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Sede Commerciale:

Via delle Mattine, 76 già Costa della Gaveta, 69 – 85100 Potenza Tel. 0971.470600 - Fax 0971.470418

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SPETTABILE C.R.O.B. I.R.C.C.S.

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85028 RIONERO IN VULTURE (PZ) --- Vs. rif. TD1417779 del 25.09.2020

OGGETTO: “FORNITURA PRODOTTI INFUNGIBILI OCCORRENTI AI LABORATORI DI RICERCA”.

OFFERTA TECNICA:

N. 1 CF – FLEXITUBE siRNA 5nmol COD. 1027417 (SUB CODICE SI04226691)

“QIAGEN”

CND N.A. – Numero di Repertorio N.A.

N. 3 CF – POLYFECT TRANSFECTION REAGENT (1ml) COD. 301105 “QIAGEN”

CND W01 – Numero di Repertorio N.A.

N. 3 CF – HIPERFECT TRANSFECTION REAGENT (1ml) COD. 301705 “QIAGEN”

CND W01 – Numero di Repertorio N.A.

Altre caratteristiche tecniche come da scheda tecnico-illustrativa in allegato.

T E C N O L I F E S.R.L.

Amministratore Delegato

Michele Laranga

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QIAGEN ® Transfection Technologies

Efficient and robust

transfection — for all

your applications

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Contents

Product Selection Guide 3

siRNA and miRNA Transfection 4

HiPerFect Transfection Reagent 4

HiPerFect HTS Reagent 6

DNA Transfection 8

Attractene Transfection Reagent 8

Plasmid DNA and siRNA/miRNA Cotransfection 9

Attractene Transfection Reagent 9

DNA Transfection of Primary Cells and Sensitive Cell Lines 10

Effectene Transfection Reagent 10

mRNA Transfection 12

TransMessenger Transfection Reagent 12

Transfection Resources 13

TransFect Protocol Database 13

Transfection Cell Database 14

Reference Database 14

Ordering Information 15

QIAGEN solutions for efficient transfection

Transfection is a commonly used tool for current genetic and molecular biology applications, as well as for researching cancer and other diseases. Several critical factors, including cell type and the nucleic acid to be transfected, must be carefully considered to ensure successful delivery into cells. To overcome major challenges and to meet specific needs for transfection of various nucleic acids, QIAGEN provides a comprehensive range of reagents for DNA, mRNA, siRNA, miRNA transfection and cotransfection into a wide variety of cell lines, including sensitive primary cells. Simply choose your transfection reagent using the Product Selection Guide and consult the reagent page for more details. Valuable transfection resources, including protocols and details about successfully transfected cell lines, are available at www.qiagen.com/TransFect-protocol and www.qiagen.com/Transfection-Cell.

Table of contents

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Product Selection Guide

Feature HiPerFect HiPerFect HTS Attractene Effectene TransMessenger

Nucleic acid siRNA, miRNA siRNA, miRNA DNA, siRNA,

or miRNA/DNA cotransfection

DNA mRNA

Benefit for transfection Low siRNA amounts minimize off-target effects

Easy to use and economical for high-throughput experiments

Efficient

transfection with very low cytotoxicity

Highly effective for primary and sensitive cells

Efficient transfection

Cell lines Eukaryotic cell lines

and primary cells Eukaryotic cell lines

and primary cells Adherent cells Eukaryotic cell lines

and primary cells Eukaryotic cell lines and primary cells Transfection in

presence of serum Yes Yes Yes Yes Yes

Reverse transfection Yes Yes Yes Yes Yes

Tested for endotoxins Yes Yes

Table 1. Key features of QIAGEN transfection reagents

Nucleic acid siRNA

miRNA DNA siRNA or

miRNA/DNA

co-transfection mRNA

Cell type

Throughput

Low High

All Adherent Primary & Adherent All

difficult-to- transfect

Recommended transfection

reagent

HiPerFect® Transfection

Reagent

Attractene Transfection

Reagent

Attractene Transfection

Reagent

TransMessenger® Transfection

Reagent HiPerfect

ReagentHTS

Effectene® Transfection

Reagent

Product Selection Guide

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siRNA and miRNA Transfection

Highly efficient siRNA and miRNA transfection

HiPerFect Transfection Reagent — experience high efficiency, with low off-target effects

n Highly efficient transfection of as little as 10 pM siRNA

n Enables use of low siRNA concentrations to minimize off-target effects

n High cell viability of even difficult-to-transfect cells

HiPerFect Transfection Reagent has been specifically developed for highly efficient transfection of eukaryotic cells with as little as 10 pM siRNA, minimizing the likelihood of off-target effects. A unique blend of cationic and neutral lipids enables effective siRNA uptake and high gene knockdown, even with low siRNA concentrations (Figures 1 and 3). In addition to siRNA, HiPerFect Transfection Reagent is highly suited for transfection of miRNA mimics, inhibitors, or target protectors.

