessenger Transfection Reagent is developed, designed, and sold for research purposes only. 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. All due care and attention should be exercised in the handling of many of the materials described in this text.

Product Warranty and Satisfaction Guarantee

QIAGEN guarantees the performance of all products in the manner described in our product literature. The purchaser must determine the suitability of the product for its particular use. Should any product fail to perform satisfactorily due to any reason other than misuse, QIAGEN will replace it free of charge or refund the purchase price. We reserve the right to change, alter, or modify any product to enhance its performance and design. If a QIAGEN product does not meet your expectations, simply call your local Technical Service Department or distributor. We will credit your account or exchange the product as you wish.

A copy of QIAGEN terms and conditions can be obtained on request, and is also provided on the back of our invoices. If you have questions about product specifications or performance, please call QIAGEN Technical Services or your local distributor (see inside front cover)
.
Safety Information

When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate material safety data sheets (MSDSs). These are available online in convenient and compact PDF format at www.qiagen.com/ts/msds.asp, where you can find, view, and print the MSDS for each QIAGEN kit and kit component.

24-hour emergency information

Emergency medical information in English, French, and German can be obtained 24 hours a day from:
Poison Information Center Mainz, Germany
Tel: +49-6131-19240.

Introduction

TransMessenger Transfection Reagent has been designed to efficiently transfect eukaryotic cells with RNA, including siRNA (see page 14). This handbook contains guidelines and protocols for the transfection of single-stranded RNA (pages 913) and double-stranded siRNA (pages 1417).

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). In the first step of TransMessengerRNA complex formation, the RNA is condensed by interaction with Enhancer R in a defined buffer system. In the second step, TransMessenger Transfection Reagent is added to the condensed RNA to produce TransMessengerRNA complexes. The TransMessengerRNA complexes are then mixed with serum-free medium and added directly to cells.

General Considerations for RNA Transfection

Transfection efficiencies are affected by a variety of parameters. The following factors should be considered carefully.

Cell culture
A healthy cell culture lays the foundation for successful transfection, particularly when transfecting cells with RNA. We strongly recommend subculturing cells for a minimum of 24 hours before transfection. This ensures normal cell metabolism and increases the likelihood of RNA uptake. Best results are obtained using the confluence levels indicated in the appropriate protocol sections. Contamination with bacteria (e.g., mycoplasma) and fungi should be avoided, since this can drastically alter transfection results. Different cells or cell types have very specific medium, serum, and supplement requirements. We recommend using cells with a low passage number (<50 splitting cycles) to ensure that the cell genotype does not become altered.

Serum
In general, the use of serum enhances transfection. However, to avoid contamination with ribonucleases (RNases), we do not recommend using serum during transfection of RNA.

RNA structure
The structure of the transfected RNA molecule can influence the efficiency of transfection by influencing the formation of the TransMessenger ReagentRNA complex. In addition, the presence of specific RNA characteristics, such as a cap, poly-A tail, or internal ribosomal entry site (IRES), may increase or decrease the efficiency of translation in a given cell type. 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. 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. A cap can be incorporated into an in vitro-transcribed RNA by adding an RNA cap-structure analog to the transcription reaction. A poly-A tail, a stretch of adenylate residues added 3' to the mRNA coding sequence, also stabilizes mRNA. 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. An IRES is a specific sequence which helps the ribosome to access the mRNA, thereby enhancing translation. However in some cell types, 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.

RNA quality
Optimal transfection results are achieved when RNA of the highest purity is used for transfection. Therefore only RNA of the highest quality, which is free of contaminating DNA and proteins, should be used. RNA purified with RNeasy Kits and Oligotex mRNA Kits is highly recommended for transfection. We strongly recommend checking RNA quality before starting the transfection. RNA concentration and purity can be determined by measuring the absorbance at 260 nm (A260) and 280 nm (A280) using a spectrophotometer. An absorbance of 1 unit at 260 nm corresponds to ~40 g RNA per milliliter. RNA integrity and size can be checked by denaturing agarose-gel electrophoresis and ethidium bromide staining. The RNA should appear as a sharp band on the stained gel. If the band is not sharp but appears as a smear containing smaller sized RNAs, it is likely that the RNA has suffered major degradation during its preparation. The functionality of the transcript can be determined by in vitro translation.

