Transfection Guidelines

© 2007 Mirus Bio Corporation. All rights reserved.

Transfection and RNAi

Technologies

B. Gopalakrishnan, Ph.D.

Mirus Bio Corporation

Madison, Wisconsin

USA

© 2007 Mirus Bio Corporation.

All rights reserved.

2

Mirus Bio Corporation

• Madison, WI- USA based biotechnology company

founded by Jon Wolff and V. Budker in 1995 that

specializes in non-viral nucleic acid delivery

• Therapeutic pipeline

– Develop efficient, non-viral gene delivery technologies

• Focused on understanding the basic mechanisms of cellular

delivery of nucleic acids both in vitro and in vivo

• Research products

– Develop novel research products including transfection reagents

and nucleic acid labeling kits



3

Presentation Overview

• Transfection– Introduction

– Methods of transfection

– Mechanism of lipid and/or polymer-mediated transfection

– Transfection optimization and critical parameters

• Applications of RNAi in the Study of Cell Biology– Introduction to RNA interference

– siRNA vs. miRNA. Roles and uses

– Profiling tissue miRNA

• Labeling miRNA & miRNA microarray

• Dynamic PolyConjugates– A New Synthetic Platform for Targeted in vivo Delivery of siRNA

• Summary



4

Nucleus

Plasmid DNA

NuclearEntry

CellularEntry

What is Transfection?

Cytoplasm



5

Nucleus

mRNA or viral RNA

CellularEntry

RNA Transfection

siRNA

No nuclear entry required

Cytoplasm



6

Transfection Methods

• Electroporation

– Delivery using high voltage, low amperage electric pulses

– Permeabilizes cell membrane to allow entry into the cell

• Calcium Phosphate Precipitation

– Addition of calcium and phosphate to DNA creates large precipitates

– Precipitates settle on cells and are taken up

• Lipid and/or Polymer-based Reagents

– Positively charged molecules that associate with nucleic acids

– Promote binding to cells and uptake by endocytosis



7

Electroporation

• Advantages

– Efficient method on many difficult to transfect cells and cell lines

– Delivers all types of nucleic acids

• Disadvantages

– Requirement

• large numbers of cells

• large amounts of nucleic acid

– Low cell survival

– Performed under serum-free conditions

– Requires expensive electroporation equipment and cuvettes



8

Calcium Phosphate Precipitation

• Advantages

– Reagents can be made in the lab

– Inexpensive

• Disadvantages

– Requires large amounts of nucleic acid

– Cumbersome protocols

– Finicky reagents

– Generally a low efficiency method



9

Lipid and/or Polymer-based Reagents

Advantages

– Simple to use

– High efficiency transfection

method

– Compatible with serum

containing media

Disadvantages

– Depending on the reagent

• Can exhibit cellular toxicity

• Can be expensive



10

Lipids Used in Transfection Reagents

• Two long alkyl chains attached to a polar head group

• Cationic lipids +/- a helper lipid

Polar

Head

Hydrophobic chains



11

Lipid Examples

H2NOPO O

-O O

O

O

( )7

)7( ( )7

( )7

O

( )7

)7( ( )7

( )7

N+ O

O

DOPE

(Polar, Helper)

DOTMA

(Cationic)

Lipofectin = 1:1 DOTMA:DOPE



12

Liposome Complexes

Cationic Lipid + Helper Lipid

Plasmid DNA

Liposome Multi-layered Liposome

Net (+) Charge

(DNA, Cationic + Helper Lipids)

(Not Drawn to Scale)



13

Cationic Lipid

Plasmid DNA

Lipoplexes

Net (+) Charge

Lipoplex Complexes




14

Cationic Lipid

Cationic Polymer

Plasmid DNA

Lipopolyplex

(DNA, Lipid, Polycation)

Net (+) Charge

Polyplex

(DNA, Polycation)

Net (+) Charge

Different Types of ComplexesPolyplexes and Lipopolyplexes




15

Nucleic acid

- ---

-

--

-- -REPULSION

Nucleus-

--

----

--

-

-

Cell

Charge Repulsion Between the Cell Surface and the

Nucleic Acid Inhibits Uptake

Cell Surface: First Transfection Barrier



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- ---

-

--

-- -

+ Transfection Reagent

+

++

+

++

+

Nucleus

Endocytosis (Cellular Uptake)

----

-+

++

+

++

+

Condensation

Cell Association

--

Transfection Reagents Promote Cell Association and Uptake

+

++

+

++

+

Endosome

+

++

+

++

+



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Nucleus

Endosomal Escape

Transfection Reagents Promote Endosomal Escape

+

++

+

++

+

+

++

+

++

+

Nuclear Entry

No evidence for promoting

nuclear entry



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Visualization of the Transfection Process

