1. 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

2. 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

3. 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

4. Loading Dye RNADNA Loading Dye RNA DNA Loading Dye RNADNA (Begin Run)

5. Staining the Gel (destain in water) RNA band (18 bases) DNA band 35 bases

6. Use of Fiber Optic Technology

7. 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/OO Intro.htm

8. Dyes Cresyl Violet Toluidine Blue O Methylene Blue Azure A Azure B Azure C Thionin Nile Blue A Brilliant Cresyl BlueCoomassie Brilliant Blue

9. General Testing of Dyes Strips of Gel Stained with Methylene Blue, Azure A, and Azure C prior to destaining RNA DNA

10. Azure B ––– RNA ––– DNA

11. Azure A ––– RNA ––– DNA

12. Azure C ––– RNA ––– DNA

13. Toluidine Blue O ––– RNA ––– DNA

14. Cresyl Violet ––– RNA ––– DNA

15. Brilliant Cresyl Blue ––– RNA ––– DNA

16. Methylene Blue

17. Thionin ––– RNA ––– DNA

18. Coomassie and Nile Blue A CoomassieNile Blue A

19. Dyes Chosen for Quantitative Analysis Cresyl Violet Toluidine Blue O Methylene Blue Azure A Azure B Azure C Thionin Nile Blue A Brilliant Cresyl BlueCoomassie Brilliant Blue

20. Thionin DNA RNA 10 – 200 picomoles

21. Azure B RNA DNA 10 – 200 picomoles

22. Cresyl Violet RNA DNA 10 – 200 picomoles

23. Methylene Blue DNA RNA DNA RNA 10 – 200 picomoles

24. Quantitative Data Azure B DNA

25. Quantitative Data Methylene Blue DNA

26. Quantitative Data Cresyl Violet DNA

27. Quantitative Data Thionin DNA

28. Corrected Thionin DNA Data

29. Quantitative Data Azure B RNA

30. Corrected Azure B RNA Data

31. Quantitative Data Cresyl Violet RNA

32. Quantitative Data Thionin RNA

33. Quantitative Data Methylene Blue RNA

34. Corrected Methylene Blue RNA Data

35. 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

36. 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

37. 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

38. Questions?