Handling RNA
Ribonucleases (RNases) are very stable and active enzymes that do not generally require cofactors to function. Since RNases are difficult to inactivate and minute amounts are sufficient to destroy RNA, do not use any plasticware, glassware, or solutions without first eliminating possible RNase contamination. Great care should be taken to avoid inadvertently introducing RNases during the transfection procedure. In order to create and maintain an RNase-free environment when working with RNA, the precautions listed below must be followed throughout the procedure.

General handling
Proper microbiological, aseptic technique should always be used when working with RNA. Hands and dust particles may carry bacteria and molds, and are the most common sources of RNase contamination. Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin. Change gloves frequently and keep tubes closed. Ensure that all glassware, plasticware, and solutions (including water and culture medium) are RNase-free.

Disposable plasticware
The use of sterile, disposable, plastic tubes throughout the procedure is recommended. These tubes are generally RNase-free and do not require pretreatment to inactivate RNases.

DNA contamination
No currently available purification method can guarantee that RNA is completely free of DNA, even when no DNA is visible on an agarose gel. For RNA transfection, digestion of the purified RNA with RNase-free DNase is recommended. The RNA can then be repurified using the RNeasy Kit.

Optimizing RNA Transfection

To achieve optimal transfection efficiency for every cell typeRNA combination, we recommend optimizing a number of parameters:

The amounts of TransMessenger Transfection Reagent, RNA, and TransMessengerRNA complexes
The cell number/confluency
The length of exposure of cells to TransMessengerRNA complexes

Once the parameters yielding maximum transfection efficiency have been determined, they should be kept constant in every experiment using a particular cell type/RNA combination.

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 8090%, while the optimal confluency for transfection of adherent cells with siRNA is 5080%. The optimal confluency should be determined for every new cell type to be transfected and kept constant in future experiments. This is achieved by counting cells prior to seeding and by keeping the time period between seeding and transfection (minimum 24 hours) constant. 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.

Table 1. Recommended number of cells to seed the day before transfection of single-stranded RNA * Be sure that cells are seeded a minimum of 24 hours before transfection of RNA. Actual cell number used depends on cell type and size. Sufficient cells should be seeded such that the culture is 8090% confluent on the day of transfection. The volume of medium used is not critical. Use a volume suitable for the cell culture format. Amount of RNA
The amount of RNA used is a critical factor for efficient transfection. Toxic effects may arise if too much RNA is used, while conversely, expression levels may be too low if insufficient RNA is used. Therefore, the amount of RNA transfected should be optimized for every new RNA and/or new cell type used. The recommended amount of RNA for transfection using 6-well plates is 2 g. A pipetting scheme for optimizing the transfection of adherent cells in 6-well plates is provided in Table 2, page 11. For transfection using other culture formats, please refer to Table 3

Amount of Enhancer R
The RNA (g) to Enhancer R (l) ratio of 1:2 given in the pipetting scheme on page 11 should not be changed. Efficient condensation of RNA with Enhancer R is determined by the mass quantity of RNA.

Ratio of TransMessenger Transfection Reagent to RNAEnhancer R mixture
The overall charge of the TransMessengerRNA complex is determined by the ratio of TransMessenger Transfection Reagent to RNAEnhancer R mixture. Optimal binding of TransMessengerRNA complexes to negatively charged groups (e.g., sialylated glycoproteins) on the cell surface requires a slightly positive net charge. The ratio of TransMessenger Transfection Reagent (l) to RNA (g) is an important factor to optimize for every new cell type and RNA used. As a starting point for optimization we recommend using an RNA:TransMessenger Transfection Reagent ratio of 2 g RNA to 8 l TransMessenger Reagent when using 6-well plates. 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, prepare separate transfection mixtures using:
The starting point RNA and TransMessenger Transfection Reagent quantities listed in Table 3,
Roughly half the listed ratios and quantities
Double the listed ratios and quantities.

