Beruflich Dokumente
Kultur Dokumente
Handbook
October 2002
© 2002 QIAGEN, all rights reserved.
QIAGEN Worldwide
QIAGEN Companies
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QIAGEN Distributors
Please see the last page for contact information for your local QIAGEN distributor.
Contents
Kit Contents 4
Storage and Stability 4
Quality Control 4
Technical Assistance 4
Product Use Limitations 5
Product Warranty and Satisfaction Guarantee 5
Safety Information 5
Introduction 6
General Considerations for RNA Transfection 6
Cell culture 6
Serum 6
RNA structure 6
RNA quality 7
Handling RNA 7
General handling 7
Disposable plasticware 8
DNA contamination 8
Optimizing RNA Transfection 9
Cell density at transfection 9
Amount of RNA 10
Amount of Enhancer R 10
Ratio of TransMessenger Transfection Reagent to RNA–Enhancer R mixture 10
Incubation period with TransMessenger–RNA complexes 10
Protocol for Transfection of Adherent Cells with RNA 12
RNA interference (RNAi), siRNA, and Transmessenger Transfection Reagent 14
Optimizing siRNA Transfection 15
Guidelines for Transfection of siRNA Duplexes Using
TransMessenger Transfection Reagent 16
Troubleshooting Guide 18
References 21
Appendix 21
Ordering Information 22
QIAGEN International Sales and Distributors 27
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).
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.
* 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 80–90% confluent on the day of
transfection. The volume of medium used is not critical. Use a volume suitable for the cell culture format.
other formats are listed in Table 3 on page 13. See Table 1 on page 9 for the recom-
mended number of cells to seed. Optimal transfection conditions should be determined
for every cell type–RNA combination to obtain the highest transfection efficiency with
TransMessenger Transfection Reagent. Please refer to the optimization guidelines on
pages 9–10.
Important notes before starting
• We strongly recommend reading this handbook carefully before starting.
• Check the integrity and functionality of the RNA (see “RNA quality”, page 7).
• The RNA should be capable of being efficiently translated in the cell type used for
transfection (e.g., IRES compatibility; see “RNA structure”, page 6).
• The cells should be in optimal physiological condition on the day of transfection.
Subculture the cells a minimum of 24 hours before transfection. The optimal
confluency for transfection is 80–90%.
Procedure
1. The day before transfection, 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.
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.
2. Incubate cells under their normal growth conditions (generally 37°C and 5% CO2).
3. On the day of transfection, dilute 4 µl Enhancer R in Buffer EC-R. Add 2 µg RNA
(minimum RNA concentration 0.1 µg/µl) and mix by vortexing for 10 s. The final
volume should be 100 µl.
For example, if the RNA concentration is 0.5 µg/µl, dilute 4 µl Enhancer R in 92 µl
Buffer EC-R, then add 4 µl 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 (15–25°C) for 5 min, then spin down the mixture for
a few seconds to collect drops from the top of the tube.
5. Add 8 µl TransMessenger Transfection Reagent to the RNA–Enhancer R mixture. Mix
by pipetting up and down 5 times, or by vortexing for 10 s.
Protocol
medium used for cell seeding.
IMPORTANT: Do not allow the cells to become dry. Minimize the time they are with-
out medium.
8. Add 900 µl 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
Volume of
Final volume of TransMessenger medium*
Culture RNA Enhancer R RNA–Enhancer R Transfection Reagent added to
format (µg) (µl) in Buffer EC-R (µl) (µl) complexes (µl)
Protocol step 3 3 3 5 8
96-well plate 0.25 0.5 15 1.5 †
25
48-well plate 0.5 1.0 30 2.5† 50
24-well plate 0.8 1.6 100 4 100
12-well plate 1.6 3.2 100 6 300
6-well plate 2.0 4.0 100 8 900
* 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 µl or 20 µl, respectively, before addition to the RNA–Enhancer R mixture prepared
in step 3.
Amount
of siRNA Ratio of siRNA to TransMessenger Transfection Reagent (µg:µl)
1:2.5 1:5 1:10
No or very small Design of siRNA The design of an siRNA can have a large effect
gene silencing sub-optimal on its gene silencing efficiency. QIAGEN offers
effect observed expert advice on siRNA design for maximum
after siRNA gene silencing effect. For more information visit
transfection www.qiagen.com/sirna.
Incubation time The gene silencing effect observed on the protein
post-transfection too level is dependent on a protein’s expression
short level and its rate of turnover within the cell. Perform
a time course experiment to determine the optimal
time point for analysis.
Problems with RNAi effects may not be seen for some genes
experimental design 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).
Appendix
Buffer Composition Storage
1x PBS (phosphate-buffered saline) 137 mM NaCl Room temperature
2.7 mM KCl
4.3 mM Na2HPO4
1.47 mM KH2PO4
Adjust to a final pH of 7.4
Related products
RNeasy Mini Kits — for isolation of up to 100 µg total RNA from animal cells or
tissues, yeast, and bacteria
RNeasy Midi Kits — for isolation of up to 1 mg total RNA from animal cells or
tissues, yeast, and bacteria
RNeasy Maxi Kits — for isolation of up to 6 mg total RNA from animal cells or
tissues, yeast, and bacteria
Oligotex mRNA Kits† — for isolation of poly A+ mRNA from total RNA
Lamin A/C siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022050
specific gene silencing in RNAi experiments
Vimentin siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022055
specific gene silencing in RNAi experiments
NuMa siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022058
specific gene silencing in RNAi experiments
Lamin B1 siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022061
specific gene silencing in RNAi experiments
GFP-22 siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022064
specific gene silencing in RNAi experiments
CAT-22 siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022067
specific gene silencing in RNAi experiments
Luciferase GL2 siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022070
specific gene silencing in RNAi experiments
Luciferase GL3 siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022073
specific gene silencing in RNAi experiments
Control (non-sil.) siRNA (5 nm) 5 nmol duplex siRNA; for use as a 1022076
non-silencing control in RNAi experiments
Control(non-sil.) siRNA, 5 nmol fluorescein-labeled duplex siRNA; 1022079
Fluorescein (5nm) for use as a non-silencing control in RNAi
experiments
Control (non-sil.) siRNA, 5 nmol rhodamine-labeled duplex siRNA; 1022083
Rhodamine (5nm) for use as a non-silencing control in RNAi
experiments
GAS41 siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022153
specific gene silencing in RNAi experiments
Lamin B2 siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022156
specific gene silencing in RNAi experiments
Emerin siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022159
specific gene silencing in RNAi experiments
LAP2 siRNA (5 nm) 5 nmol duplex siRNA; functionally tested for 1022162
specific gene silencing in RNAi experiments
All library siRNAs are also available in 10 and 20 nmol amounts; please inquire.
Numerous 3' and 5' modifications are available on request. For an up-to-date list, and information on how to
order custom siRNA, please visit www.qiagen.com/sirna.
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