Small RNA sequencing

By using this technique, it is possible to discriminate small RNAs from the larger RNA family to better understand their functions in the cell and in gene expression.

This step is very critical and important for any molecular-based technique since it ensures that the small RNA fragments found in the samples to be analyzed are characterized by a good level of purity and quality.

After the adapters are ligated to both ends of the small RNAs, retrotranscription occurs producing complementary DNA molecules (cDNAs) which will be, eventually, amplified by different amplification techniques depending on the sequencing protocol that is being followed (Ion Torrent exploits the emulsion PCR, while Illumina requires a bridge PCR) in order to obtain up to billions of amplicons to be sequenced.

Small RNA bearing one or more of these modifications are often inefficiently and incompletely converted into cDNAs, leading to challenges with their detection and quantitation by deep sequencing, which can be overcome by enzyme (such as PNK and AlkB) pre-treatment.

The small RNAs are finally quantified by assigning molecules to transcript annotations from different databases (Mirbase, GtRNAdb and Gencode).