standardizing electrophoresis conditions: how to … · migration of dna during electrophoresis ......

Standardizing electrophoresis

conditions: How to eliminate a major

source of error in the comet assay

Gunnar Brunborg

Norwegian Institute of Public Health,

Department of Chemicals and radiation,

Oslo, Norway

ICAW 2015 Antwerp Sept 2015

There is still too much variation in

comet assay results

Our ambition is to contribute to reducing such

variations

Approach in this project: Analysis of critical

parameters, and revision of protocols if

needed.


The comet assay: biology, chemistry

and physics

• The degree of DNA damage formed in a cell

depends on biology and chemistry.

• The conversion of this DNA damage into

comet tails is determined by the rate of

migration of DNA during electrophoresis

• Electrophoresis is first of all a physical

process


Analysing the physics of comet tail

formation through electrophoresis

In principle,

• Total DNA-migration = Tail%DNA

= f(dragging forces; retarding forces)

Here,

• Dragging forces = Constant x U[V/cm] x time

• Retarding forces = Constant x Viscosity

(temperature; matrix (agarose

concentration))


The conditions known to affect DNA

migrations

1. Electric potential (Voltage gradient, V/cm)

2. Time

3. The matrix (agarose concentration affects

the pore size)

4. Temperature

5. Electrophoresis solution (if the pH is low)


Some points to note

• What is the significance of the electric current?

– Answer: None (but it depends on the volume of

the liquid).

• What is the inter-relationship between the applied

voltage and the current?

– Answer: It’s a constant (= related to the electric

conductance of the liquid in the tank)

• How can you alter the current?

– By adding more liquid

– By changing the conductance of the liquid

– By using another tank, with different dimensions


The current is determined by the volume of the

electrophoresis liquid (the voltage is kept constant)


The current is determined by the voltage (when the volume

is kept constant)

• The voltage has to be

above a certain level

(i.e. the potential of

the electrode, which

for platinum is close to

2 volts). Above 2 V,

the current increases

almost linearly


96 minigel format (4 microliter per gel)

Gutzkow KB,

Langleite TM, Meier

S, Graupner A, Collins

AR, Brunborg G.

High-throughput

comet assay using 96

minigels.

Mutagenesis, May

2013; 28(3):333-40


0 Gy

10 Gy

with

circu

latio

n

with

out circu

latio

n

0

20

40

60

80

% T

ail

DN

A inte

nsity

We reported substantial variations between technical

replicates in neighbouring gels processed in the same

electrophoresis (Gutzkow et al., Mutagenesis 2013).

The 96 minigel format confronted us with a

potential problem in comet electrophoresis


Our understanding of the principles of

electrophoresis should allow us to

explain the cause of these variations

• Hypothesis: There are larger variations in

local voltage without than with circulation of

the electrophoresis solution


Local voltage measured using a

multi-electrode

Electrophoresis tank with platform, with unit containing 19 thin

platinum electrodes. They are covered with plastic so that only the

lower 1 mm part is exposed to the solution. Their distance to the

platform surface may be adjusted.

V

Pt plastic


In reality the unit looks like this…


Tank with multi-electrode


Voltages measured in all electrodes,

every 10 seconds

• A device (Agilent 34972A, with the

34901A Multiplexer) scans all

electrodes during 1 second, at a

preset interval (every 10 seconds).

• Accurate determinations of time-

dependent local voltages are thus

made.

• One channel is used to record the

temperature in the solution.


http://cp.home.agilent.com/agilent7/s7viewers/flash/genericzoom.swf?logo2=false&serverUrl=/agilent7/is/image/&contentRoot=/agilent7/skins/&locale=en&config=Agilent/AGILENT-IMGSET&image=Agilent/PROD-1756491-IS

http://cp.home.agilent.com/agilent7/s7viewers/flash/genericzoom.swf?logo2=false&serverUrl=/agilent7/is/image/&contentRoot=/agilent7/skins/&locale=en&config=Agilent/AGILENT-IMGSET&image=Agilent/PROD-1000000083.epsg.pro-IS

Electrodes placed in a thin agarose gel


Circulation rate 3 Measurements during 25 minutes.

