Sequential subtractive approach facilitates identification of differentially expressed genes

In order to identify the genes expressed during calcium oxalate crystal formation, we devised a sequential subtractive protocol that allows for efficient isolation of rare differentially expressed transcripts. This new protocol combines the beneficial elements of suppression subtractive hybridization (SSH), subtractive hybridization (SH), and differential screening into a single method. The protocol was designed to increase retention of rare transcripts, efficiently remove unwanted transcripts, have the capacity to analyze numerous transcripts simultaneously, and reduce the number of false positives. The potential for this method was demonstrated through the identification of several transcripts that were differentially expressed in two calcium oxalate defective (cod) Medicago truncatula mutants: cod 4 (which has abundant crystal accumulation) and cod 5 (which lacks crystal accumulation). Transcripts were isolated that showed higher steady-state levels in cod 4 than cod 5. The observed differences in transcript levels appeared to occur via two different mechanisms. In most cases, the higher transcript levels were a result of an increase in the amount of transcript present in cod 4. In one instance, however, the higher steady-state transcript level resulted from a decrease in the amount of transcript present in cod 5 rather than an increase in cod 4. The protocol described is well suited for the identification of rare transcripts from complex transcript pools