**3. Steps in RNA sequencing**

The procedure of RNA sequencing is performed by three steps:


The first step in RNA sequencing is the isolation of RNA from the sample provided. The main requirement is that the sample should possess RNA of sufficient *RNA Sequencing in Potentially Malignant Disorders DOI: http://dx.doi.org/10.5772/intechopen.97712*

**Figure 1.** *Steps in RNA sequencing.*

quality to enable library sequencing. Based on the quality of the RNA, measured by a bioanalyser an RNA integrity number is given ranging from 1 to 10. The number 10 shows least degradation of RNA with highest quality. RNA of low quality may result in erroneous sequencing.

The next step is the selection of RNA species from the pool of total RNA including mRNA, tRNA, rRNA etc. in this pool, around 95% is contributed by rRNA and are removed before sequencing as it may have the tendency to overshadow the read of other types of RNAs. This can be achieved by many techniques, like selecting poly A RNAs by targeted reaction with poly-T- oligos in magnetic beads. There are also commercially available kits which deplete the rRNA like Ribozero or Ribominus. In another method, the sample is treated with the enzyme, DNAse to reduce the quantity of the genetic material (DNA) in it. The quantity of RNA is determined by either capillary or gel electrophoresis. In the final step, the isolated RNA is then transcribed to DNA. DNA is more stable than RNA and thereby facilitates techniques of amplification without undergoing damage. Also, most of the sequencing libraries require DNA. This cDNA is then utilized in next generation sequencing (**Figure 1**) [8].
