Advances in molecular biology and biochemistry have provided tools with which to explore and increase our understanding of mediators that regulate development and pathological changes in the pulmonary circulation. Combinations of techniques have allowed researchers to start to unravel complex biological pathways that have direct relevance to pulmonary research, such as those that contribute to cellular proliferation and vascular permeability. The aim of this chapter is to describe some of the established protocols to quantify DNA, RNA, and protein expression, discuss advances in these techniques, and illustrate how they can be applied. Expression levels of cell signaling components can be detected at the level of DNA, RNA, and protein. Southern blotting (also termed "Southern hybridization" and named for Edwin Southern, who developed the technique) allows for the detection of a specific DNA sequence in a preparation of DNA, typically from cellular nuclei. This method was modified and extended later for the detection of RNA and protein, named Northern and Western blots, respectively. All three of these protocols are based on similar principles and utilize similar methods. Alternatively, measurement of gene expression [using messenger RNA (mRNA) as a starting material for the generation of complementary DNA (cDNA)] can also be performed via reverse-transcription (RT) polymerase chain reaction (PCR), followed by agarose gel electrophoresis. A modification of this technique, real-time PCR (also termed "quantitative PCR"), allows for quantitative analysis of gene copy number. A number of other high-throughput techniques exist for the profiling of gene and protein expression, such as by use of DNA and protein microarrays; however, these will be discussed in more detail in subsequent sections. Many variations exist for the protocols outlined below and most buffers can be made using a variety of recipes or can be purchased from a number of suppliers. However, the principles remain the same; understanding the stages in each process and their purpose is key. This chapter outlines the methods that have proven to be versatile, reliable, and sensitive.