Left axis: Voltage, Right axis: Temp (deg)

-10,0

-8,0

-6,0

-4,0

-2,0

0,0

2,0

4,0

6,0

8,0

10,0

0,0

1,0

2,0

3,0

4,0

5,0

6,0

7,0

8,0

9,0

10,0

0 20 40 60 80 100 120 140

102 (VDC)103 (VDC)104 (VDC)105 (VDC)106 (VDC)107 (VDC)108 (VDC)109 (VDC)110 (VDC)111 (VDC)112 (VDC)113 (VDC)114 (VDC)115 (VDC)116 (VDC)117 (VDC)118 (VDC)119 (VDC)


Temperature

0,0

2,0

4,0

6,0

8,0

10,0

12,0

0,0

1,0

2,0

3,0

4,0

5,0

6,0

7,0

8,0

9,0

10,0

0 20 40 60 80 100 120 140

102 (VDC)103 (VDC)104 (VDC)105 (VDC)106 (VDC)107 (VDC)108 (VDC)109 (VDC)110 (VDC)111 (VDC)112 (VDC)113 (VDC)114 (VDC)115 (VDC)116 (VDC)117 (VDC)118 (VDC)119 (VDC)

No Circulation Measurements for 25 minutes.

Left axis: Voltage, Right axis: Temp (deg)


Are these curves so different?


Yes indeed, when you transform the

measurements into local voltage (V/cm) and its

variation with time!

No circulation, uncorrected

measurements

With circulation rate 3 uncorrected measurements

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

0 50 100 150

0

0,2

0,4

0,6

0,8

1

1,2

1,4

0 20 40 60 80 100 120 140


Some variation in the spacing of the

electrodes


Results are corrected to account for varying

spacing of the electrodes

No circulation

corrected measurements

With circulation rate 3

corrected measurements

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

0 50 100 1500

0,2

0,4

0,6

0,8

1

1,2

1,4

0 50 100 150ICAW 2015 Antwerp Sept 2015

Calculations

• Voltage recordings used to calculate time-

integrated «dragging force»

– (Voltage per cm x minutes)

• At each electrode location.


Expected comet tail

(=time-integrated electric potential) With circulation rate 3

Mean: 4.96

CV (%) = 0.25

No circulation

Mean: 5.01

CV (%) = 12.21

0,00

1,00

2,00

3,00

4,00

5,00

6,00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Corrected electrode voltages

0,00

1,00

2,00

3,00

4,00

5,00

6,00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Corrected electrode voltages


Is there an optimal rate of

circulation?

0

5

10

15

20

25

30

0 20 40 60

Uncorrected

Corrected

Circulation: ml per minute

0,00

1,00

2,00

3,00

4,00

5,00

6,00

0 10 20 30 40 50 60

Uncorrected

Corrected

Mean time-integrated

electric potential

CV (%) time-integrated

electric potential


Comet assay can be

easy and cheap • Refrigerator

• External pump

• Circulation of electrophoresis

solution through heat

exchanger in ice/water

A new concept


A new concept

Electro4ease


Large gels (e.g. on glass slides) are most likely

subjected to the same error as small ones!

1 cm

3 cm

• Do you score comets over all the

surface of a large gel (often 10,000

comets), in a systematic way? – If the answer is yes, then your only problem is

probably that your comet tails within one

sample vary more than necessary

– If the anser is no, then you introduce significant

errors

– In both cases, you may not be aware of the

problem!

• Do you use small gels (2 – 10 microliter

of agarose, 100 – 400 comets)? – You probably score comets over the whole

surface, and you often score all of them

– You probably observe rather large variations

between parallell samples (technical

replicates).

A comet assay gel may cover

a substantial portion of a glass

slide (2.5 x 7.5 cm).

The gel surface hence spans

several of the electrode

positions for which we have

measured large voltage

variations


In addition to possible experimental

variations in your cell work…

• Are you prepared for errors introduced in

your results, by the electrophoresis alone,

of probably

+/- 25%


Collaborators

• Norwegian Institute of Public Health

– Hildegunn Dahl

– Lena Sareisian

– Anne Graupner

– Kristine Bjerve Gutzkow

– Ann-Karin Olsen

– Jan Zahlin

• University of Oslo

– Andrew Collins ICAW 2015 Antwerp Sept 2015

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