Transfection can be performed by seeding the cells 24 hours prior to transfection, or cell seeding and transfection can be performed on the same day, which is the standard procedure for reverse transfection (Figure 2).

Figure 1. nA HiPerFect Transfection Reagent consists of a unique blend of cationic and neutral lipids that optimizes transfection complex formation and release of siRNA in cells. nB Other

suboptimal lipid reagents do not efficiently release siRNA in cells, leading to inefficient knockdown.

HiPerFect Reagent

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Cationic lipid Neutral lipid siRNA A

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Chaptertitle X.XX siRNA and miRNA Transfection

Easy-to-follow procedure, for any cell type

HiPerFect Transfection Reagent is provided as a ready-to-use solution — just add the reagent to your diluted siRNA/miRNA, mix, incubate, and pipet the complexes onto the cells. Transfections can be performed in the presence of serum, eliminating the need to remove complexes from cells.

A wide range of adherent and suspension cells, as well as primary cell lines have been successfully transfected with the HiPerFect Transfection Reagent (for an overview of selected cell lines, refer to Table 2). Take the guesswork out of transfection with cell-specific protocols, available in the TransFect Protocol Database (www.qiagen.com/Transfect-protocol).

Transfection of siRNA can result in off-target effects, which may produce misleading results in RNAi experiments. These effects can be largely avoided by using low siRNA concentrations. With HiPerFect Transfection Reagent, highly efficient transfection and silencing have been observed with as little as 10 pM siRNA, allowing more experiments with each set of siRNA (Figure 3).

Figure 2. Reverse transfection using HiPerFect Transfection Reagent.

Figure 3. Efficient transfection of picomolar siRNA amounts.

HeLa S3 cells were transfected with a range of amounts of CDC2 siRNA and with a nonsilencing control siRNA (Control) using HiPerFect Transfection Reagent (3 µl) in a 24-well plate format. After 48 hours, CDC2 expression was analyzed by quantitative, real-time RT-PCR.

Cell line Cell type siRNA concentration used

(% knockdown achieved)

K562 Human chronic myeloid leukemia 5 nM (85%)

Jurkat Human T-cell 75 nM (83%)

D1.1 Human T-cell 50 nM (84%)

RAW 264.7 Mouse macrophage 25 nM (77 %)

J774.A1 Mouse macrophage 50 nM (97 %)

PMA-differentiated

THP-1 Human acute monocytic

leukemia cells 5 nM (82 %)

MEL Murine erythroleukemia cells 5–20 nM (50–70 %)

Table 2. Successfully transfected cell lines

Reverse transfection using HiPerFect Transfection Reagent

Step 1 siRNA

Step 2 HiPerFect Reagent Step 3 Cells

120 90 60 30 0

10 nM CDC25 nM CDC21 nM CDC2

100 pM CDC210 pM CDC2 Contro

l Untransfected Relative expression of CDC2 mRNA (%)

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siRNA and miRNA Transfection

Figure 4. Reproducibly high transfection and silencing efficiency. MCF-7 cells were transfected with CDC2 siRNA (10 nM, 20 nM, or 30 nM) or AllStars Negative Control siRNA (30 nM) using HiPerFect HTS Reagent in 96-well plates. Each transfection was performed in tripli- cate. Gene silencing was analyzed by real-time, quantitative RT-PCR. All samples transfected with CDC2 siRNA demon- strated consistently high CDC2 knockdown in all replicates.

Figure 5. High-throughput transfection using HiPerFect HTS Reagent.

Relative CDC2 mRNA expression

1.8 1.5 1.2 0.9 0.6 0.3

0 1 2 3

30 nM CDC2 1 2 3 1 2 3 1 2 3

20 nM CDC2 10 nM CDC2 30 nM AllStars Blank

Complex formation:

Spot siRNA, add HiPerFect

HTS Reagent

Transfection:

Add cells

HiPerFect HTS Reagent — for high-throughput RNAi experiments

In high-throughput pathway and miRNA functional analyses or drug discovery, easy-to-handle processes, reliable results, and cost effectiveness are key priorities. HiPerFect HTS Reagent has been developed especially to meet these needs in RNAi experiments. It is highly stable and easy to use, and provides robust and reliable transfection of siRNA and miRNA, ensuring consistent and reproducible data (Figure 4).

HiPerFect HTS Reagent provides:

n A one-day, one-plate procedure

n Economical RNAi experiments

n Reproducible, high transfection efficiency of siRNA/miRNA

n Ease of handling and stability

Reliable transfection for reproducible results

Reductions in time-to-result, labor, and consumable use significantly improve the workflow of high-throughput experiments. With HiPerFect HTS Reagent, cells are seeded and transfected on the same day, saving time and effort. Just one plate is used for both complex formation and transfection, minimizing consumable use and pipetting steps (Figure 5).

Low volumes of HiPerFect HTS Reagent are used for each transfection, ensuring maximum cost-efficiency without compromising on the reproducibility and reliability of RNAi results. With HiPerFect HTS Reagent, efficient transfection is ensured in a wide range of cell lines, including difficult-to-transfect primary cells (Figure 6).

