The Viromer Factbook. Transfection. of sirna, mrna and plasmids 2015/2016

Size: px

Start display at page:

Download "The Viromer Factbook. Transfection. of sirna, mrna and plasmids 2015/2016"

Melinda Thompson
5 years ago
Views:

1 The Viromer Factbook Transfection of sirna, mrna and plasmids 215/216

2 Welcome to Viromer Transfection Reagents! We provide: Transfection of sirna and mirna High transfection efficiency Great safety Easy and fast transfection with consistent results Due to an active escape of Viromer complexes from the endosome Because Viromer complexes are non-charged, gentle on cells and compatible with serum and antibiotics Ascribed to straightforward protocol including initial optimization All features combined generated excellent results in challenging cells such as primary- and suspension cultures, macrophages and stem cells shown in this booklet. We thank our supporters for sharing their valuable data with us and the growing community of Viromer users. New to Viromers? Our Start Positive controls make it easy to begin with, see page 16 Want to learn about the Viromer technology? page 2 has the answers Product information, support and our list of distributors to be found on pages Viromer blue Versatile for standard and challenging cells Viromer GREEN Selected for specific cells such as THP-1, fibroblasts, and colon carcinoma Viromer BLACK Optimized for very challenging cells such as neural stem cells and primary keratinocytes, unique for transfection of antagomir 2 3

Viromer in IMMUNOLOGY RESEARCH Viromer BLUE Very Efficacious Knock-Down in Macrophages and THP-1 Cells RAW264.

6 nm,1,1 1 1 1 THP-1 EC 5 = 15nM nm sirna THP-1: Human monocytic cell line (AML),1,1 1 1 1 nm sirna Reduction of AHA-1 mrna using its sirna and Viromer BLUE.

Data collected by Axolabs, Kulmbach, Germany Human primary macrophages number of endosomes per cell (ref.

using sirna targeting EEA1, which leads to its reduction on endosomes. Viromer BLUE effectively transfects human primary macrophages in a HTS setup.

Bickle, MPI CBG Dresden, Gemanny Mock Viromer BLUE and GREEN Neutral Charge of Transfection Complex results in No Aggregation and High Performance in Suspension Cells SSC-H / SSC-Height SSC-H /

3 Viromer in IMMUNOLOGY RESEARCH Viromer BLUE Very Efficacious Knock-Down in Macrophages and THP-1 Cells RAW264.7: Mouse macrophage-like cell line % of target mrna % of target mrna RAW EC 5 =.6 nm,1, THP-1 EC 5 = 15nM nm sirna THP-1: Human monocytic cell line (AML),1, nm sirna Reduction of AHA-1 mrna using its sirna and Viromer BLUE. Concentrations on the x-axis in nm, AHA-1 sirna and control sirna as filled and open symbols, respectively. Data collected by Axolabs, Kulmbach, Germany Human primary macrophages number of endosomes per cell (ref. to mock-treated cells) EEA1 sirna [nm] control sirna [nm] Primary macrophages were freshly isolated from buffy coat PBMC and transfected in a 384-well plate format using sirna targeting EEA1, which leads to its reduction on endosomes. Viromer BLUE effectively transfects human primary macrophages in a HTS setup. Reduction of EEA1 on endosomes was followed by image analysis. Z-scores are -12 and lower. We are absolutely delighted! M. Bickle, MPI CBG Dresden, Gemanny Mock Viromer BLUE and GREEN Neutral Charge of Transfection Complex results in No Aggregation and High Performance in Suspension Cells SSC-H / SSC-Height SSC-H / SSC-Height 1,K ,K Monocytes-Granolytes 15, , K FSC-H / FSC-Height Monocytes-Granolytes 16, , K FSC-H / FSC-Height PBMCs FL1-H / FL1-Height FL1-H / FL1-Height Q1 Q , K Q1 Q4 FSC-H / FSC-Height FSC-H / FSC-Height Q2,11 Q3 1, Q2,97 Q3 3, , K I used Viromer GREEN for transfection of PBMCs from buffy coats with a FITC labeled sirna getting a very high transfection efficiency of 95% after 24h. In sum I like your product very much. The application is very easy and the results much better compared to Lipofectamine which I had used before. M. Ballbach, University Tübingen, Germany Promyoblast macrophage cell line KG-1a Transfection efficiency in KG-1a cells after transfection with a labeled sirna using Viromer BLUE. The result was exciting and encouraging. The delivery efficiency is over 9% with good fluorescence intensity. J. Lung, C. Lung Gang Memorial Hospital, Chia-Yi, Conjoint Laboratory, Chiayi, Taiwan Viromer in IMMUNOLOGY RESEARCH 4 5

