RNA sequencing

RNA sequencing(RNA-Seq for short) is a molecular biology method that can be used to determine:

• which genes are active in a cell or tissue
• how strongly these genes are expressed
• which RNA variants (isoforms, mutations) are present.

RNA sequencing makes the transcriptome visible (the transcriptome is the entirety of all RNA molecules that are present in a cell at a certain point in time).

DNA contains the genetic information of a cell. However, at any given time, only some of the genes are read and transcribed into RNA (this is a measure of the current gene activity). RNA sequencing therefore does not measure the "potential" (DNA), but the actual gene activity, i.e. the frequency with which the DNA is transcribed at a certain point in time. RNA sequencing typically takes place in several steps.

RNA isolation: The entire RNA is isolated from a sample (e.g. blood, skin biopsy, tape tear with genetic material, tumor tissue). This contains

• mRNA (coding RNA)
• rRNA
• tRNA
• lncRNA, miRNA etc.

In general, people are mainly interested in mRNA because it reflects the activity of genes.

Transcription into a cDNA: In a next step, the mRNA is transcribed into a cDNA. The reason for this intermediate step is that the mRNA is unstable. Therefore, it cannot be sequenced directly. It is therefore transcribed into cDNA (complementary DNA) by means of reverse transcription. This is the same process as RT-PCR (Reverse Transcriptase Polymerase Chain Reaction).

Library production: The cDNA is fragmented, provided with special adapter sequences and amplified. This creates a so-called sequencing library.

Sequencing (e.g. ^Illumina®): In the next step, the cDNA fragments are read in high-throughput sequencing devices. This involves millions of short sequences ("reads"). A powerful detection platform that can process these quantities of reads is, for example, the Illumina NovaSeq system

Bioinformatic evaluation: In the digital processing procedure, the analyzed reads are compared with the reference genome. The analyzed DNA reads can now be assigned to specific genes and counted. The result allows a statement to be made on the following question:

• Which genes are upregulated?
• Which are down-regulated?
• Which signaling pathways are active?

Difference to microarrays: In the past, microarrays were mainly used. The RNA-Seq is superior, as all genes can be examined in one analysis order with extremely fast turnover.

Applications

In medicine:

• Tumor profiling
• Inflammatory processes with the aim of personalized medicine and the establishment of precision medicine
• Analysis of immune responses
• Biomarker search
• Effects of drugs on the activity of certain genes

In research:

• The molecular comparison of sick vs. healthy
• Developmental biology
• Characterizing cell types
• Discovery of new, previously unknown transcripts