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Relative CDC2 mRNA expression 1.2 0.9 0.6 0.3 0

10 nM

Lot A

5 nM 1 nM 10 nM 10 nM

Lot B

5 nM 1 nM 10 nM 10 nM

Lot C

5 nM 1 nM 10 nM 10 nM

Lot D

5 nM 1 nM 10 nM 10 nM

Lot E

5 nM 1 nM 10 nM

CDC2 siRNA AllStars

Blank

siRNA and miRNA Transfection

Ease-of-use and stability facilitates workflow

The use of a robust, high-performing reagent is essential for a standardized, smooth-running, high-throughput RNAi transfection workflow, where high numbers of samples are processed simultaneously. HiPerFect HTS Reagent offers many benefits, including high lot-to-lot consistency and high stability in dilution (Figures 7 and 8). These qualities minimize experimental artifacts and make experimental setup easier. For example, HiPerFect HTS Reagent can be diluted in the morning and used for complex formation throughout the day without any decrease in performance.

Figure 6. Efficient transfec- tion in human and mouse cell lines. Various cell types were transfected with Lamin A/C siRNA or AllStars Negative Control siRNA at the concentra- tions indicated using HiPerFect HTS Reagent in 384-well plates. Gene silencing was analyzed by real-time, quantitative RT-PCR. HiPerFect HTS Reagent provided high Lamin A/C knockdown for all concentrations tested in all cell types.

Figure 7. High lot-to-lot consistency. HeLa S3 cells were transfected with CDC2 siRNA or AllStars Negative Control siRNA at the concentrations indicated using HiPerFect HTS Reagent from 5 different lots in 96-well plates. Gene silencing was analyzed by real-time, quantitative RT-PCR. All samples transfected with CDC2 siRNA demon- strated consistently high CDC2 knockdown for all reagent lots.

1.2 1.0 0.8 0.6

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DNA Transfection

Robust and efficient DNA transfection

Attractene Transfection Reagent — for all adherent eukaryotic cells

n Highly efficient transfection with extremely low cytotoxicity

n Reagent of choice for all adherent cells, including difficult-to-transfect cells

n Rapid Fast-Forward Protocol

n Highly suited for cotransfection and vector-based RNAi

Attractene Transfection Reagent represents a new generation in lipid technology, ensuring highly efficient DNA transfection of eukaryotic cells in the presence of serum. It is a nonliposomal lipid that enables transfection of all adherent cells, including difficult-to-transfect cell types such as HaCaT, MonoMac6, and HCT116, and some suspension cell types (Jurkat, K562).

It is also highly suitable for cotransfection of DNA with siRNA or miRNA mimics, inhibitors, or target protectors.

Extremely low cytotoxicity

A critical factor for successful transfection experiments is ensuring that the transfection reagent and transfection process do not cause cytotoxicity, which would result in unreliable data that is difficult to interpret. Stressed cells have altered gene expression patterns compared to unstressed cells. In addition, the presence of dead cells makes it difficult to observe changes in cell phenotype. The exceptionally low cytotoxicity of Attractene Transfection Reagent ensures that results are due to the nucleic acid transfected and not to the transfection process itself (Figure 9).

Figure 9. Healthy cells after transfection using Attractene Transfection Reagent. HepG2 cells were transfected with DNA (pGFP) using Attractene Transfection Reagent or Reagent L from another supplier according to the manufacturer’s instructions. FACS® analysis confirmed equal numbers of transfected cells in both cultures. Two days after transfection, cells were examined by light microscopy. Cells transfected using Attractene Transfection Reagent were healthy and viable. In contrast, cells transfected using Reagent L displayed high levels of cell death. Cell viability was measured using a CellTiter-Blue® assay (Promega).

Viability was significantly lower in cells transfected using Reagent L compared to cells transfected using Attractene Transfection Reagent (set at 100%).

Attractene Reagent Reagent L

A B

C

Attractene Reagent Reagent L

A B

C

Attractene Reagent Reagent L

A B

C

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Plasmid DNA and siRNA/miRNA Cotransfection

Flexible Fast-Forward Protocol saves time

Attractene Transfection Reagent is highly suited for rapid fast-forward DNA transfection. In the Fast-Forward Protocol, cells are seeded and transfected on the same day (Figures 10 and 11). This is faster, saves labor, and increases experimental flexibility compared to protocols where cells are seeded the day before transfection.

Efficient cotransfection of plasmid DNA with siRNA or miRNA

Attractene Transfection Reagent can be used for a broad range of applications, including transient or stable transfection or cotransfection of plasmid DNA with siRNA or miRNA. Ease and flexibility of handling enables preparation and storage of transfection complexes, making Attractene Transfection Reagent suitable for use with automated systems.

Transfection of shRNA (short-hairpin RNA) vectors for gene silencing experiments can be achieved with high efficiency (Figure 12). Attractene Transfection Reagent is also highly suitable for cotransfection of DNA with siRNA or miRNA mimics, inhibitors, or target protectors.

