Expression Profiling by iAFLP: A PCR-Based Method for Genome-Wide Gene Expression Profiling

摘要

The availability of comprehensive sets of genes has prompted the researchers to carry out systematic collection of gene expression data. RT–PCR has the highest specificity and sensitivity for transcript detection among all available methods. Low throughput, especially when quantitative data are desired, has precluded RT–PCR from genome-wide application. Here we report a PCR-based expression profiling method, introduced amplified fragment length polymorphism (iAFLP), that has the same specificity and sensitivity as RT–PCR and a throughput level comparable to that of DNA–microarray hybridization. In this method, restricted ends of total cDNAs from six sources were ligated with adaptors having various length of short insertions to a common sequence (polymorphic adaptors). Amplification of a pool of these differentially adapted cDNAs with a gene-specific primer and an adaptor primer allows us to quantitate the abundance of any transcript in six mRNA sources. Using three different primer colors this technique allows quantitation of expression of 864 genes across six different sources per day with a single autosequencer, which is comparable to the throughput of microarray analysis in terms of number of genes × number of sources.