Next generation sequencing basics – RNA

RNA-sequencing (RNA-seq) is a technique that utilises next-generation sequencing to investigate gene expression in cells by quantifying the presence and amount of RNA reads within a sample. This allows scientist to explore the cellular transcriptome (total cellular RNA content including mRNA, ncRNA, miRNA, etc.), evaluate changes in gene expression (e.g. caused by changes in environmental conditions) and study alternative splicing events (differences in the maturation of mRNA).

The basic workflow of RNA-seq experiments consists of several steps, each with important considerations that will affect the quality of generated data.

Basic steps are:

  1. Experimental design (define the research question and how to address it)
  2. RNA extraction and processing
  3. Library preparation (conversion of RNA to cDNA, adapter ligation etc.)
  4. Next-generation sequencing
  5. Data processing and statistical data analysis


Overall, RNA-seq is the gold standard to explore cellular transcriptomes and how genes are expressed, with high reproducibility and low levels of noise.

Author: Nikolaus Virgolini (ESR8)