Figure 12. Effective knockdown after shRNA vector transfection. Using Attractene Transfection Reagent, HEK293 cells were transfected with a plasmid expressing green fluorescent protein (pGFP) only, or cotransfected with pGFP and a plasmid expressing an shRNA targeted against the green fluorescent protein gene (pGFP + pshGFP), or cotransfected with pGFP and a negative control plasmid expressing a scrambled shRNA (pGFP + pNegative). After cotransfection of GFP and the shRNA vector pshGFP, the green- fluorescent protein was effectively silenced, indicating efficient cotransfection.

Figure 10. Attractene workflow. Simply mix reagent and DNA, incubate, and then add to cells. Cells can be seeded the day before or the day of transfection.

Figure 11. Flexible, rapid Fast-Forward Protocols.

Traditionally, cells are seeded the day before transfection.

Using the Fast-Forward Protocol, seeding and transfection take place on the same day.

DNA

Incubate 10 min at RT

20 min Attractene

Reagent

Add medium and apply to cells

pGFP pGFP + pshGFP pGFP + pNegative

A B C

pGFP pGFP + pshGFP pGFP + pNegative

A B C

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DNA Transfection of Primary Cells and Sensitive Cell Lines

Effectene Transfection Reagent — for primary cells and sensitive cell lines

n Far lower toxicity and gentler than many alternatives

n Highly efficient transfection with low DNA amounts and in the presence of serum

n Low endotoxin level, with <10 EU/ml

Low cytotoxicity — highly-suited for transfection of sensitive cells

Effectene Transfection Reagent is an innovative, non-liposomal lipid formulation that is used in conjunction with a special DNA-condensing enhancer and optimized buffer to achieve high transfection efficiencies in sensitive cells. Because transfection can be performed in the presence of serum and requires low amounts of DNA, cytotoxicity is minimal, making Effectene Transfection Reagent highly suitable for transfection of sensitive cell types such as primary cells (Figures 13 and 14), and offering significant advantages compared to other transfection reagents (Figure15).

Figure 13. Transfection efficiency of 40% in primary cells.

Expression of green fluorescent protein (GFP) in primary rabbit aortic smooth muscle cells transfected using Effectene Reagent. One day prior to transfection, 1 x 105 cells were seeded, and transfections were performed in 6-well plates using 0.4 µg of a GFP-reporter plasmid and 10 µl Effectene Transfection Reagent per well. Cells were viewed 24 hours post-transfection by fluorescence microscopy.

Approximately 40% of the cells were transfected, as determined by FACS analysis.

(Data provided by K. Veit, 2nd Medical Clinic, Dept.

Clinical Pharmacology, Mainz, Germany.)

Figure 14. Efficient transfection of primary neuronal cells using Effectene Transfection Reagent. Primary neuronal cells, cultivated on coverslips in 24-well plates, were transfected with plasmid DNA expressing green fluorescent protein. A range of plasmid DNA to Effectene Transfection Reagent ratios was transfected. Transfection efficiencies are expressed as the percentage of cells that were GFP positive. Highest transfection efficiencies were obtained using 200 ng of DNA with 2–5 µl of Effectene Transfection Reagent.

(Data provided by J. Meier, I. Strömel, R. Iosub, S.

Schmidt, and R. Grantyn, Humboldt University Medical School [Charité], Berlin, Germany.)

200 16

12 8 4

0

400 800

DNA (ng)

Transfection effeciency (%)

Effectene Reagent:DNA

= 10:1 (µl:µg) Effectene Reagent:DNA

= 25:1 (µl:µg) Effectene Reagent:DNA

= 50:1 (µl:µg)

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DNA Transfection of Primary Cells and Sensitive Cell Lines

Easy, straightforward procedure

The Effectene procedure enables fast and straightforward transfection, saving time and effort (see flowchart). DNA is first mixed with DNA- condensing enhancer and buffer. Next, Effectene Transfection Reagent is added and complexes are formed. Complexes are mixed with medium (which can contain serum and antibiotics) and are added to cells — removal of complexes after transfection is not necessary for most cell lines. The advanced Effectene technology allows efficient transfection of even small amounts of DNA in the presence of serum.

Effectene outperforms other lipid-based reagents

Due to extremely low cytotoxicity, Effectene Transfection Reagent is highly suitable for transfection of sensitive cell types, such as primary cells. Compared to other, commonly used lipid-based reagents, Effectene delivers higher transfection efficiency (Figure 15).

Figure 15. Comparison of transfection efficiencies using Effectene Transfection Reagent and two commonly used lipid-based reagents. Murine teratocarcinoma F9 cells (5 x 105) were transfected in 6-well plates with a luciferase-reporter plasmid using optimized conditions based on the manufacturer’s instructions for each reagent. Transfection efficiencies were determined by measuring luciferase activity 48 hours post-transfection, and are given as relative light units (RLU).

(Data provided by I. Clavereau, D. Petitprez, and I. Van Seuningen, Unité INSERM 377, Place de Verdun, Lille Cedex, France.)

