RNA-seq: The New Gene Expression Microarray

Source: Nagalakshmi et al (2008). The Transcriptional Landscape of the Yeast Genome Defined by RNA Sequencing. Science 320:1344-49.

Enfin, the long anticipated RNA-seq paper. Throughout the last decade, gene-expression microarrays have been vastly employed for reporting changes in the expression level of all the transcripts. However, higher efficiency and lower prices in the sequencing market may very well render these methods obsolete in a matter of years. cDNA microarray chips don't provide any information regarding the 5' and 3' end of the transcript (important for e.g. alternative splicing). Although, one might argue the usage of tiling arrays for such matters, 97 slides for a human genome is out of question.

In the RNA-seq method, the mRNAs are extracted (using oligo-dT columns), converted to cDNA, subjected to fragmentation and massive high-throughput sequencing (Illumina sequencing which gives ~35 nucleotides from one end). I'll not go through the proof-of-principle steps, but suffices to say that the method works quite decently. 91.5% of the genes were active with 20% of them reported as highly expressed (biosynthetic pathways and ion transporters). In this type of data, a sharp decline in the number of reads denotes that 5' and 3' ends. The authors have calibrated their parameters using 5' RACE followed by sequencing for 125 genes.

Aside from boundary information, this approach enables us to comment on many other issues (e.g. uORFs, polyA sites and ...). As I said, my guess is that this approach will become mainstream in molecular biology in near future.