ABSTRACT Title of Document: NUCLEIC ACID EXTRACTION AND DETECTION ACROSS TWO-DIMENSIONAL TISSUE SAMPLES Michael Daniel Armani, Doctor of Philosophy, 2010 Directed By (co-advisors): Professor Benjamin Shapiro, Fischell Department of Bioengineering, & Professor Elisabeth Smela, Department of Mechanical Engineering. Visualizing genetic changes throughout tissues can explain basic biological functions and molecular pathways in disease. However, over 90% of mammalian messenger RNA (mRNA) is in low abundance (<15 copies per cell) making them hard to see with existing techniques, such as in-situ hybridization (ISH). In the example of diagnosing cancer, a disease caused by genetic mutations, only a few cancer- associated mRNAs can be visualized in the clinic due to the poor sensitivity of ISH. To improve the detection of low-abundance mRNA, many researchers combine the cells across a tissue sample by taking a scrape. Mixing cells provides only one data point and masks the inherent heterogeneity of tissues. To address these challenges, we invented a sensitive method for mapping nucleic acids across tissues called 2D- PCR. 2D-PCR transfers a tissue section into an array of wells, confining and separating the tissue into subregions. Chemical steps are then used to free nucleic acids from the tissues subregions. If the freed genetic material is mRNA, a purification step is also performed. One or more nucleic acids are then amplified using PCR and detected across the tissue to produce a map. As an initial proof of concept, a DNA map was made from a frozen tissue section using 2D-PCR at the resolution of 1.6 mm per well. The technique was improved to perform the more challenging task of mapping three mRNA molecules from a frozen tissue section. Because the majority of clinical tissues are stored using formalin fixation and not freezing, 2D-PCR was improved once more to detect up to 24 mRNAs from formalin- fixed tissue microarrays. This last approach was used to validate genetic profiles in human normal and tumor prostate samples faster than with existing techniques. In conclusion, 2D-PCR is a robust method for detecting genetic changes across tissues or from many tissue samples. 2D-PCR can be used today for studying differences in nucleic acids between tumor and normal specimens or differences in subregions of the brain. NUCLEIC ACID EXTRACTION AND DETECTION ACROSS TWO- DIMENSIONAL TISSUE SAMPLES By Michael Daniel Armani Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2010 Advisory Committee: Professor Benjamin Shapiro, Chair Professor Elisabeth Smela Professor Yu Chen Professor Joonil Seog Professor Jason D. Kahn, Dean’s Representative Dr. Michael W. Phelan, M.D. © Copyright by Michael Daniel Armani 2010 Dedication To my loving parents, for all their hard work and motivation. ii Acknowledgements I am especially thankful for my wonderful advisors, Dr. Benjamin Shapiro, Dr. Elisabeth Smela, and Dr. Michael Emmert-Buck for guiding me for so many years. They taught me how question the status quo, formulate my own objectives logically, and keep from being biased by my own assumptions. Although I had three advisors at the same time, I am certain this created a unique mindset and proved that too many cooks do not spoil the broth. The skills they taught me branched out into all aspects of my life. I would like to thank Dr. Steven Hewitt and the TARP lab members Kris Ylaya and Jennifer Martinez for helpful discussions, sharing their time, and providing research support. A very special thanks also goes to Michael Tangrea, who shared countless hallway conversations and brainstorming sessions with me, resulting in many ideas that I pursued. I would also like to acknowledge the past and present members of Dr. Emmert-Buck’s lab (John Gillespie, Skye Kim, Brian Yang, Jaime Rodriguez- Canales, Jeff Hansen, Rodrigo Chuaqui, Heidi Erickson, Liang Zhu, Wusheng Yan, Sumana Mukherjee, Anneley Richardson, Alex Rosenberg), for discussing ideas with me, teaching me how to use the current molecular biology tools, doing experimental work with me, and for kindly sharing reagents or space. iii A very special thanks also goes to Belhu Metaferia, who essentially taught me a crash-course in organic chemistry, synthetic chemistry, and hydrogel chemistry even though he was not associated with our project or lab officially. I also must acknowledge many other colleagues, who helped me develop my ideas and supported my project in many ways including Disha Pant, Jeff Burke, Marc Dandin, Mark Kujawski, Mario Urdaneta, and Bavani Balakrisnan, from Dr. Smela’s lab; also Roland Probst, Pramod Mathai, Alek Nacev, Nir Kalush, and Derek Metzer from Dr. Shapiro’s lab. A special thanks to Eddie Chen and Maura Manion who were actually the first ones to work on 2D-PCR and who passed their project to me. I would also like to thank many more people who helped facilitate this project. This includes Tom Loughran and Jonathan Hummel at the UMD FabLab, Howie Grossenbacher at the UMD aerospace machine shop, Connie Newcome at Adhesives Research, who supplied us with specialty adhesive samples that were essential to this project, and Kelley Banfield at SAIC-Frederick who ran our Fluidigm experiments. I would like to thank people outside of my work who helped me and supported me, especially Sarah, and many others. Lastly, I have to thank the U.S. tax-payers, as their financial support made it to this project one way or another. iv Table of Contents DEDICATION..............................................................................................................II ACKNOWLEDGEMENTS........................................................................................ III TABLE OF CONTENTS............................................................................................. V LIST OF FIGURES .................................................................................................... IX LIST OF TABLES....................................................................................................XIII CHAPTER 1 : MOTIVATION AND BACKGROUND.............................................. 1 1.1 Introduction................................................................................................... 1 1.2 Background................................................................................................... 4 1.2.1 Nucleic acids......................................................................................... 4 1.2.2 Changes in nucleic acids....................................................................... 6 1.2.3 Detecting genetic changes by the polymerase chain reaction (PCR) ... 7 1.2.4 Typical methods of tissue sample preservation and storage............... 17 1.2.5 Typical methods for recovering nucleic acids .................................... 22 1.2.6 A common lab workflow to study changes in nucleic acids............... 28 1.2.7 Summary............................................................................................. 29 1.3 Current methods for localizing nucleic acids in tissue ............................... 29 1.3.1 In-situ hybridization (ISH).................................................................. 29 1.3.2 In-situ PCR (ISP)................................................................................ 33 1.3.3 Laser microdissection & PCR............................................................. 35 1.3.4 Macrodissection or voxelation & PCR............................................... 39 1.3.5 Immunohistochemistry (IHC)............................................................. 42 1.3.6 Summary............................................................................................. 43 1.4 Aims............................................................................................................ 44 1.4.1 A robust nucleic acid mapping method .............................................. 44 1.4.2 2D-PCR addresses all of these needs.................................................. 49 1.4.3 Aim 1 – Map DNA using 2D-PCR..................................................... 51 1.4.4 Aim 2 – Map RNA using 2D-PCR..................................................... 52 1.4.5 Aim 3 – Mapping total RNA in FFPE tissue using 2D-PCR.............. 52 CHAPTER 2 : DNA MAPS FROM FROZEN TISSUE ............................................ 55 2.1 Abstract....................................................................................................... 56 2.2 Introduction................................................................................................. 56 2.3 DNA Mapping Methodology...................................................................... 59 2.4 Materials and Methods................................................................................ 63 2.4.1 Device fabrication............................................................................... 63 2.4.2 Tissue sectioning and transfer............................................................. 64 2.4.3 Device heating and sealing ................................................................. 65 2.4.4 DNA amplification protocol............................................................... 67 2.4.5 Visualization....................................................................................... 67 2.5 Results........................................................................................................