Add medium and apply to cells

Effectene procedure

Add Effectene Transfection Reagent

Mix and incubate 5–10 min at RT

Add Enhancer

Mix and incubate 2–5 min at RT DNA in Buffer EC

Incubate to allow expression of the transfected 0 gene

10 20 30 40 50

Effectene

Reagent Reagent L Reagent F Transfection reagent

Transfection efficiency (104 x RLU)

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mRNA Transfection

Highly efficient transfection of mRNA

TransMessenger Transfection Reagent

n Highly efficient mRNA transfection

n Specially developed lipid formulation ensures reproducible results

n Efficient transfection of primary neuronal cells

TransMessenger Transfection Reagent is a non-liposomal lipid that works in combination with a nucleic acid-condensing enhancer to form small transfection complexes that are more easily taken up into cells.

RNA molecules are condensed by the enhancer, and then coated by TransMessenger Transfection Reagent, for efficient transfer into eukaryotic cells. This allows highly efficient mRNA transfection, even in the presence of serum (Figures 16 and 17). The reagent can also be used for efficient transfection of neuronal cells (1).

Optimal results are achieved using high-purity RNA that is free of DNA, proteins, and other contaminants. RNA purified with RNeasy Kits is highly recommended. Since the amount of RNA is a critical factor for successful transfection, we recommend optimizing the amounts of RNA and TransMessenger Transfection Reagent for every cell type–RNA combination (Figure 17).

Transfection of cells with RNA, rather than DNA, provides an alternative approach that offers many possibilities for transfection experiments. For example, RNA transfection could be useful for studying cells that are not efficiently transfected with plasmid DNA, and could allow direct studies of RNA function. Transfected RNA sequences are expressed in the absence of transcription and in a promoter-independent manner. In addition, protein expression usually occurs sooner following transfection of RNA than following transfection of DNA.

Reference

1. Krichevsky A.M. and Kosik K.S. (2002) RNAi functions in cultured mammalian neurons. Proc. Natl. Acad. Sci. USA 99, 11926.

For additional references using TransMessenger Transfection Reagent in neuronal cells, see www.qiagen.com/RefDB/search.asp.

Figure 16. Efficient transfection using TransMessenger Transfection Reagent. Green fluorescent protein (GFP) was expressed in HeLa S3 cells. In this experiment, 8 x 104 cells were seeded into 48-well plates and transfected 24 hours later with 0.5 µg of an in vitro-transcribed GFP- encoding RNA (transcribed from P7ASP-GFP/Mlu) using 1 µl Enhancer R and 2.5 µl TransMessenger Transfection Reagent. Cells were analyzed 24 hours post-transfection by fluorescence microscopy. Approximately 50% of the cells were successfully transfected.

(P7ASP-GFP/Mlu provided by J. Bogenberger, Stanford University Blood Center, Palo Alto, CA, USA.)

Figure 17. Amount of RNA and TransMessenger Transfection Reagent versus transfection efficiency. In optimization experiments using CHO-K1 cells, 2 x 104 cells were seeded in quadruplicate into 96-well plates and transfected 24 hours later with an in vitro-transcribed CAT-encoding RNA using nA increasing amounts of RNA with 1.5 µl TransMessenger Transfection Reagent and nB increasing amounts of TransMessenger Transfection Reagent with 0.25 µg RNA, as described in the TransMessenger Transfection Reagent Handbook.

CAT activity was measured 24 hours post-transfection.

0 0.125 µg

0.75 µl 1.5 µl 2.25 µl

0.25 µg 0.5 µg

0.2 0.4 0.6 0.8 1.0 1.2 1.4

0 0.2 0.4 0.6 0.8 1.0

Amount of RNA

CAT activity (ng CAT/ml)CAT activity (ng CAT/ml)

Amount of TransMessenger Reagent A

B

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Transfection Resources

Transfection Resources

TransFect Protocol Database

n Protocols for specific cell types

n Selectable protocols for plate format and nucleic acid

n Reverse-transfection and Fast-Forward Protocols

n Easy-to-access online database

The TransFect Protocol Database is an invaluable resource for transfection experiments that takes the guesswork out of transfection protocols. Rather than adapting existing protocols to fit a certain cell type or plate format, the database provides the exact protocol needed, saving time and effort.

Simply visit www.qiagen.com/TransFect-protocol and enter the cell type, nucleic acid, and plate format to receive a QIAGEN transfection protocol to print out or download in convenient PDF format (Figure 18). Use of the TransFect Protocol Database is free of charge and no registration is required. Protocols can be generated for hundreds of cell types, multiple plate formats, and DNA, RNA, and siRNA.

Figure 18. Easily search for and download a protocol using the TransFect Protocol Database.

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Chaptertitle X.XX Transfection Resources

References from your fellow researchers

Access to peer-reviewed publications that use transfection reagents from QIAGEN can be gained from the online reference database at www.qiagen.com/RefDB/search.asp.