4 Viromer in metabolic RESEARCH Viromer BLUE Very Effective Knock-Down in Metabolic Cells Viromer BLACK Adipocytes Primary hepatocytes Excellent Results 1.5 preadipocytes: mature adipocytes: mature adipocytes: 1 % in Primary 72h post transfection 48h post transfection 72h post transfection Keratinocytes fold of si control % of target mrna , 1, 75, 5, 25,, si control si target Viromer BLUE si control si target Viromer BLUE,1, nm sirna Data generated by C. Weigert, University of Tübingen si control si target Viromer BLUE The results show that for our cells, Viromer Blue is considerably superior to other transfection reagents. A. Fender, University Hospital Düsseldorf, Germany Thanks for providing a fantastic reagent. I am looking forward to work with your chemistry. P. Hallenborg, University of Southern Denmark, Sweden Primary skeletal myoblasts ppar alpha mrna expression relative to ctrl sirna 8 % 6 % 4 % 2 % % ctrl 1nM ppar 1nM ppar 1nM Freshly isolated mouse hepatocytes were transfected with Viromer BLUE and ppar-alpha (blue bars) or control sirna (ligth blue bar). 24h later prepared for RNA analysis. 1nM yielded a complete knock-down. Data generated by M. Matz-Soja, University of Leipzig, Germany C2C12 myoblasts protein X ß-actin target sirna + Knock-down of protein X in C2C12 myoblasts cells transfected with Viromer BLUE. Data generated by Prof. J. Hall, J. Zagalak - ETH Zurich, Institute for Pharmaceutical Science, Switzerland Primary keratinocytes p63 [fold expression] sictl sip63 We are very happy with the results. Now Viromer Black is a very good alternative to our standard transfection method which is associated with the use of high sirna concentration and significant cell loss. The toxic effect with Viromers is very low and we get a very good cell yield. University of Regensburg, Germany Viromer BLUE and GREEN Strong Knock-Down in Cancer Cells Melanoma cells relative mrna expression Viromer BLUE Lipofectamine 2 Knock-down efficiency in a melanoma cell line after transfection with Viromer BLUE. Rel. GAPDH mrna expression ctrl sirna sirna1 sirna2 "The Viromer transfection has functioned wonderfully University Hospital Essen, Germany Hs74T: Gastric carcinoma Hs746T cells were transfected with GAPDH sirna (25nM) for 72h NEG Luc PLK1 gapdh Viromer BLUE efficiently and safely transfected Hs746T cells. Expression of GAPDH mrna was reduced by 9% without any signs of toxicity. Data generated by Gaither, Novartis, USA Glioblastoma cells Percentage (%) 1 NCH no sirna Viromer BLUE NCH149 no sirna Viromer BLUE Transfection efficiency (%, light blue bars) and cell death (%, dark blue bars) of glioblastoma cell lines after transfection of sirnas (at 1nM) using Viromer BLUE. The results are more than satisfying, considering that we were not able to transfect these cell lines with any other transfection reagent that is on the market. I. Dokic, DKFZ Heidelberg, Germany CT26: Colorectal carcinoma Western Blot shows total reduction of Stat3 protein using its sirna complexed to Viromer GREEN. Scrambled control sirna has no effect on Stat3 protein levels. + + Stat3 total protein ß-Actin control sirna Stat3-siRNA We used both Viromers for knocking down Stat3 in CT26 cells and are satisfied with knock-down efficiency. We are delighted from your Viromer Green and would like to test your following products. F. Greten, Georg-Speyer-Haus Frankfurt, Germany Viromer in oncology RESEARCH 6 7

Viromer in stem cell RESEARCH Viromer BLUE Outperforms Standard Reagents Superior Transfection and Knock-Down Efficiency in Primary Human Mesenchymal Stem Cells fsc-h fold of si control 1 8 6 4 2 WT

Viromer BLUE in comparison to untransfected (WT) cells. 1.2 1..8.5.4 ** Knock-down of protein X in MSC transfected with Viromer BLUE.

