Getting the Stain IN
A Quantitative Analysis of DNA and RNA Staining with Various Dyes
Dr. Amber Charlebois
Teaching Assistant: Jeremy Tang
Rupak Bhuyan
Lauren Dai
Tyler Davenport
Bruce Easop
Arka Mallela
Kenneth Pu
Shireen Rudina
Tina Sankhla
Margaret Yang
Purpose
To identify dyes that:
 Can differentiate between DNA and RNA in gel
electrophoresis
 Are safe, inexpensive, and effective
 Have a linear relationship between amount of nucleic
acid and absorbance
Project Overview
Nucleic Acid – Negatively Charged
Polyacrylamide Gel Electrophoresis (PAGE)
http://en.wikipedia.org/rna
(2007). Retrieved July 29, 2008, from Southern Illinois
University School of Medicine Web site:
http://web.siumed.edu/~bbartholomew/images/chapter6/F0621.jpg
(Begin Run)
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Dye
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Dye
RNA
DNA
RNA
DNA
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Dye
RNA
DNA
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Dye
RNA
(destain
water)
Staininginthe
Gel
DNA band
35 bases
RNA band (18
bases)
Use of Fiber Optic Technology
Use of Fiber Optic Technology
Halogen Source
Detector
(2006, September 26). Introduction to the Ocean Optics
Spectrometer System . Retrieved July 29, 2008, from
Truman State University Chemistry Laboratory Web site:
http://chemlab.truman.edu/Instrumentation/OceanOptics/OO
Intro.htm
Dyes
Cresyl Violet
Toluidine Blue O
Methylene Blue
N
CH3
S
NH2
N
N
H3C
H2N
O
NH2
CH3COO
Azure A
Coomassie Brilliant Blue
N
Cl
CH3
Azure B
Nile Blue A
H3C N
CH3
Azure C
S
N
Cl
CH3
Thionin
Brilliant Cresyl Blue
CH3
General Testing of Dyes
DNA
RNA
Strips of Gel Stained with Methylene Blue,
Azure A, and Azure C prior to destaining
Azure B
––– RNA
––– DNA
Azure A
––– RNA
––– DNA
Azure C
––– RNA
––– DNA
N
H3C N
CH3
S
Cl
N
CH3
CH3
Toluidine Blue O
––– RNA
––– DNA
N
H2N
O
NH2
CH3COO
Cresyl Violet
––– RNA
––– DNA
Brilliant Cresyl Blue
––– RNA
––– DNA
H3C
N
CH3
N
CH3
S
NH2
Cl
Methylene Blue
––– RNA
––– DNA
Thionin
––– RNA
––– DNA
Coomassie and Nile Blue A
Coomassie
Nile Blue A
Dyes Chosen for Quantitative Analysis
Cresyl Violet
Toluidine Blue O
Methylene Blue
N
CH3
S
NH2
N
N
H3C
H2N
O
NH2
CH3COO
Azure A
Coomassie Brilliant Blue
N
Cl
CH3
Azure B
Nile Blue A
H3C N
CH3
Azure C
S
N
Cl
CH3
Thionin
Brilliant Cresyl Blue
CH3
Thionin
DNA
10 – 200 picomoles
RNA
Azure B
DNA
10 – 200 picomoles
RNA
Cresyl Violet
DNA
10 – 200 picomoles
RNA
Methylene Blue
DNA
10 – 200 picomoles
DNA
RNA
RNA
Quantitative Data Azure B DNA
0.5
Absorbance vs. Amount of DNA for Azure B at 601.43 nm
0.45
0.4
A b s o rb a n c e
0.35
y = 0.0013x + 0.1825
R² = 0.866
0.3
0.25
0.2
0.15
0.1
0.05
0
0
50
100
150
Amount (pmol)
200
250
Quantitative Data Methylene Blue DNA
0.6
Absorbance vs. Amount of DNA for Methylene Blue at 670.54 nm
A b s o rb a n c e
0.5
y = 0.0006x + 0.3429
R² = 0.6061
0.4
0.3
y = 0.0006x + 0.24
R² = 0.3414
0.2
Trial 1
0.1
Trial 2
0
0
50
100
150
Amount (pmol)
200
250
300
Quantitative Data Cresyl Violet DNA
0.3
Absorbance vs. Amount of DNA for C resyl Violet at 560 nm
A b s o rb a n c e
0.25
y = 0.0005x + 0.145
R² = 0.861
0.2
y = 0.0003x + 0.1338
R² = 0.3152
0.15
Trial 1
0.1
Trial 2
0.05
0
0
50
100
150
Amount (pmol)