Incubation period with TransMessengerRNA complexes
Remove TransMessengerRNA complexes 3 hours after their addition to the cells, wash the cells with PBS, and add fresh medium. If no cytotoxic effects are observed, the incubation time can be increased up to 4 hours.

Table 2. Pipetting scheme for optimizing the transfection of adherent cells in 6-well plates
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. For transfection of siRNA, see page 14. As a starting point, use 2 g RNA for transfection in 6-well plates. Starting points for optimizing transfection in other formats are listed in Table 3 on page 13. See Table 1 on page 9 for the recommended number of cells to seed. Optimal transfection conditions should be determined for every cell typeRNA combination to obtain the highest transfection efficiency with TransMessenger Transfection Reagent.

Important notes before starting
We strongly recommend reading this handbook carefully before starting. Check the integrity and functionality of the RNA (see RNA quality). The RNA should be capable of being efficiently translated in the cell type used for transfection (e.g., IRES compatibility; see RNA structure).
The cells should be in optimal physiological condition on the day of transfection. Subculture the cells a minimum of 24 hours before transfection. The ptimal confluency for transfection is 8090%. Procedure 1. The day before transfection, seed 46 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.
Note: Make sure that cells are in good condition and are seeded at least 24 h before transfection. Cells should be 8090% confluent on the day of transfection. 2. Incubate cells under their normal growth conditions (generally 37C and 5% CO2). 3. On the day of transfection, dilute 4 l Enhancer R in Buffer EC-R. Add 2 g RNA (minimum RNA concentration 0.1 g/l) and mix by vortexing for 10 s. The final volume should be 100 l.
For example, if the RNA concentration is 0.5 g/l, dilute 4 l Enhancer R in 92 l Buffer EC-R, then add 4 l RNA solution.
IMPORTANT:
Always mix Enhancer R with Buffer EC-R before adding RNA.
Always keep the ratio of RNA to Enhancer R constant.
Note:
The best results are achieved when RNA of the highest purity is used for transfection. RNA purified with RNeasy Kits and Oligotex mRNA Kits is highly recommended. 4. Incubate at room temperature (1525C) for 5 min, then spin down the mixture for a few seconds to collect drops from the top of the tube. 5. Add 8 l TransMessenger Transfection Reagent to the RNAEnhancer R mixture. Mix by pipetting up and down 5 times, or by vortexing for 10 s. 6. Incubate the samples for 10 min at room temperature (1525C) to allow transfectioncomplex formation. 7. While complex formation takes place, gently aspirate the growth medium from the plate, and carefully wash cells once with sterile PBS using 1.52 times the volume of medium used for cell seeding.
IMPORTANT:
Do not allow the cells to become dry. Minimize the time they are without medium. 8. Add 900 l cell growth medium without serum or antibiotics to the tube containing the transfection complexes. Mix by pipetting up and down twice, then immediately add the transfection complexes drop-wise onto the cells. Gently swirl the plate to ensure uniform distribution of the transfection complexes.
IMPORTANT:
Use medium without serum or antibiotics to avoid RNase contamination. 9. Incubate cells with the transfection complexes for 3 h under their normal growth conditions.
Note:
If no cytotoxic effects are observed, the incubation time can be increased up to 4 h. 10. Remove the complexes from the cells, wash cells once with PBS, then add 2 ml fresh medium containing serum and antibiotics to the cells. 11. Incubate cells under their normal growth conditions to allow for protein expression. Incubation time is determined by the assay and RNA used.
Note: Compared to DNA transfection, optimal expression levels may be obtained earlier following transfection with RNA. 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. Table 3. Starting points for optimizing the transfection of adherent cells in different formats * We recommend using medium without serum or antibiotics to avoid possible RNase contamination.
If transfections are performed in 96- or 48-well plates, dilute TransMessenger Transfection Reagent with Buffer EC-R to a total volume of 10 l or 20 l, respectively, before addition to the RNAEnhancer R mixture prepared in step 3.