By using a FITC labeled control sirna I got a 92 % transfection efficiency using Viromer Blue. This is an enormous difference compared to promega reagent and lipofectamine. J.

5 Viromer in stem cell RESEARCH Viromer BLUE Outperforms Standard Reagents Superior Transfection and Knock-Down Efficiency in Primary Human Mesenchymal Stem Cells fsc-h fold of si control WT - 2,4% fl1-h Lipofectamine RNAiMAX - 2,% fl1-h Delivery of a fluorescein-labeled oligo in primary mesenchymal stem cells with Lipofectamine RNAiMAX, Viafect, and Viromer BLUE in comparison to untransfected (WT) cells ** Knock-down of protein X in MSC transfected with Viromer BLUE. n = 3 ViaFect -,8% Viromer BLUE - 91,4% fl1-h fl1-h The Viromer reagent is very encouraging for MSC. By using a FITC labeled control sirna I got a 92 % transfection efficiency using Viromer Blue. This is an enormous difference compared to promega reagent and lipofectamine. J. Luetzkendorf, University Hospital Halle, Germany The Viromer reagent is very appropriate to transfect MSC with sirna. I was able to achieve a nearly 4-fold target gene knock down. S. Werner, Institute for Physiological Chemistry, Halle, Germany Viromer BLUE Strong and Specific Knock-Down in Primary Microglia Cells siglo-red RQ sictrl sistat1 trans Transfection efficiency in primary microglia cells transfected with siglo-red using Viromer BLUE. RT-PCR Western Blot CTRL sictrl 24 h 48 h sistat1 sistat3 sictrl sistat1 sistat3 Viromer BLACK yields 7% Transfection in Neural Stem Cells Non transfected counts counts gfp-log_hight Transfected Region Total gfp-log_hight Region Total Count Count % Hist 1..7 % Hist Viromer in neuronal RESEARCH.2. si control Viromer BLUE si target Viromer BLUE * p.5 ** p.1 *** p.5 We are very satisfied with the results. We see good silencing both at the mrna and protein levels. Cells are viable in 8-9% under the condition that we change medium after 4h. M. Maleszewska, Nencki Institute of Experimental Biology Warsaw, Russia Viromer Black reaches it transfection efficiency at as high as 69.34% compared to our standard transfection method at around 3%. N. Li, UCL Institute of Neurology London 8 9

Transfection of plasmid DNA and mrna outperforms Major Competitors on Hard-to-Transfect Cancer Cells C6: Glioma cells Brightfield Fluorescence

fluorescence [RFU] 12, 1, 8, 6, 4, 2, MCF-7 cells RLU x 1 2 18 16 14 12 1 8 5 6 4 2 MCF-7 Luciferase Activity [RLU] 125 1 75 25 Viability [%] Viromer

for specific cells such as primary cardiomyocytes and hepatocytes Given the efficiency we saw, your product might be uniquely poised to address many

Viromer Red showed faster and better transfection efficiency in Glioma cell line as compared to Lipofectamines.

6 Transfection of plasmid DNA and mrna outperforms Major Competitors on Hard-to-Transfect Cancer Cells C6: Glioma cells Brightfield Fluorescence Brightfield Fluorescence Lipofectamine-3 Lipofectamine-LTX 24 Hour 72 Hour SH-SY5Y: Neuroblastoma cells FuGene HD % positive cells / mean fluorescence [RFU] 12, 1, 8, 6, 4, 2, MCF-7 cells RLU x MCF-7 Luciferase Activity [RLU] Viability [%] Viromer in oncology RESEARCH, FuGene HD TransIT-X2 FuGene HD Lipofectamine 2 Viromer red Versatile for standard and challenging cells Viromer YELLOW Selected for specific cells such as primary cardiomyocytes and hepatocytes Given the efficiency we saw, your product might be uniquely poised to address many research questions using this clinically relevant cell line. Viromer Red showed faster and better transfection efficiency in Glioma cell line as compared to Lipofectamines. 214 Pearse Lab, Miami Project to Cure Paralysis, Dr. S. Rao, USA 71,5 % 1,6 % Positive Cells 18,6 % 4,4 % 17,9 % 4,2 % Geo-Mean FL Data generated by H. Cynis, Fraunhofer Institute for Cell Therapy and Immunology, Halle, Germany. Cell viability [%] Data generated by Lipocalyx, Halle, Germany. 1 11