200
250
300
Quantitative Data Thionin DNA
Absorbance vs. Amount of DNA for Thionin at 560 nm
0.45
y = 0.0008x + 0.2441
R² = 0.6753
0.4
A b s o rb a n c e
0.35
0.3
y = 0.0009x + 0.1818
R² = 0.8071
0.25
0.2
Trial 1
0.15
0.1
Trial 2
0.05
0
0
50
100
150
Amount (pmol)
200
250
300
Corrected Thionin DNA Data
Absorbance vs. Amount of DNA for Thionin at 560 nm (C orrected)
0.45
y = 0.0022x + 0.1775
R² = 0.9906
0.4
0.35
A b s o rb a n c e
y = 3E -05x + 0.3645
R ² = 0.75
y = 0.0005x + 0.2647
R² = 0.9979
0.3
0.25
0.2
y = 0.003x + 0.0703
R² = 0.9832
0.15
0.1
0.05
0
0
50
100
150
Amount (pmol)
200
250
300
Quantitative Data Azure B RNA
Absorbance vs. Amount of RNA for Azure B at 600.85 nm
0.6
y = 0.002x + 0.1082
R² = 0.9876
A b s o rb a n c e
0.5
0.4
y = 0.0009x + 0.2181
R² = 0.6103
0.3
Trial 1
0.2
Trial 2
0.1
0
0
50
100
150
Amount (pmol)
200
250
300
Corrected Azure B RNA Data
Absorbance vs. Amount of RNA for Azure B at 600.85 nm
(C orrected)
0.6
A b s o rb a n c e
0.5
y = 0.0029x + 0.0925
R² = 0.9826
0.4
y = 0.002x + 0.1082
2
R = 0.9876
0.3
Trial 1 (corrected)
0.2
Trial 2
0.1
0
0
50
100
150
Amount (pmol)
200
250
Quantitative Data Cresyl Violet RNA
Absorbance vs. Amount of RNA for C resyl Violet at 560 nm
0.4
0.35
y = 0.0011x + 0.0957
R² = 0.8671
A b s o rb a n c e
0.3
y = 0.0008x + 0.1219
R² = 0.9552
0.25
0.2
0.15
Trial 1
0.1
Trial 2
0.05
0
0
50
100
150
Amount (pmol)
200
250
300
Quantitative Data Thionin RNA
Absorbance vs. Amount of RNA for Thionin at 560.04 nm
0.5
y = 0.0017x + 0.1176
R² = 0.8422
0.45
A b s o rb a n c e
0.4
Trial 1
0.35
Trial 2
0.3
y = 0.0002x + 0.1259
R² = 0.1756
0.25
0.2
0.15
0.1
0.05
0
0
50
100
150
Amount (pmol)
200
250
300
Quantitative Data Methylene Blue RNA
Absorbance vs. Amount of RNA for Methylene Blue
0.5
y = 0.0017x + 0.1162
R² = 0.9374
y = 0.0014x + 0.165
R² = 0.8657
0.45
A b s o rb a n c e
0.4
0.35
0.3
0.25
672 nm- Trial 2
y = 0.0009x + 0.1939
R² = 0.9054
601 nm- Trial 1
y = 0.001x + 0.1485
2
R = 0.9638
0.2
0.15
601 nm- Trial 2
0.1
672 nm-Trial 1
0.05
0
0
50
100
150
Amount (pmol)
200
250
300
Corrected Methylene Blue RNA Data
Absorbance vs. Amount of RNA for Methylene Blue (C orrected)
0.35
A b s o rb a n c e
0.3
y = 0.0017x + 0.144
R² = 0.9894
y = 0.0027x + 0.1136
R² = 0.9335
0.25
601 nm- Trial 1
y = 0.0022x + 0.0966
0.2
2
R = 0.9569
0.15
601 nm- Trial 2
y = 0.0016x + 0.1145
2
R = 0.9874
0.1
672 nm- Trial 1
0.05
672 nm- Trial 2
0
0
20
40
60
Amount (pmol)
80
100
120
Conclusions
 Methylene Blue: metachromatic, linear for RNA
up to 100 picomoles
 Azure B: shows promise for linearity for RNA
 Thionin: abnormal pattern for DNA
 Cresyl Violet: needs further research
 All other dyes except Nile Blue and Coomassie
show promise in nucleic acid staining
Future Research
 More careful pipetting
 The effect of dye band area on absorbance
values
 Mixing Dyes
 Use of other statistical methods to
determine linearity
 Multi-linear regression pattern
Acknowledgements
Dr. Miyamoto
Dr. Paul Quinn
Counselors
Schering-Plough Foundation
Novartis
The Dorr Foundation
The Jennifer A. Chalsty Foundation
The Edward W. and Stella C. Van Houten Memorial Fund
The Jewish Communal Fund
Laura and John Overdeck
NJGSS Alumni and Parents 1984-2008
Questions?