Imagination at work.

MyBioApp

Determining transfection conditions with Cytell™ Cell Imaging System & siGLO™ Transfection Indicators

2

Project Background

To explore the use of Dharmacon™ reagents with cell imaging and analysis products.

In this initial test, a plate was set up to measure uptake of a fluorescent transfection control at different concentrations with increasing amounts of DharmaFECT™ 1 transfection reagent.

siGLO™ Red Transfection Indicator and siGLO Green Transfection Indicator

Novel FAM- and DY-547-labeled RISC-independent controls, modified to localize to the nucleus as a clear signal of transfection success.

Useful for determination of optimal transfection conditions for siRNA in mammalian cells.

DharmaFECT 1

All-purpose transfection reagent, demonstrating efficient, low-toxicity delivery to over 80% of validated cell types.

DharmaFECT 2, 3, 4 provide chemically distinct alternatives when DharmaFECT 1 is not optimal for a particular cell type or density.siGLO transfection | 3 February 2015

3

Experiment

• A549 cells were plated 24h before transfection at 10,000 cells/well in medium containing Ham’s F-12, 2 mM L-Glutamine, 1.5 g/L Sodium Bicarbonate and 10% FBS.

• siGLO™ Transfection Indicacors and DharmaFECT™ 1 transfection reagent were mixed in serum-free medium (HyClone™), incubated for 20 min at room temperature, full growth medium added to the transfection mix and then added to the cells.

• Incubate for 24hr, fix, and stain with Hoechst 33342.• Image on Cytell™ Cell Imaging System using siGLO Red or

siGLO Green BioApp build with MyBioApp creator. Blue Channel – All Cells Orange or Green Channel for siGLO Transfection Indicator (nuclear

reference segmentation)

siGLO transfection | 3 February 2015

4

Experiment Setup

siGLO transfection | 3 February 2015

siGLO™ Transfection Indicator DharmaFECT™ 1

Mixture incubated for 20min at 25ºC in Hyclone™ serum free media to allow formation of lipid/nucleic acid complexFull

GrowthMedium

Add to cells

10,000 cells grown for 24h

Treated cells grown for 24h

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Plate Map for siGLO™ Green and siGLO Red Transfection Indicator

nM

siG

LO R

NA

nM

siG

LO R

NA

siGLO transfection | 3 February 2015

µL/wellDharmaFECT™ 1

µL/wellDharmaFECT 1

6

siGLO Red

Setting the threshold for siGLO™ BioApp

siGLO transfection | 3 February 2015

Positive Control

Positive Control

Threshold is set just ahead of the histogram peak of a strong positive sample

siGLO Green

Negative Control

Negative Control

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Results for siGLO™ Green BioAppnM

siG

LO R

NA

nM

siG

LO R

NA

DharmaFECT 1

Negative

Controls

Favorable Transfection Conditions

Increased transfection increases with both siGLO and DharmaFECT™ 1 concentration.

siGLO transfection | 3 February 2015

8

Results for siGLO™ Green Transfection Indicator

siGLO transfection | 3 February 2015

9

Results for siGLO™ Red BioAppnM

siG

LO R

NA

nM

siG

LO R

NA

DharmaFECT 1

Negative

Controls

Increased transfection increases with both siGLO and DharmaFECT™ 1 concentration.

siGLO transfection | 3 February 2015

Favorable transfection conditions

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Results for siGLO™ Red Transfection Indicator

siGLO transfection | 3 February 2015

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Consistent results for siGLO™ Green and siGLO Red Transfection Indicators

siGLO transfection | 3 February 2015

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.450

20

40

60

80

100

120

Transfection EfficiencysiGLO Red

100nM50nM25nM12.5nM

DharmaFECT 1

% T

ransfe

cte

d

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.450

20

40

60

80

100

120

Transfection EfficiencysiGLO Green

100nM50nM25nM12.5nM

DharmaFECT™ 1

% T

ransfe

cte

d

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Importance of transfection optimization

Identifying optimal cell densities, transfection reagents, and concentrations is essential for effective implementation of RNA interference (RNAi) in cell-based experiments. • Poor delivery = poor gene knockdown

Not all cell types are amenable to the same transfection conditions. • Optimization should be carried out in each new cell type to

maximize siRNA delivery and minimize toxicity.

 

siGLO™ Transfection Indicators are non RISC-engaging molecules that localize to the nucleus, providing a distinct visual indication of transfection success. • Available with either DY-547 (Red) or 6-FAM (Green).

siGLO transfection | 3 February 2015

http://dharmacon.gelifesciences.com/rnai-and-custom-rna-synthesis/rnai-controls/siglo-rnai-controls/

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Summary

• Cytell™ Cell Imaging System was able to image and analyze siGLO™ Green and siGLO Red Transfection Indicators

• The BioApps created provided easily readable output tables that enable a user to determine transfection conditions (DharmaFECT™ formulation, amount of DhamaFECT and cell density) that are suitable for subsequent siRNA experiments

• Cytell™ Cell Imaging System and siGLO Transfection Indicators enable easy optimization of conditions for efficient siRNA delivery into cells

siGLO transfection | 3 February 2015

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www.gelifesciences.com/Cytell/BioApps

siGLO transfection | 3 February 2015

http://dharmacon.gelifesciences.com/transfection/

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