Viromer in IMMUNOLOGY RESEARCH transfects 9% of Human Primary Monocytes with mrna SSCA FITCA 1 75 5 25 1e3 1e2 1e1 1-1 %

$93\P1\P2 25 5 5 fsc-a 75 P1\P2\U 1,66%-T 2,24%-# P1\P2\L 72,57%-T 97,76%-# 75 1 1 Data generated by Miltenyi Biotec,$ $68\P1\P2 P1\P2\U 6,93%-T 89,72%-# 1 P1\P2\L 6,98%-T 1,28%-# -1 25 5 75 1 fsc-a GFP expression of live cells (FITC-A Mean$ P1/P2) 4 35 3 25 2 15 1 5 relative cell viability (% P2 of Total in Counts) 2 ng 1 ng 5 ng 25 ng Medium mrna complexed to

P1/P2) 4 35 3 25 2 15 1 5 relative cell viability (% P2 of Total in Counts) 2 ng 1 ng 5 ng 25 ng Medium mrna complexed to

cells/96-well treatment with various doses of GFPmRNA GFP detection via MACSQuant Sum: 9% pos.

compared to Standard Reagents Primary human macrophages not transfected GFP-plasmid + jetpei Macrophage + + GFP-mRNA

Weigert, University Hospital Frankfurt, Germany Transfection of RAW 264.

24h after transfection efficiency was monitored using fluorescence microscopy. Data generated by by H.

7 Viromer in IMMUNOLOGY RESEARCH transfects 9% of Human Primary Monocytes with mrna SSCA FITCA e3 1e2 1e1 1-1 % -# P1 78,5%-T 78,5%-# Medium MarJ _ fsc-a MarJ _ \P1\P fsc-a 75 P1\P2\U 1,66%-T 2,24%-# P1\P2\L 72,57%-T 97,76%-# Data generated by Miltenyi Biotec, Bergisch Gladbach, Germany FITCA 1e3 1e2 1e1 : 1ng GFP MRNA P1 88,17%-T 88,17%-# MarJ _ % -# fsc-a MarJ _ \P1\P2 P1\P2\U 6,93%-T 89,72%-# 1 P1\P2\L 6,98%-T 1,28%-# fsc-a GFP expression of live cells (FITC-A Mean P1/P2) relative cell viability (% P2 of Total in Counts) 2 ng 1 ng 5 ng 25 ng Medium mrna complexed to Viromer Red CD14 purified monocytes from hpbmcs (Buffy Coat) 4. cells/96-well treatment with various doses of GFPmRNA GFP detection via MACSQuant Sum: 9% pos. transfected cells, no visible toxicity % GFP positive cells (UR 3) Unsurpassed Transfection of Macrophages compared to Standard Reagents Primary human macrophages not transfected GFP-plasmid + jetpei Macrophage + + GFP-mRNA Plasmid (target) Plasmid (non-target) HA actin Data generated by A. Weigert, University Hospital Frankfurt, Germany Transfection of RAW 264.7: Mouse macrophages with: Transfection of RAW cells with GFP-plasmid DNA and GFP-mRNA using. RAW cells were transfected with pcmv-gfp plasmid and GFP-mRNA using. 24h after transfection efficiency was monitored using fluorescence microscopy. Data generated by by H. Cynis, Fraunhofer Institute for Cell Therapy and Immunology, Halle, Germany + Transfection of plasmid DNA into RAW 264.7: Mouse macrophages % positive cells / mean fluorescence [RFU] 7, 6, 5, 4, 3, 2, 1,, 64, % 43,1 % Positive Cells TransIT-X2 FuGene HD 15,6 % 3,5 % TransIT-X2 11,4 % 2,8 % Geo-Mean FL Data generated by H. Cynis, Fraunhofer Institute for Cell Therapy and Immunology, Halle, Germany Viromer in IMMUNOLOGY RESEARCH 12 13

Viromer in METABOLIC RESEARCH Excellent Transfection Efficiency in Primary Keratinocytes, C2C12

efficiency 1 1 1 1 2 1 3 1 4 fl1-h Expression of GFP in primary keratinocytes transfected with.

