[15] READS: A method for display of 3′-end fragments of restriction enzyme-digested cDNAs for analysis of differential gene expression

This chapter discusses a gel-based method for analysis of differential gene expression called READS 2 or restriction endonucleolytic analysis of differentially expressed sequences. This method uses stringent polymerase chain reaction (PCR) conditions to amplify only the extreme Y-end fragment of a restriction endonuclease-digested cDNA molecule without employing methods for physical separation of the fragments before or after PCR amplification. A comprehensive analysis of differentially expressed mRNAs between two physiological states can be done by comparing the gel patterns of their 3'-end cDNA restriction fragments. One of the important features of READS is the reproducibility of gel patterns in repeat experiments. Because the method relies on specific amplification of only the 3'-end restriction fragments under stringent PCR conditions, reproducibility of gel patterns can be expected because the set of cDNA molecules synthesized from a particular total RNA by a given anchored primer will always be the same and will, therefore, always generate the same pattern of 3'-end restriction fragments. The chapter provides a detailed description of the method used to perform READS analysis on control and activated Jurkat T cells.