Question

How does RNA sequencing work? Please provide a visual.

Answer 1

RNA sequencing works by transforming the information in the RNA into double stranded DNA, that is because RNA is not a stable enough molecule for processes like sequencing.

The steps are just a few and is very similar to DNA sequencing, only one step is special for RNA sequencing. You firs need to stablish your target RNA molecules, you need to extract them and isolate such molecules. Once you have your RNA we start with the special step: converting RNA into cDNA.

The only process that can transform RNA into its complemetary DNA is Reverse Transcription, done precisely by the enzyme called Reverse Transcriptase. The first DNA strand uses a DNA oligonucleotide primer, this one will attach to its complementary region in RNA and will allow the transcriptase to generate the first DNA strand. The second strand is generated by a DNA polymerase that works on the already created first strand, this polymerase also needs a primer. Now we have a DNA double strand that is complementary to the RNA initial sample. This DNA molecule can go into Sanger sequencing.

Let us illustrate the cDNA creation: