Optimization of cDNA representational difference analysis for the identification of differentially expressed mRNAs

Representational difference analysis (RDA) is a powerful and sensitive tool for identification of differentially expressed genes (M. Hubank and D. G. Schatz, 1999, Methods Enzymol. 303, 325-349; 1994, Nucleic Acids Res. 22, 5 640-5648) that will identify both up- and downregulated genes differentiall y expressed between two cDNA populations. This manuscript provides a thorou gh description of an optimized RDA method. This procedure while still based on the traditional RDA originally developed by Lisitsyn and co-workers (N. A. Lisitsyn, 1995, Trends Genet. 11, 303-307; N. A. Lisitsyn, F. S. Leach, B. Vogelstein, and M. H. Wigler, 1994, Cold Spring Harbor Symp. Quant. Bio l. 59, 585-587; N. Lisitsyn, N. Lisitsyn, and M. Wigler, 1993, 259, 946-951 ) and modified by Hubank and Schatz for RNA (1994, Nucleic Acids Res. 22, 5 640-5648) is improved and requires less starting material than many existin g methods. Several key modifications are included (1). Size-exclusion gel-f iltration microspin columns are used throughout the procedure to remove the primers and low molecular weight cDNAs. This results in reducing the numbe r of ethanol precipitations required and in improving the yield of desirabl e amplification products (2). Elimination of the mung bean nuclease treatme nt in favor of a simple dilution of PCR serves as a means of markedly reduc ing the single-stranded cDNAs that can interfere with the amplification of differentially expressed products (3). The use of up to six unique noninter acting primers ensures that no anomalous amplification occurs due to carryo ver of primers or incomplete digestion from the ends of the cDNAs (4). A se t of cDNA standards was developed and various concentrations were used to b etter characterize the ability of representational difference analysis to i dentify rare messages in a complex cDNA population (5). Integral to this ma nuscript, a detailed laboratory protocol is available from the authors (cra ig.byus@ucr.edu) and provides a step-by-step description of the modified pr ocedure. (C) 2000 Academic Press.