CLONING DIFFERENTIALLY EXPRESSED MESSENGER-RNAS

Differential gene expression occurs in the process of development, mai ntenance, injury, and death of unicellular as well as complex organism s. Differentially expressed genes are usually identified by comparing steady-state mRNA concentrations. Electronic subtraction (ES), subtrac tive hybridization (SH), and differential display (DD) are methods com monly used for this purpose. A rigorous examination has been lacking a nd therefore quantitative aspects of these methods remain speculative. We compare these methods by identifying a total of 58 unique differen tially expressed mRNAs within the same experimental system (HeLa cells treated with interferon-gamma), ES yields digital, reusable data that quantitated steady-state mRNA concentrations but only identified abun dant mRNAs (seven were identified), which represent a small fraction o f the total number of differentially expressed mRNAs. SH and DD identi fied abundant and rare mRNAs (33 and 23 unique mRNAs respectively) wit h redundancy. The redundancy is mRNA abundance-dependent for SH and pr imer-dependent for DD. We conclude that DD is the method of choice bec ause it identifies mRNAs independent of prevalence, uses small amounts of RNA, identifies increases and decreases of mRNA steady-state level s simultaneously, and has rapid output.