Introduction to small RNA-sequencing
Small RNA sequencing enriches and sequences short RNA molecules, including microRNAs (miRNAs) and other compatible small-RNA species, such as tRNAs, small interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs) and more. It enables discovery or quantification of small RNAs that are generally not captured efficiently by standard poly(A)-selected mRNA sequencing.
Use small RNA-seq when miRNAs or another short RNA class is a primary biological readout—for example, when studying post-transcriptional regulation, extracellular or vesicle-associated small RNAs, or condition-dependent changes in miRNA abundance.
Small RNA-seq is not a substitute for conventional mRNA-seq when the main objective is broad protein-coding gene expression. The selection for small RNA species will specifically deplete longer mRNA transcripts.
Compatible sample types
Projects generally begin with purified total RNA or a small-RNA-enriched fraction (some RNA extraction kits specifically isolate small RNA species). Compatibility depends on RNA quantity, extraction method, preservation, sample composition, and the small-RNA species of interest. Investigators should avoid RNA extraction methods that unintentionally exclude the small-RNA fraction (specifically no poly-A based isolation methods).
Genomics CoLab offerings
NEBnext Small RNA Library Preparation: Intended for miRNA and other compatible
small-RNA discovery and quantification.
See our small RNA-sequencing pricing.
Design decisions
When you start your project you will discuss with Genomics CoLab term the following items to assure we can meet your project needs:
- The specific RNA classes of interest.
- RNA extraction and preservation method.
- Whether discovery or quantification of a defined set of miRNAs is the priority.
- Biological replication and batch balance.
- Whether matched mRNA expression data are also needed.
- Species, reference annotations, adapter trimming, and sequencing depth.
What the CoLab performs
The CoLab reviews sample quality and project compatibility, prepares and quality controls small-RNA libraries, pools libraries, coordinates sequencing, and provides the agreed data-processing or analysis services.
What the investigator supplies
The investigator supplies RNA prepared using a compatible extraction method, sample metadata, biological groups, species information, and the small-RNA classes or hypotheses of interest.
Data and analysis delivered
Deliverables may include raw reads, and may be expanded to include quality-control summaries, annotated small-RNA counts, normalized abundance tables, differential-abundance results, standard visualizations, predicted targets and pathway analysis. The precise annotation and analysis scope depends on the organism and project goals.
Pricing and sequencing considerations
Small RNA libraries generally require a sequencing configuration and analysis workflow different from bulk mRNA-seq. Estimates should separate library preparation, sequencing, and analysis. Projects combining small RNA and mRNA require separate library preparations and sequencing allocations for the two data types.
To start a project
Please download our project description form, fill it in with the as much detail as you can and then send it along with a consultation request to the [email protected] email address.