Breeding of yeast strains with higher RNA content is important because yeast RNA is a significant source of 5'-ribonucleotides, which have considerable use in both the food and pharmaceutical industries. Ribosomal RNA (rRNA) is an important source of yeast RNA as it accounts for about 80 % of total RNA content. We previously reported a dominant suppressor mutant of an rrn10 disruptant named SupE, which displays the ability not only to restore diminished RNA content caused by rrn10 disruption but also to increase the transcription level of ribosomal protein (RP) genes on an a dagger rrn10 background in Saccharomyces cerevisiae. Here, to construct an S. cerevisiae strain with higher RNA content, we investigated the effect of increasing the copy number of the rDNA gene on a a dagger rrn10 SUPE background. We successfully constructed a SupE strain with two copies of the rDNA cluster (ca. 300 rDNA genes) by using chromosome-splitting technology. The RNA content of this strain was 61 % higher than that of the SupE strain with a single copy of the rDNA cluster (ca. 150 rDNA genes), and 40 % higher than that of the wild-type strain with two copies of the rDNA cluster. A further increase in RNA content of 47 % was achieved by multicopy expression of the RPL40A gene in the SupE strain with two copies of the rDNA cluster. These observations suggest that we have constructed an S. cerevisiae strain with two copies of the rDNA cluster, which has achieved a considerably higher RNA content. Furthermore, the strategy taken in this study provides an effective approach to constructing S. cerevisiae strains with high potential for yeast food biotechnology.