Small RNA Sequencing Library Prep Kits

Small RNA sequencing (small RNA-seq) provides a comprehensive view of the short RNA molecules present in a sample. These molecules are generally shorter than 200 nucleotides, with many of the most studied species falling in the 15-90 nucleotide range. Classes of small RNAs include microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), transfer RNAs (tRNAs) and their derived fragments (tRFs), Y-RNA fragments, and a wide variety of other non-coding RNAs. Although diverse in origin, they share important biological roles: they regulate gene expression, control cellular stress responses, and circulate in biofluids where they can be measured as stable biomarkers. By enabling systematic detection and quantification of these molecules, small RNA sequencing has become an essential tool for both basic research and translational applications.

Main applications for small RNA-seq:

miRNA expression profiling

Small RNA sequencing allows measurement of mature miRNAs and their variants, known as isomiRs. Profiling these molecules makes it possible to compare expression across conditions or time points, and to link miRNA changes to pathways and biological targets.

tRNA-derived fragments and Y-RNA fragments

Transfer RNAs and Y-RNAs can be processed into shorter fragments with distinct regulatory roles. Sequencing enables the classification of tRNA fragments such as tRF-5, tRF-3, and tiRNAs, as well as 5′ and 3′ Y-RNA fragments. This helps to clarify their biogenesis, tissue specificity, and contribution to cellular processes.

piRNAs and other germline small RNA

PIWI-interacting RNAs form a specialized class of small RNAs enriched in reproductive tissues and stem cells. They are involved in transposon silencing and germline maintenance. Sequencing offers the ability to characterize entire piRNA repertoires, revealing sequence diversity, length distributions, and their potential functional impact.

Extracellular and extracellular vesicle-associated small RNAs

Small RNAs are also found outside cells, either freely circulating in plasma and serum or packaged within extracellular vesicles. These extracellular RNAs are of particular interest as non-invasive biomarkers that can reflect disease states or physiological changes. Small RNA sequencing enables their detection and quantification, supporting biomarker discovery and longitudinal monitoring of patients over time.

Revvity provides a complete portfolio to support small RNA-seq on Illumina® and Element Biosciences® platforms, including a widely adopted library prep kit , targeted blockers designed to remove highly abundant species and spike-in controls that enable quantitation of miRNA copy number. Explore below to identify which components best match your laboratory’s workflow.

For research use only. Not for use in diagnostic procedures.

Small RNA library prep and accessory products

Small RNA sequencing library prep

Unlike long RNA workflows , small RNA-seq library prep requires specialized chemistry to efficiently capture very short RNA molecules and to minimize the formation of adapter dimers, which can otherwise dominate libraries.

In most workflows the process begins with the ligation of a 3′ adapter to the small RNAs. This adapter is usually adenylated, which reduces the risk of unwanted ligation events such as dimers or concatemers and improves the specificity of ligation to the RNA target. Once the 3′ adapter is in place, a 5′ adapter is ligated to RNAs that carry a 5′ phosphate group, creating the priming sites needed for downstream cDNA synthesis.

After adapter ligation, the RNA molecules are converted into cDNA by reverse transcription, followed by PCR amplification with indexed primers. This step not only increases the quantity of material available for sequencing but also introduces the barcodes required to multiplex multiple samples in a single sequencing run. A critical part of small RNA library preparation is the subsequent size selection step. Because the inserts are extremely short, size selection ensures that only molecules containing a genuine small RNA insert are retained. For example, libraries prepared for microRNA analysis typically yield a final product of about 140–160 base pairs, depending on the kit chemistry used. This enrichment is essential to reduce background noise from adapter dimers and to achieve accurate representation of small RNA species in the sequencing data.

Depletion of dominant species

Small RNA libraries are frequently dominated by a few species (tRNA, tRNA-derived fragments, Y-RNA fragments, a handful of highly expressed miRNAs), consuming most of sequencing reads and masking low-abundance signals. To increase library diversity, we have developed a depletion strategy based on targeted blockers. These blockers are short, sequence-specific oligos that hybridize to abundant small RNAs during the earliest ligation step. This prevents adapter addition and reduces their carryover without affecting unrelated species.

Spike-in controls

Small RNA spike-in controls are synthetic RNA oligonucleotides that mimic the properties of endogenous microRNAs but are not present in natural samples. By adding them in known concentrations before library prep, they serve as internal standards that allow normalization across samples and enable the calculation of absolute copy numbers for individual miRNAs. Using spike-ins provides a reliable baseline for comparing data between experiments, cohorts, or laboratories, ensuring that biological conclusions are based on true changes in small RNA abundance rather than technical variability

Broad coverage of small RNA classes

small RNA classes, ensuring that researchers can capture the full complexity of the short RNA transcriptome. These include canonical miRNAs and their isomiR variants, piRNAs that play key roles in genome defense, and tRFs that reflect stress responses and translational control. The kit also supports the analysis of Y-RNA fragments, small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), as well as extracellular RNA species circulating in plasma, serum, or extracellular vesicles. This broad compatibility allows laboratories to move beyond miRNA profiling and explore the diverse spectrum of small RNAs involved in cellular regulation, intercellular communication, and biomarker discovery.

Selection guide

Item	Sequencers	Revvity solution
Blocker of abundant human miRNA found in blood	Illumina^® and Element^® Platforms	NEXTFLEX blood miRNA blocker NEXTFLEX blood miRNA blockers Discover
Custom small RNA Blockers		NEXTFLEX Custom Small RNA blockers NEXTFLEX Custom Small RNA Blockers Discover
Spike-in controls		miND^® Spike-in Controls
Small RNA library prep		NEXTFLEX Small RNA-seq Kit v4 NEXTFLEX Small RNA Sequencing Kit V4 Discover