RNA Sequencing

Sequencing RNA at two given physiological conditions and comparing the abundance of these molecules against each other has the capacity to show the transcriptomic alterations between these conditions. If a given treatment is applied to an organism and RNA sampled at given time points and compared to the steady state, such an analysis has the capacity to reveal the effect such a treatment has on the organism and the genes that bring about such a change. The abundance of RNA sampled at these given conditions shows the gene regulation at the transcriptomic level. This is referred to as differential expression between points under consideration. The data analysis relies on the availability of a sequenced and annotated genome for the organism being studied.

High throughput sequencing of mRNA in a controlled time point experiment, following a given physiological change with normalized conditions is referred to as differential transcriptome analysis, commonly referred to as RNA-Seq. Transcriptomic regulation can be verified using quantitative real time PCR, which remains the gold standard for this purpose.

Whereas RNA-Seq for differential transcriptome analysis relies on a sequenced genome, whole transcriptome sequencing does not. In this application total RNA is isolated followed by depletion of rRNA, then the material is then used for library preparation. Deep sequencing and de novo assembly of the data has the capacity to show the whole transcriptome content in a qualitative analysis. The result generates RNA transcripts in different stages of maturation for all genes being transcribed at the time of sampling. The application does not require an assembled genome and as such will give transcript isoforms, full length genes, tRNA, miRNA, ncRNA etc depending on the depth of coverage used.