C2C12 myoblasts GFP-1 Phase contrast Transfection efficiency after transfection of GFP-N1

Primary pancreatic tumor cells I used the Viromer to transfect our primary pancreatic cancer

with lipofectamine and infect with lentivirus.

B. Sainz, University of Madrid, Spain Viromer YELLOW Outperforms Standard Transfectants in

Lipofectamine 2 Lipofectamine 3 I was very happy with the results.

I have tried incubating the myocytes with the Yellow reagent for 4 h as suggested and the

Kaludercic, University of Padova, Italy Viromer yellow Hepatocyte cell line FAO Expression of

In comparison to 3 other transfection reagents Viromer YELLOW was most sensitive with highest

Klingler, University Hospital Tübingen, Germany Viromers in METABOLIC RESEARCH We have tried the

8 Viromer in METABOLIC RESEARCH Excellent Transfection Efficiency in Primary Keratinocytes, C2C12 myoblasts, and Pancreatic Tumor Cells Primary keratinocytes Counts % transfection efficiency fl1-h Expression of GFP in primary keratinocytes transfected with. Data generated by M. Podgórska; Prof. S. Smola: University Hospital Homburg/Saar, Germany. C2C12 myoblasts GFP-1 Phase contrast Transfection efficiency after transfection of GFP-N1 plasmid into undifferentiated C2C12 cells using. Primary pancreatic tumor cells I used the Viromer to transfect our primary pancreatic cancer cells, which are derived directly from patient tumors and they are VERY difficult to transfect with lipofectamine and infect with lentivirus. The tumor responded very well and we got very good transfection efficiency with Viromer Red. Dr. B. Sainz, University of Madrid, Spain Viromer YELLOW Outperforms Standard Transfectants in Primary Cardiomyocytes and Hepatocyte Cell Line FAO Primary cardiomyocytes Viromer yellow Lipofectamine 2 Lipofectamine 3 I was very happy with the results. So far I had good results with the standard protocol and lowest concentration of Viromer Yellow. I have tried incubating the myocytes with the Yellow reagent for 4 h as suggested and the results were very good. N. Kaludercic, University of Padova, Italy Viromer yellow Hepatocyte cell line FAO Expression of GFP in rat hepatocyte cell line FAO transfected with Viromer YELLOW. In comparison to 3 other transfection reagents Viromer YELLOW was most sensitive with highest transfection efficiency 48h post transfection. C. Klingler, University Hospital Tübingen, Germany Viromers in METABOLIC RESEARCH We have tried the Viromer on undifferentiated C2C12. The myotube remains being transfected and it works great. Your transfection product (Viromer YELLOW) works much better than the other reagents we have used before to transfect neonatal rat cardiomyocytes. We obtain 5% efficiency with low toxicity. Dr. CL Tse, University of Oklahoma, Health Science Center, Dept. of Physiology, USA A. Castellano, University of Seville, Spain 14 15

Transfection using Start Positive Controls Start Positive controls are preformulated, ready-to-use transfection complexes.

Start Positive controls for / YELLOW comprise a pdna and one mrna sample each and facilitate comparative studies between these

Start Positive Controls Strong Transfection of Challenging Cells with plasmid DNA and mrna SH-SY5Y RAW 264.

Viromer BLUE sirna controls GAPDH-siRNA complexed to Viromer BLUE non-targeted sirna labeled with Cy3 complexed to Viromer BLUE pdna

5kb) plasmid complexed to Viromer YELLOW GFP mrna complexed to Viromer YELLOW Challenging cell lines were transfected with pcmv-gfp