Transfection Cell Database

n Comprehensive list of transfected cell lines with experimental details

n Wide range of protocols

n Easy-to-access online database

Visit the Transfection Cell Database (www.qiagen.com/Transfection-Cell), where fellow researchers have provided useful data and practical experimental details such as growth conditions, nucleic acid concentration, plate format, cell number, and incubation time for a wide range of successfully transfected cell lines (Figure 19). View a complete, detailed list of all cell lines or search for results for your cell line of interest.

Figure 19. Useful protocols and data for a wide range of cell lines. The transfection cell database provides practical experimental details to help you in your research.

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Chaptertitle X.XX Ordering Information

Ordering Information

Product Contents Cat. no.

siRNA and miRNA transfection

HiPerFect Transfection Reagent (0.1 ml)* Trial kit: Reagent for up to 33 transfections in 24-well plates or up to 133 transfections in 96-well plates

301702

HiPerFect Transfection Reagent (1 ml)* Reagent for up to 333 transfections in 24-well plates or up to 1333 transfections in 96-well plates

301705

HiPerFect HTS Reagent (0.1 ml)* Trial kit: Reagent for transfections in 4–10 x 96-well plates 301802

HiPerFect HTS Reagent (2 x 1 ml)* Reagent for transfections in 80–200 x 96-well plates 301806

DNA transfection

Attractene Transfection Reagent (0.1 ml)* Trial kit: Reagent for up to 66 transfections in 24-well plates

1051561

Attractene Transfection Reagent (1 ml)* Reagent for up to 660 transfections in 24-well plates 301005

Effectene Transfection Reagent (0.3 ml)* Trial kit: Reagent, Enhancer, Buffer for 12 transfections in 60 mm dishes or 53 transfections in 12-well plates

1054250

Effectene Transfection Reagent (1 ml)* Reagent, Enhancer, Buffer for 40 transfections in 60 mm dishes or 160 transfections in 12-well plates

301425

mRNA transfection

TransMessenger Transfection Reagent (0.5 ml) Reagent, Enhancer, Buffer for 60 transfections in 6-well plates or 80 transfections in 12-well plates

301525

For bulk quantities (e.g., 100 ml), please inquire.

* Additional sizes available. Also suitable for DNA/siRNA or miRNA cotransfection.

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.

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USA n Orders 800-426-8157 n Fax 800-718-2056 n Technical 800-DNA-PREP (800-362-7737)

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Flexible RNAi Technologies You Can Rely On

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RNAi is an invaluable tool for functional genomics and drug discovery research. In this brochure, you can discover QIAGEN’s RNAi product portfolio, consult recommendations for control experiments, and read about critical factors. Whatever your throughput level, QIAGEN provides flexible, innovative technologies to facilitate success in your research!

Critical parameters for successful RNAi 4

siRNA design 6

siRNA delivery 7

RNAi controls 8

QIAGEN’s GeneGlobe® Web portal 9

RNAi selection guide 11

RNAi product descriptions 12

Ordering information 19

Resources

GeneGlobe Web portal: source of gene- and pathway-specific products www.qiagen.com/GeneGlobe

RNAi products and resources

www.qiagen.com/siRNA

TransFect Protocol Database for cell-type specific transfection protocols

www.qiagen.com/TransFect

Transfection Cell Database: a comprehensive list of transfected cell lines with experimental details

www.qiagen.com/TransfectionCellDatabase QIAGEN AllStars RNAi Controls page:

information about appropriate RNAi controls

www.qiagen.com/AllStars

Literature, journal references, tips and tools, and more

www.qiagen.com/Support QIAGEN ProductFinder

www.qiagen.com/ProductFinder

Introduction

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In functional genomics research, chemically synthesized short interfering RNAs (siRNAs) are transfected into cells. An RNA-induced silencing complex (RISC) is assembled, siRNAs unwind, and a single strand of RNA remains bound to the RISC. The RISC targets mRNA transcripts that have complementary sequences to the bound RNA and cleaves the homologous mRNA, preventing translation.

miRNAs are first transcribed in the nucleus as long, primary miRNAs (pri-miRNA) which are then processed into precursor miRNAs (pre-miRNA) by the nuclear microprocessor complex (Drosha and DGCR8). Pre-miRNAs are transported into the cytosol by Exportin-5. Dicer and TRBP process the pre-miRNAs into mature miRNAs that are incorporated into the RISC. When the RISC identifies the complementary or partially complementary mRNA target, it inhibits gene expression by translational repression or by mRNA degradation.

Transfection of siRNA as a research tool for functional genomics

TRBP DGCR8

Introduction

siRNA and miRNA pathways

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Critical parameters

Critical parameters for successful RNAi

Planning an RNAi experiment begins with a biological question or hypothesis, and this helps to define many of the parameters incorporated into the experimental design. These parameters include the type and number of gene targets, the choice of cell line and method of siRNA delivery, the downstream assay, appropriate controls (see page 8), time points for analysis, and finally, the statistical methodology for data analysis.

