The ABCs (and ATCGs) of RNA-Seq

DNA has often been likened to a book, with a set of instructions written in A’s, T’s, C’s, and G’s. But even if the contents of the book are known (which is increasingly the case as more and more genomes are sequenced), the where, when, or how loudly, the book is read is not known.

After all, genes may be dormant, ferociously active, or anything in between. A gene’s transcription to RNA is influenced by the type of cell housing the gene, the stage of development of the organism, and the particular conditions that are currently encountered. So although sequencing a genome is certainly invaluable and informative, studying the transcriptome, the full set of all RNA molecules and their quantity in an organism, is crucial for studying genes in action and illuminating their relevance in development and disease.

RNA-Seq(uencing) is a relatively recent approach to documenting the transcriptome and has become a widely used tool in the biological community. It is an actively developing field with many different approaches, depending on the needs of the experiment. In general, however, RNA-Seq works by “reverse-transcribing” RNA in the lab to its DNA complement (known as “cDNA”), which is then fragmented into short sequences. (Alternatively, the RNA might be fragmented first, then reverse-transcribed.) Sequence adaptors are then added to the ends of the cDNA fragments, and then they are ready for their sequences to be read.¹

This brings us to why RNA-Seq is somewhat of a newcomer. The demand for sequencing that can be performed quickly and at low cost drove the development of new, next generation technologies that hold advantages over the traditional method. Various novel methods allow many sequences to analyzed concurrently. This enables, to a high degree of accuracy, the multiple reading of the same section of DNA (“deep sequencing”). With this technology now on the scene, it has become more feasible to sequencing all the cDNA that comes with reverse transcribing RNA from cells. In fact, this high demand for RNA-Seq and other sequencing methods are illustrated through the many vendors at Scientist that offer expertise in next generation sequencing.

RNA-Seq boasts several favorable properties. For one, the genome sequence of the species does not have to be known, a plus for researchers working with organisms a bit off the beaten path. Another bonus is that the background signal is low to non-existent. There is no worry of reaching an upper limit either, and as a result, transcripts can be measured in a range spanning as many as five orders of magnitude.² The clarity of RNA-Seq also means that person-to-person variability in transcript composition can often be picked up.

These desirable features and more are cause for the current intense interest in RNA-Seq. The dynamic transcriptome in all its variation means that genes are no simple book to be read, but RNA-Seq helps with the comprehension.