9 Transfection using Start Positive Controls Start Positive controls are preformulated, ready-to-use transfection complexes. Use them for transfection of new cell types or as reference material. Start Positive controls for / YELLOW comprise a pdna and one mrna sample each and facilitate comparative studies between these genetic cargoes. See results on page 17. Start Positive Controls Strong Transfection of Challenging Cells with plasmid DNA and mrna SH-SY5Y RAW HUVEC C2C12 mrna transfection plasmid DNA transfection Transfection using Start Positive Controls. Viromer BLUE sirna controls GAPDH-siRNA complexed to Viromer BLUE non-targeted sirna labeled with Cy3 complexed to Viromer BLUE pdna / mrna controls pcmv-gfp (3.5kb) plasmid complexed to GFP mrna complexed to Viromer YELLOW pdna / mrna controls pcmv-gfp (3.5kb) plasmid complexed to Viromer YELLOW GFP mrna complexed to Viromer YELLOW Challenging cell lines were transfected with pcmv-gfp plasmid and GFP-mRNA using Start Positive controls of. Transfection was monitored using fluorescence microscopy. Between pdna and mrna we typically observe faster, homogeneous and stronger expression from mrna. We attribute this to the instant availability of the transcript. Data generated by by H. Cynis, Fraunhofer Institute for Cell Therapy and Immunology, Halle, Germany 16 17

Start Positive Controls match Performance of Standard Viromer Reagent raw HUVEC raw HUVEC Viromer Transfection Protocol

Positive Control 1 2 1 2 Viromer Dilute sirna, pdna or mrna and provide Viromer droplet Combine dilution to Viromer droplet,

transfection mrna transfection Transfection using Start Positive Control and Viromer.

reagent. Transfection was monitored using fluorescence microscopy.

10 Start Positive Controls match Performance of Standard Viromer Reagent raw HUVEC raw HUVEC Viromer Transfection Protocol Straightforward and simple workflow for all plate formats Working with Viromer Working with Start Positive Controls Start Positive Control Viromer Dilute sirna, pdna or mrna and provide Viromer droplet Combine dilution to Viromer droplet, mix, and wait for 15 min Rehydrate lyophilized complexes and wait for 1 min Add to your cells in fresh media plasmid DNA transfection mrna transfection Transfection using Start Positive Control and Viromer. Challenging cell lines were transfected with pcmv-gfp plasmid and GFP-mRNA using Start Positive controls or standard Viromer reagent. Transfection was monitored using fluorescence microscopy. Start Positive controls and Viromer show equal performance. Data generated by by H. Cynis, Fraunhofer Institute for Cell Therapy and Immunology, Halle, Germany Add to your cells in fresh media View results View results 18 19

THE VIROMER TECHNOLOGY ph 7 ph 5 endosome ph 7 ph 5 H+ cytosol Viromer : A novel polymer based transfection reagent mimicking the viral infection process by an active endosome

Viromers mimic the influenza mechanism, but use a polymer instead of a fusion peptide. Uncharged (grey) and charged (blue) groups regulate membrane transfer.

3:h late endosome Viromers accumulate near the nucleus, ongoing acidification. THE VIROMER TECHNOLOGY Fatty acids (red) resemble Influenza s GLU, alkyl (grey) are similar to ALA.

11 THE VIROMER TECHNOLOGY ph 7 ph 5 endosome ph 7 ph 5 H+ cytosol Viromer : A novel polymer based transfection reagent mimicking the viral infection process by an active endosome escape mechanism. In influenza the ph-sensitive fusion peptide inserts into the endosomal membrane. Mechanism relies on protonation of GLU, balanced by hydrophobic ALA. Viromers mimic the influenza mechanism, but use a polymer instead of a fusion peptide. Uncharged (grey) and charged (blue) groups regulate membrane transfer. H+ The Viromer Uptake Pathway: The Active Escape Process 1: h early endosome Viromers are taken up at the cell surface. 3:h late endosome Viromers accumulate near the nucleus, ongoing acidification. THE VIROMER TECHNOLOGY Fatty acids (red) resemble Influenza s GLU, alkyl (grey) are similar to ALA. 4:3h arrival in the cytosol H+ As a result, both influenza and Viromer promote an active endosome escape leading to cytosolic delivery. Discharge of sirna from endosomes and starting diffusion. endosome cytosol Model: HeLa, Viromer GREEN, labelled sirna. Data courtesy of Chromotek. 2 21