Gene and siRNA selection

RNAi experiments range in scale from the knockdown of a single gene of interest, to small numbers of related genes or genes within a common pathway, to genomewide high-throughput screens. In addition to individual siRNAs in tubes, QIAGEN provides FlexiPlate siRNA, which allows you to pick and choose gene targets for medium- to high- throughput experiments and to custom array the siRNAs in plates (page 13). QIAGEN has also developed and validated several control siRNAs which should be included in each RNAi experiment to ensure reliable interpretation of results (page 15).

Optimized transfection

The cell type chosen for RNAi experiments should allow efficient siRNA delivery. Transfection must be optimized to ensure that siRNA is transfected at the lowest effective concentration to avoid unwanted nonspecific effects. The transfection method selected should provide high efficiency and low cytotoxicity, and should be amenable to the experimental setup, for example, suitable for reverse transfection in an automated format.

Robust phenotypic analysis

Phenotypic analysis often involves cell-based assays, which are designed to measure a variety of cellular parameters, including viability, production of detectable products, changes in protein localization, ion concentration, cellular motility, and morphology. Characterization of changes in protein localization and protein modification can provide important clues about gene function (see Qproteome® Kits, page 18). Cell-based assays measuring pathway activity can reveal information about gene function and regulation (see Cignal Reporter System at www.SABiosciences.com).

Knockdown verification

QIAGEN recommends using at least 2 siRNAs that target different regions within the same transcript.

The probability that the same off-target phenotype is induced by 2 independent siRNAs for the same target gene is extremely low. Therefore, confirming a phenotype with distinct siRNAs is an easily applied and convincing way to verify that the observed phenotype is specific to the target gene knockdown (Figure 1). Confirmation with multiple siRNAs has become a requirement for publication of RNAi results in many peer-reviewed journals. Knockdown of the target gene can be verified at the mRNA level by real-time RT-PCR or at the protein level by western blotting (page 18).

In addition to confirmation with multiple siRNAs, a rescue experiment can be performed to provide conclusive evidence that knockdown of a targeted gene, and not an off-target effect, is responsible for an observed phenotype (page 8).

Untransfected cells

Nonsilencing siRNA

QIAGEN siRNA 1

QIAGEN siRNA 2

Figure 1. Confirmation of phenotype with 2 siRNAs. A549 cells were examined 24 hours after transfection with PLK1 siRNAs or nonsilencing siRNA. PLK1 knockdown characteristically results in the accumulation of rounded, apparently mitotic cells. This phenotype is clearly visible after transfection of each of the 2 siRNAs designed by QIAGEN. (Data kindly provided by Ralph Graeser, Sarah Umber, Michaela Brosig, and Michael H.G. Kubbutat, ProQinase GmbH, Freiburg, Germany.)

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RNAi workfl ow

RNA purification

Protein purification Phenotypic assay

siRNA selection

siRNA transfection

cDNA preparation

Western blot Rescue experiment

Counts Counts

Counts Counts

500 400 300 200 100 0

Propidium iodide400 600 800 1000

0 200

Untransfected G2 16.2%

QIAgenes + CDC2 siRNA

CDC2 siRNA only

QIAgenes + nonsilencing siRNA 700

560 420 280 140 0

Propidium iodide400 600 800 1000

0 200

G2 36.3%

850 680 510 340 170 0

Propidium iodide400 600 800 1000

0 200

G2 23.4%

950 760 570 380 190 0

Propidium iodide400 600 800 1000

0 200

G2 14.9%

G0/G1 S G2 G0/G1 S G2

G0/G1 S G2 G0/G1 S G2

FlexiTube siRNA FlexiTube siRNA Premix FlexiPlate siRNA

SureSilencingshRNA Plasmids

QIAGEN AllStars RNAi Controls

HiPerFect® Transfection Reagent

Qproteome Kits RNeasy® Kits

AllPrep® Kits miRNeasy Kits

QuantiTect® Reverse

Transcription Kit Rescue

siRNA Cignal Reporter System

QIAgenes Expression Kits FastLane® Kits

Biology-on-ArraySystems

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siRNA design

siRNA design

siRNAs from QIAGEN are designed using cutting-edge HP OnGuard siRNA Design, which delivers potent and specific siRNA. Multiple siRNAs for every human, mouse, and rat gene in the EntrezGene database can be ordered at the QIAGEN GeneGlobe Web portal (page 9). HP OnGuard siRNA Design incorporates many unique and advanced features (Table 1).

Feature Description Benefit References

Neural-network

technology siRNA design uses the BioPredsi neural network

which is based on an extremely large RNAi data set. Potent siRNA 1–3

The world’s largest siRNA validation project

Data from this project, in which QIAGEN scientists proved the effectiveness of thousands of siRNAs, were used to reinforce and improve the design process.

A large number of druggable genome siRNAs have been proven in wet-lab experiments to provide at least 70% knockdown during this project; insights from the project enhanced design of potent siRNA

4

Homology analysis A proprietary tool and an up-to-date,

nonredundant sequence database are used. Decreases off-target effects Affymetrix®

GeneChip® analysis Genomewide analysis enabled development of siRNA design improvements that minimize off-target effects.