1 hour 4 hours

8 hours 24 hours

Autofluorescing cells

Rhodamine-labeled DNA

Yellow Fluorescent Protein

COS-7 Cells



19

General Transfection Reagent Protocol



20

Transfection Optimization

• Amount of reagent and nucleic acid

• Complex formation time

• Cell confluency (density) at transfection

• Post-transfection incubation time



21

Transfection Optimization:DNA & Reagent Amount

0.E+00

1.E+07

2.E+07

3.E+07

4.E+07

5.E+07

6.E+07

7.E+07

8.E+07

COS-7 CHO-K1 HEK 293

Lu

cif

era

se

Ac

tiv

ity

(R

LU

s) 3 µl reagent/1 µg DNA

1.2 µl reagent/0.4 µg DNA

TransIT®-LT1 Reagent

COS-7 HEK 293CHO-K1



22








23

0.E+00

4.E+07

8.E+07

1.E+08

2.E+08

2.E+08

0 10 20 30 40 50 60

Complex Formation Time (minutes)

Lu

cif

era

se A

cti

vit

y (

RL

Us)

Transfection Optimization:Complex Formation Time

CHO-K1 Cells - TransIT®-LT1 Reagent

0.E+107

4.0E+107

8.0E+107

1.2E+108

1.6E+108

2.0E+108



24








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Transfection Optimization:Cell Confluency at Transfection

0.E+00

2.E+07

4.E+07

6.E+07

8.E+07

2 3 4 2 3 4

Lu

cif

era

se A

cti

vit

y (

RL

Us)

75% Confluency

100% Confluency

Amount of TransIT®-LT1 Reagent/µg DNA

NIH3T3

Cells



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Transfection Optimization:Post-transfection Incubation Time

0

500,000

1,000,000

1,500,000

2,000,000

2,500,000

3,000,000

3,500,000

0 6 12 18 24 30 36 42 48

Hours Post Transfection

Fir

efl

y L

ucif

erase E

xpressio

n (

RLU

s)

Firefly luc mRNA Delivered to NIH3T3 Cells using TransIT®-mRNA Kit

Lu

cif

era

se

Ac

tivit

y (

RL

Us)



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Other Critical Parameters• Mycoplasma contamination

– Contamination is difficult to detect visually

– Can affect transfection results – decreased viability

• Use of endotoxin-free DNA

– Endotoxin can decrease cell viability and inhibit transfection

• Transfection of Cells at Consistent Passage Numbers

– Cells change during passage

– Changes can affect transfection results

• Transfection Comparisons to be done on the Same Day

– Transfection performance can vary day-to-day



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Transfection Efficiency varies with Cell Line (e.g.TransIT®-LT1 Reagent)



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Efficiency varies with transfection reagents

HeLa Cells Plated per 12-well (x 103)

Pe

rce

nt C

on

flu

en

cy (

line

s)

Lu

cife

rase A

ctivity (

RL

Us x

10

6)

(b

ars

)Transfection Reagent - 1 Reagent -2 Confluency at Transfection

0

1

2

3

4

5

6

7

25 50 75 100 125 150 200 250 300 350

0

20

40

60

80

100



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0.00E+00

2.00E+07

4.00E+07

6.00E+07

8.00E+07

1.00E+08

1.20E+08

1.40E+08

3 4.5 6 3 4.5 6 10 15 20

TransIT-LT1 Reagent TransIT-293 Reagent PolyFect

Reagent Amount (ul) per 1.5 ug DNA

Lu

cif

era

se A

cti

vit

y (

RL

Us)

Transfection Results Vary Day-to-Day

Day 1 Transfections

Day 2 Transfections

1.4E+108

2.0E+107

4.0E+107

6.0E+107

8.0E+107

1.2E+108

1.0E+108

Lu

cif

era

se A

cti

vit

y (

RL

Us

)

0.E+107

Reagent-A Reagent-B Reagent-C



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0.00E+00

2.00E+07

4.00E+07

6.00E+07

8.00E+07

1.00E+08

1.20E+08

1.40E+08

3 4.5 6 3 4.5 6 10 15 20

TransIT-LT1

Reagent

TransIT-293

Reagent

PolyFect

0.00E+00

1.00E+07

2.00E+07

3.00E+07

4.00E+07

5.00E+07

6.00E+07

3 4.5 6 3 4.5 6 10 15 20

TransIT-LT1

Reagent

TransIT-293

Reagent

PolyFect

Performance Trends Remain Similar

Day 1 Transfections Day 2 Transfections

1.4E+108

2.0E+107

4.0E+107

6.0E+107

8.0E+107

1.2E+108

1.0E+108

Lu

cif

era

se A

cti

vit

y (

RL

Us

)

0.E+107

Reagent Amount (µl) per 1.5 µg DNA


Reagent Amount (µl) per 1.5 µg DNA


1.0E+107

2.0E+107

3.0E+107

4.0E+107

6.0E+107

5.0E+108

0.E+107



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Transfection Summary

• Transfection efficiency is highly dependent on cell line

• Optimize transfection efficiency by testing five key variables

– Reagent and DNA amount, complex formation time, confluency, and

post-transfection incubation time

• For Consistent Results

‒ Maintain similar passage numbers between experiments

– Perform critical comparisons in the same experiment

• Challenges

‒ Uniformity in efficiency

‒ Hard-to-transfect Cell lines

Documents

Transfection Guidelines