RNA interference (RNAi), siRNA, and TransMessenger Transfection Reagent

Scientists at QIAGEN have successfully performed RNAi experiments using TransMessenger Transfection Reagent and a gene-specific siRNA (1). Below are some guidelines based on information from current literature (25), and our own experiences.

Controls for RNAi experiments
Controls should always be included in RNAi experiments. An siRNA with a scrambled oligonucleotide sequence can be used as a negative control. 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. Fluorescently labeled siRNA allows transfection efficiency to be easily followed.

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. For more information visit www.qiagen.com/sirna.

Measuring the gene-silencing effect
The gene-silencing effect can be monitored at the protein level by western blotting, immunofluorescence, gene functional analysis, or FACS. Silencing can also be monitored at the mRNA level by real-time RT-PCR. 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. All expression data should be compared to levels of a housekeeping gene to exclude the possibility of non-specific effects.

General recommendations
The guidelines and recommendations below are based on RNAi experiments performed in 24-well plates. Please keep in mind that these are only guidelines, and the efficiency of transfection can be dependent on many different parameters, such as siRNA quality, cell type, passage number, and confluency of the cells at the time of transfection. 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. If you would like to discuss optimization of your conditions please contact our Technical Services Department (see inside front cover).

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. Visit the Transfection Tools site for answers to your RNAi questions www.qiagen.com/transfectiontools/.

Optimizing siRNA Transfection

To achieve the best results in siRNA transfection we recommend optimizing the following parameters.

Amount of siRNA and Enhancer R
The amount of siRNA used is a critical factor for efficient transfection and gene silencing. The recommended starting amount for transfection of siRNA in 24-well plates is 0.8 g. 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. Always use a 1:2 ratio of nucleic acid (g) to Enhancer R (l).

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. As a starting point for optimization we recommend using an siRNA to TransMessenger Transfection Reagent ratio (g:l) of 1:5 when using 24-well plates. To optimize siRNA transfection in 24-well format, 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.

Cell density at transfection
The optimal confluency for transfection of adherent cells with siRNA is 5080%. The optimal confluency should be determined for every new cell type to be transfected and kept constant in future experiments. This is achieved by counting cells prior to seeding and by keeping the time period between seeding and transfection (minimum 24 hours) constant. 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.

Table 4. Pipetting scheme for optimization of siRNA transfection in 24-well plates

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. (2). 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. Please be sure to read the background information and the protocol notes on pages 9 and 10 of this handbook before starting. 1. The day before transfection, seed 50,000 to 100,000 cells (depending on cell type and the time point of analysis) per well of a 24-well plate in 0.5 ml appropriate growth medium containing serum and antibiotics.
Make sure that cells are in good condition and are seeded 24 h before transfection. Cells should be 5080% confluent on the day of transfection. 2. Incubate cells under their normal growth conditions (typically 37C and 5% CO2). 3. On the day of transfection, dilute 1.6 l Enhancer R in Buffer EC-R. Add 0.8 g siRNA (minimum siRNA concentration, 0.1 g/l) and mix by vortexing. The final volume should be 100 l.
IMPORTANT:
Always mix Enhancer R with Buffer EC-R before adding RNA. Always keep the ratio (g:l) of siRNA to Enhancer R constant (1:2). 4. Incubate at room temperature (1525C) for 5 min, and then centrifuge the mixture for a few seconds to collect drops from the top of the tube. 5. Add 4 l TransMessenger Transfection Reagent to the siRNAEnhancer R mixture. Mix by pipetting up and down 5 times, or vortexing for 10 s. 6. Incubate the samples for 10 min at room temperature (1525C) to allow transfectioncomplex formation. 7. While complex formation is taking place, gently aspirate the growth medium from the plate, and carefully wash cells once with sterile PBS using 1.52 times the volume of media used for cell seeding.
IMPORTANT:
Do not allow the cells to become dry. Minimize the time they are without medium. 8. Add 300 l cell growth medium (without serum or antibiotics) to the tube containing the siRNATransMessenger Reagent complexes. Mix by pipetting up and down twice, then immediately add the transfection complexes drop-wise onto the cells. Gently swirl the plate to ensure uniform distribution of the transfection complexes.
Note:
Use of media without serum or antibiotics avoids the potential introduction of RNases. It is possible that transfection in the presence of serum may give improved results. Double-stranded siRNA is more resistant to degradation by RNAses than singlestranded RNA and therefore, if RNase contamination is not a concern, or if your serum is validated to have no detectable RNase activity, medium containing serum can be used. 9. Incubate cells with the transfection complexes for 3 h under their normal growth conditions.
Note:
If no cytotoxic effects are observed, the incubation time can be increased up to 4 h. 10. Remove complexes from the cells, wash cells once with PBS, and then add 500 l fresh medium containing serum and antibiotics to the cells. 11. Incubate cells under their normal growth conditions and monitor gene silencing after an appropriate incubation time. Change medium as required.
Note:
The optimal time point for gene silencing analysis is dependent on cell type, the gene targeted, and method of analysis. A time course experiment should be performed to determine the appropriate incubation time.