Insights from the analysis enhanced design of specific siRNA

Up-to-date siRNA

target sequences Current data from NCBI databases

ensure accurate design. Accurate siRNA design

Asymmetry siRNAs are designed with unequal stabilities of the base pairs at the 5' end of the antisense strand. This enables the antisense strand, which is less tightly bound at its 5' end, to enter RISC, while the sense strand is degraded.

Produces highly functional siRNAs and reduces the risk of off-target effects caused by the incorrect

strand entering RISC

5, 6

3' UTR/seed

region analysis Intelligently weighted, multi-parameter searches for matches of the seed region of the siRNA antisense strand with the 3' untranslated region of unintended mRNA targets are performed.

Minimizes risk of off-target effects caused by

siRNA acting like an miRNA 7–12

SNP avoidance The RefSNP database is used to exclude siRNAs

which span single nucleotide polymorphisms (SNPs). Increases siRNA potency, as an siRNA spanning a SNP will vary in its effectiveness Interferon motif

avoidance siRNAs are screened for multiple sequence motifs known to result in an interferon response. siRNAs with such motifs are rejected.

Reduces risk of off-target effects caused by an

interferon response 13, 14

Table 1. HP OnGuard siRNA Design features

References

1. Huesken, D. et al. (2005) Design of a genome-wide siRNA library using an artificial neural network. Nat. Biotechnol. 23, 995.

2. Mukherji, M. et al. (2006) Genome-wide functional analysis of human cell-cycle regulators. Proc. Natl. Acad. Sci. 103, 14819.

3. Matveeva, O. et al. (2007) Comparison of approaches for rational siRNA design leading to a new efficient and transparent method.

Nucleic Acids Res. 35, e63.

4. Krueger, U. et al. (2007) Insights into effective RNAi gained from large-scale siRNA validation screening. Oligonucleotides 17, 237.

5. Aza-Blanc, P. et al. (2003) Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol. Cell 12, 627.

6. Schwarz, D.S. et al. (2003) Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199.

7. Farh, K.K. et al. (2005) The widespread impact of mammalian microRNAs on mRNA repression and evolution. Science 310, 1817.

8. Grimson, A. et al. (2007) MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell 27, 91.

9. Jackson, A.L. et al. (2003) Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol. 21, 635.

10. Lewis, B.P., Burge, C.B., and Bartel, D.P. (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15.

11. Lim, L.P. et al. (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769.

12. Saxena, S., Jónsson, Z.O., and Dutta, A. (2003) Small RNAs with imperfect match to endogenous mRNA repress translation. Implications for off-target activity of small inhibitory RNA in mammalian cells. J. Biol. Chem. 278, 44312.

13. Judge, A.D., Sood, V., Shaw, J.R., Fang, D., McClintock, K., and MacLachlan, I. (2005) Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat. Biotechnol. 23, 457.

14. Hornung, V. et al. (2005) Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11, 263.

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siRNA delivery

siRNA delivery — key questions and answers

How can I optimize siRNA transfection?

siRNA transfection should be optimized when establishing RNAi in the lab and for every new cell line studied. The HiPerFect Transfection Reagent Handbook contains useful information on optimization (www.qiagen.com/HB/HiPerFect).

In optimization experiments, transfection efficiency can be monitored by transfection of a positive control, such as AllStars Cell Death Control siRNA (page 15).

Transfection factors that should be optimized include:

Reagent and siRNA amounts

Cell confluency at transfection

Incubation time of cells and complexes prior to analysis

Why is it important to transfect low siRNA amounts?

Transfection of siRNA at high concentrations can result in off-target effects, in which siRNAs affect the expression of partially homologous or nonhomologous genes. Research suggests that off-target effects, which may produce misleading results in RNAi experiments, can be largely avoided by using low siRNA concentrations (1, 2). HiPerFect Transfection Reagent facilitates highly effective siRNA uptake, which enables gene silencing using low siRNA concentrations without compromising knockdown efficiency.

Why should cytotoxicity be avoided during siRNA transfection?

Cytotoxicity caused by siRNA delivery interferes with data analysis after RNAi due to unreliable phenotypic analysis. Factors affecting data analysis include changes in phenotype caused by the transfection reagent, changes in gene expression caused by stress, and the presence of dead cells which should not be used for analysis. HiPerFect Transfection Reagent is not toxic to cells and does not cause vacuole formation (which indicates disruption of cellular processes). Detailed cell cycle and cell morphology analyses show no differences between mock-transfected cells (HiPerFect Transfection Reagent alone) and untransfected cells.

What is the difference between forward and reverse transfection?

In reverse-transfection protocols, siRNA is added to plate wells first, followed by transfection reagent. After complex formation, cells are added. This is in contrast to forward transfection, where cells are added to plate wells first, and complexes are added afterwards. Reverse transfection is widely used, especially for high-throughput experiments, because it is easily automatable, siRNAs

Riferimenti

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