The next-generation sequencing technologies are being rapidly applied in biological research. Tens of millions of short sequences generated in a single experiment provide us enormous information on genome composition, genetic variants, gene expression levels and protein binding sites depending on the applications. Various methods are being developed for analyzing the data generated by these technologies. However, the relevant experimental design issues have rarely been discussed. In this review, we use RNA-seq as an example to bring this topic into focus and to discuss experimental design and validation issues pertaining to next-generation sequencing in the quantification of transcripts.