Troubleshooting Guide

The following troubleshooting guide is helpful if lower transfection efficiencies, higher cytotoxicities, or lower gene-silencing effects than expected are observed. Comments and suggestions are listed in the order in which they should be considered. Observation Possible cause Comments and suggestions

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Low transfection
efficiency Sub-optimal
TransMessenger
Reagent:RNA ratio If the ratio of TransMessenger Transfection Reagent to RNA is sub-optimal, the overall
charge of the complexes may be negative, neutral, or strongly positive, which can lead to
inefficient adsorption to the cell surface. Optimize the TransMessenger Transfection
Reagent to RNA ratio according to the optimization guide. Insufficient
TransMessenger
RNA complex If the transfection efficiency is lower than expected and cytoxicity is acceptably low, increase the overall amount of TransMessengerRNA complex added to the cells. Refer to the pipetting scheme in Table 2. Sub-optimal
incubation time for
protein expression Different cell types achieve maximal expression levels at different times post-transfection. This should be kept in mind when determining the length of incubation after transfection. If the time point of maximal expression is not known for a particular cell typeRNA combination, a time course experiment may be necessary. Influence of the RNA Factors such as the size of the RNA and special features contained within it (e.g., IRES, cap, or poly-A tail) can influence its stability and/or the expression rate. Some RNA features may not be compatible with every cell type. Sub-optimal cell
density If cell density at the time of TransMessenger RNA complex addition is not at an optimal
level, cells may not be in the optimal growth phase. This can lead to insufficient uptake of
complexes into the cells or inefficient processing of the RNA of interest. For adherent cells, the optimal confluency for transfection is 8090% (for single-stranded RNA) or 5080% (for siRNA). Be sure to seed cells a minimum of 24 h prior to transfection. Cell typeRNA
combination RNA species with features such as a cap, poly-A tail, or IRES will not necessarily be processed in every cell type. For example, an RNA with an IRES may not be translated in COS-7 and HeLa cells. If no expression is observed change the cell type or, if possible, the RNA. Poor RNA quality RNA used for transfection should be of high quality. Impurities present in RNA preparations can potentially lower transfection efficiency. Degradation of RNA leads to decreased expression levels. RNA should be checked for degradation before transfection. We recommend purifying RNA using RNeasy Kits, Oligotex mRNA Kits, or QIAGEN DNA/RNA Kits. Reporter assay
problem Include positive controls to ensure that the reporter assay is working properly. Serum Serum may contain RNases. We therefore recommend transfection in the absence of
serum. Excessive
cell death Excessive exposure
of cells to
TransMessengerRNA
complexes If excessive cell death is observed after 3 h incubation, reduce the time of cellcomplex
incubation to 12 h. In addition, wash particularly sensitive cells (e.g., primary cells)
with 1.52 volumes of medium without serum instead of PBS after removal of the
TransMessengerRNA complexes. Concentration of
TransMessengerRNA
complexes is too high
Cells are stressed If cell death is still excessive after reducing the exposure time, decrease the amount of TransMessengerRNA complexes added to cells. Avoid stressing cells with temperature shifts and long periods without medium during washing steps. It is particularly important for RNA transfection that the cells are in good condition. Therefore, ensure that cell density is not too low at transfection. For adherent cells, the optimal confluency for transfection is 8090% (for singlestranded RNA) or 5080% (for siRNA). Be sure to seed cells a minimum of 24 h before transfection. Poor RNA quality RNA used for transfection should