What is RNA Sequencing? and its working.

What the term RNA Sequencing?   

RNA-seq provides researchers a window into the RNA environment of a cell during different physiological or pathological states or during different stages of development to work out cellular responses to those changes. RNA-seq allows for top throughput NGS, providing both qualitative and quantitative information about the various RNA species present during a given sample.

There are various types of RNA-Seq stated follow:

Direct RNA-seq sequences the RNA during a cell directly. 

Total RNA-seq sequences all RNAs present during a sample. 3’-mRNA seq produces a summary of the organic phenomenon within a cell.

Small RNA-seq involves a size selection step during RNA isolation and appears at important non-coding RNA transcripts like cell-free RNA and miRNAs.

Single-cell RNA-seq provides an expression profile on the single-cell level to avoid potential biases from sequencing mixed groups of cells.

How does RNA-seq work?

Early RNA-seq techniques used Sanger sequencing technology, a way that although innovative at the time, was also low-throughput, costly, and inaccurate. it's only recently, with the arrival and proliferation of NGS technology, have we been ready to fully cash in of RNA-seq’s potential4.

The first step within the technique involves converting the population of RNA to be sequenced into cDNA fragments (a cDNA library). this enables the RNA to be put into an NGS workflow. Adapters are then added to every end of the fragments. These adapters contain functional elements which enable sequencing; for instance , the amplification element and therefore the primary sequencing site. The cDNA library is then analyzed by NGS, producing short sequences which correspond to either one or both ends of the fragment. The depth to which the library is sequenced varies counting on the techniques that the output data are going to be used for. The sequencing often follows either single-read or paired-end sequencing methods. Single-read sequencing may be a cheaper and faster technique (for reference, about 1% of the value of Sanger sequencing) that sequences the cDNA from only one end, whilst paired-end methods sequence from both ends, and are therefore costlier and time-consuming5,6.

A further choice must be made between strand-specific and non-strand-specific protocols. the previous method means the knowledge about which DNA strand was transcribed is retained. the worth of additional information obtained from strand-specific protocols makes them the favorable option.

These reads, of which there'll be many millions by the top of the workflow, can then be aligned to a genome of reference and assembled to supply an RNA sequence map that spans the transcriptome7.

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