12 Viromer Neutral Charge of Transfection Complexes are proven safe and effective in Suspension or 3D Cell Cultures Viromer are proven superior to Lipofectamine with 3D spheroids and show advanced performance in THP-1, PBMCs, or primary microglia cells. (see page 4, 5 and 9) Viromer Safe Transfection with a Wide Working Range The outcome of RNAi experiments depends on the ratio between the sirna signal and any unspecific noise. Viromers achieve signal: noise ratios between 2 and 3, much better than six leading competitors. Neutral Charge 3D culture of cancer stem cells (mammospheres) Viromer BLUE phenotypic assay Signal: noise ratios of transfectants % in class relative mrna levels Target GAPDH Target GAPDH % cell viability 1 5 3x signal: noise ratio mv Viromer GREEN Lipofectamine Target Specific sirna Control sirna Knockdown of target and GAPDH control in cancer mammospheres.,1 1 1 nm sirna 5 Interferin RNAiMAX siport NeoFX TransIT-TKO Ribocellin Dharmafect Viromer BLUE Viromer GREEN And thank you very much for letting us test the Viromer system, we were satisfied with the efficiency of knockdown. J. Holland, MDC Berlin Very low background in a phenotypic assay. Cell cycle of HeLa was arrested using PLK-1 sirna (filled symbols, the signal) or a control sirna (open symbols, the noise). For Viromer BLUE, the EC5 values differ by a factor of 3. Signal: noise ratio for various transfectants. Signal: noise is defined as the ratio of EC5 values for the active and control sirna

13 Products & Ordering Viromer for sirna/mirna applications Product Number Transfections in 24-well Viromer blue VB-1LB- 5 Viromer blue VB-1LB-1 5 Viromer blue VB-1LB-3 3 x 5 Viromer blue GAPDH / CY3 sirna controls VB-siBUNDLE-1 Viromer green VG-1LB- 5 Viromer green VG-1LB-1 5 Viromer green VG-1LB-3 3 x 5 Viromer black VBk-1LB- 8 Viromer black VBk-1LB-1 5 Viromer black VBk-1LB-3 3 x 5 Technical Support, Customer Service and Orders Bettina Weber bettina.weber@lipocalyx.de Dr. Olivia Zabel olivia.zabel@lipocalyx.de Dr. Sandra Lagauzère sandra.lagauzere@lipocalyx.de US - Customers Ashley Rae Kark ashley.kark@lipocalyx.com Orders order@lipocalyx.de Fax: Viromer for plasmid/mrna applications Product Number Transfections in 24-well Viromer red VR-1LB- 5 Viromer red VR-1LB-1 6 Viromer red VR-1LB-3 3 x 6 Viromer red pdna- / mrna-gfp controls VR-BUNDLE-1 Viromer yellow VY-1LB- 5 Viromer yellow VY-1LB-1 1 x 6 Viromer yellow VY-1LB-3 3 x 6 Viromer yellow pdna- / mrna-gfp controls Webshop: VY-BUNDLE

International Distributors Australia & New Zealand Austria Brasilia China Korea Poland TrendBio Pty Ltd www.trendbio.com.au Biozym Biotech Trading GmbH (AUT) www.biozym.

com France Germany Italy Japan United Kingdom & Ireland USA Interchim S.A. www.interchim.com Biozym Scientific GmbH (DE) www.biozym.com Voden Medical Instruments S.p.a. www.vodenmedical.

14 International Distributors Australia & New Zealand Austria Brasilia China Korea Poland TrendBio Pty Ltd Biozym Biotech Trading GmbH (AUT) Sinapse Biotechnologia Maibio CO., Ltd. Dong In Biotech CO., Ltd. BLIRT S.A. France Germany Italy Japan United Kingdom & Ireland USA Interchim S.A. Biozym Scientific GmbH (DE) Voden Medical Instruments S.p.a. Nakamura Scientific Instruments Industry CO., Ltd. Cambridge Bioscience Ltd. OriGene Technologies, Inc

15 Lipocalyx GmbH Weinbergweg Halle Germany Lipocalyx US 1 Jill Ct. Building 16 Unit 1 Hillsborough, NJ 8844 USA viromer blue viromer green viromer black viromer red viromer YELLOW

The Viromer Factbook. sirna and plasmid transfection. Fall 2014

The Viromer Factbook. sirna and plasmid transfection. Fall 2014 The Viromer Factbook sirna and plasmid transfection Fall 214 Viromers Features and Benefits Active escape Zero charge Stable particles Lipid free Reverse transfection Maximized transfection efficacy and