be of high quality. Impurities present in RNA preparations can potentially lower transfection efficiency. Degradation of RNA leads to decreased expression levels. RNA should be checked for degradation before transfection. We recommend purifying RNA using RNeasy Kits, Oligotex mRNA Kits, or QIAGEN DNA/RNA Kits. RNA-related effects Toxic effects may arise if RNA encoding a toxic protein is transfected, or if too much RNA is used. In contrast, if insufficient RNA with a low translation rate is used, transfection efficiency may be too low. Optimize the amount of RNA according to the optimization guide for each new RNA and/or cell type used. Key gene is silenced If the gene targeted in an RNAi experiment is key to the survival of the cell, silencing this gene will lead to cell death. Variable
transfection
efficiencies in
replicate
experiments Inconsistent cell
confluencies in
replicate experiments Count cells prior to seeding to ensure that the same number of cells is seeded for each
experiment. Keep incubation times between seeding and complex addition consistent
between experiments. Cells should be seeded no later than 24 h before transfection. Possible mycoplasma
contamination Mycoplasma contamination influences transfection efficiency. Variations in the growth behavior of mycoplasma-infected cells will lead to different transfection efficiencies between replicate experiments. Cells have been
passaged too
many times Cells that have been passaged a large number of times tend to change their growth behavior, morphology, and transfectability. When cells with high passage numbers are used for replicate experiments, decreased transfection efficiencies may be observed in later experiments. We recommend using cells with a low passage number (<50 splitting cycles). No or very small
gene silencing
effect observed
after siRNA
transfection Design of siRNA
sub-optimal The design of an siRNA can have a large effect on its gene silencing efficiency. QIAGEN offers expert advice on siRNA design for maximum gene silencing effect. For more information visit www.qiagen.com/sirna. Incubation time
post-transfection too
short The gene silencing effect observed on the protein level is dependent on a proteins expression level and its rate of turnover within the cell. Perform a time course experiment to determine the optimal time point for analysis. Problems with
experimental design RNAi effects may not be seen for some genes targeted with certain siRNAs in some cell types. If possible, repeat experiments using a different cell type and/or siRNA. Include where possible both positive and negative controls in your experiments. QIAGEN offers siRNA that has been functionally tested for specific gene silencing (see ordering information). References

QIAGEN maintains a large, up-to-date online database of scientific publications utilizing QIAGEN products. Comprehensive search options allow you to find the articles you need, either by a simple keyword search or by specifying the application, research area, title, etc. For a complete list of references, visit the QIAGEN Reference Database online at www.qiagen.com/RefDB/search.asp or contact QIAGEN Technical Services or your local distributor.
1. Dennig, J. and Konrad, J. (2002) TransMessenger Transfection Reagent enables targeted gene silencing in Hela-S3 cells through RNA interference. QIAGEN News, 2002 No. 2, 7.
2. Elbashir, S.M., et al. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494.
3. Caplen, N.J., Parrish, S.,Imani, F., Fire, A., and Morgan, R.A. (2001) Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA 98, 9742.
4. Krichevsky, A.M., and Kosik, K.S. (2002) RNAi functions in cultured mammalian neurons. Proc. Natl. Acad. Sci. USA 99, 11926.
5. Demeterco, C., Itkin-Ansari, P., Tyrberg, B., Ford, L., Jarvis, R., and Levine, F. (2002) c-Myc controls proliferation versus differentiation in human pancreatic endocrine cells. J. Clin. Endocrinol. Metab. 87, 3475.

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