Getting the Stain INA Quantitative Analysis of DNA and RNA Staining with Various Dyes

Dr. Amber Charlebois

Teaching Assistant: Jeremy Tang

Rupak BhuyanLauren DaiTyler Davenport Bruce EasopArka MallelaKenneth PuShireen RudinaTina 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)

(2007). Retrieved July 29, 2008, from Southern Illinois University School of Medicine Web site: http://web.siumed.edu/~bbartholomew/images/chapter6/F06-21.jpg

http://en.wikipedia.org/rna

Loading Dye RNA DNA

Loading Dye

Loading Dye RNA

RNADNA

Loading Dye

RNA DNA

(Begin Run)

Staining the Gel(destain in water)

RNA band (18 bases)

DNA band 35 bases

Use of Fiber Optic Technology

Detector

Halogen Source

Use of Fiber Optic Technology

(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/OOIntro.htm

Dyes

S

N

NH3C

CH3Cl

N

CH3

CH3S

N CH3

NH2NH3C

CH3Cl

Cresyl Violet Toluidine Blue OMethylene Blue

O

N

NH2H2NCH3COO

Azure A Azure B Azure C Thionin

Nile Blue A Brilliant Cresyl BlueCoomassie Brilliant Blue

General Testing of Dyes

Strips of Gel Stained with Methylene Blue, Azure A, and Azure C prior to destaining

RNA

DNA

Azure B

––– RNA

––– DNA

Azure A

––– RNA

––– DNA

Azure C

––– RNA

––– DNA

Toluidine Blue O

––– RNA

––– DNA

S

N

NH3C

CH3Cl

N

CH3

CH3

Cresyl Violet

––– RNA

––– DNA

O

N

NH2H2NCH3COO

Brilliant Cresyl Blue

––– RNA

––– DNA

Methylene Blue

––– RNA

––– DNA

S

N CH3

NH2NH3C

CH3Cl

Thionin

––– RNA

––– DNA

Coomassie and Nile Blue A

Coomassie Nile Blue A

Dyes Chosen for Quantitative Analysis

S

N

NH3C

CH3Cl

N

CH3

CH3S

N CH3

NH2NH3C

CH3Cl

Cresyl Violet Toluidine Blue OMethylene Blue

O

N

NH2H2NCH3COO

Azure A Azure B Azure C Thionin

Nile Blue A Brilliant Cresyl BlueCoomassie Brilliant Blue

Thionin

DNA

RNA

10 – 200 picomoles

Azure B

RNA

DNA

10 – 200 picomoles

Cresyl Violet

RNA

DNA

10 – 200 picomoles

Methylene Blue

DNA

RNA

DNARNA

10 – 200 picomoles

Quantitative Data Azure B DNAAbs orbanc e vs . Amount of DNA for Azure B at 601.43 nm

y = 0.0013x + 0.1825R ² = 0.866

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 50 100 150 200 250Amount (pmol)

Abso

rbanc

e

Quantitative Data Methylene Blue DNAAbs orbanc e vs . Amount of DNA for Methylene B lue at 670.54 nm

y = 0.0006x + 0.3429R ² = 0.6061

y = 0.0006x + 0.24R ² = 0.3414

0

0.1

0.2

0.3

0.4

0.5

0.6

0 50 100 150 200 250 300

Amount (pmol)

Abso

rban

ce

Trial 1

Trial 2

Quantitative Data Cresyl Violet DNAAbs orbanc e vs . Amount of DNA for C res yl Violet at 560 nm

y = 0.0003x + 0.1338R ² = 0.3152

y = 0.0005x + 0.145R ² = 0.861

0

0.05

0.1

0.15

0.2

0.25

0.3

0 50 100 150 200 250 300Amount (pmol)

Abso

rban

ce

Trial 1

Trial 2

Quantitative Data Thionin DNAAbs orbanc e vs . Amount of DNA for T hionin at 560 nm

y = 0.0009x + 0.1818R ² = 0.8071

y = 0.0008x + 0.2441R ² = 0.6753

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 50 100 150 200 250 300

Amount (pmol)

Abso

rban

ce

Trial 1

Trial 2

Corrected Thionin DNA DataAbs orbanc e vs . Amount of DNA for T hionin at 560 nm (C orrec ted)

y = 0.0005x + 0.2647R ² = 0.9979

y = 3E -05x + 0.3645R ² = 0.75

y = 0.003x + 0.0703R ² = 0.9832

y = 0.0022x + 0.1775R ² = 0.9906

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 50 100 150 200 250 300

Amount (pmol)

Abso

rban

ce

Quantitative Data Azure B RNAAbs orbanc e vs . Amount of R NA for Azure B at 600.85 nm

y = 0.0009x + 0.2181R ² = 0.6103

y = 0.002x + 0.1082R ² = 0.9876

0

0.1

0.2

0.3

0.4

0.5

0.6

0 50 100 150 200 250 300

Amount (pmol)

Abso

rban

ce

Trial 1

Trial 2

Corrected Azure B RNA DataAbs orbanc e vs . Amount of R NA for Azure B at 600.85 nm

(C orrec ted)

y = 0.0029x + 0.0925R ² = 0.9826

y = 0.002x + 0.1082R 2 = 0.9876

0

0.1

0.2

0.3

0.4

0.5

0.6

0 50 100 150 200 250

Amount (pmol)

Abso

rban

ce

Trial 1 (corrected)

Trial 2

Quantitative Data Cresyl Violet RNA

Abs orbanc e vs . Amount of R NA for C res yl Violet at 560 nm

y = 0.0008x + 0.1219R ² = 0.9552

y = 0.0011x + 0.0957R ² = 0.8671

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 50 100 150 200 250 300

Amount (pmol)

Abso

rban

ce

Trial 1

Trial 2

Quantitative Data Thionin RNAAbs orbanc e vs . Amount of R NA for T hionin at 560.04 nm

y = 0.0002x + 0.1259R ² = 0.1756

y = 0.0017x + 0.1176R ² = 0.8422

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 50 100 150 200 250 300

Amount (pmol)

Abso

rban

ce

Trial 1

Trial 2

Quantitative Data Methylene Blue RNAAbs orbanc e vs . Amount of R NA for Methylene B lue

y = 0.0017x + 0.1162R ² = 0.9374

y = 0.0009x + 0.1939R ² = 0.9054

y = 0.0014x + 0.165R ² = 0.8657

y = 0.001x + 0.1485R 2 = 0.9638

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 50 100 150 200 250 300

Amount (pmol)

Abso

rban

ce

672 nm- Trial 2

601 nm- Trial 1

601 nm- Trial 2

672 nm-Trial 1

Corrected Methylene Blue RNA DataAbs orbanc e vs . Amount of R NA for Methylene B lue (C orrec ted)

y = 0.0017x + 0.144R ² = 0.9894y = 0.0027x + 0.1136

R ² = 0.9335

y = 0.0016x + 0.1145R 2 = 0.9874

y = 0.0022x + 0.0966R 2 = 0.9569

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 20 40 60 80 100 120

Amount (pmol)

Abso

rban

ce

601 nm- Trial 1

601 nm- Trial 2

672 nm- Trial 1

672 nm- Trial 2

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

AcknowledgementsDr. MiyamotoDr. Paul Quinn

CounselorsSchering-Plough Foundation

NovartisThe Dorr Foundation

The Jennifer A. Chalsty FoundationThe Edward W. and Stella C. Van Houten Memorial Fund

The Jewish Communal Fund Laura and John Overdeck

NJGSS Alumni and Parents 1984-2008

Questions?