Small RNA Sequencing Library Prep Kits & Accessories
Small RNA-seq library preparation enables robust detection and quantitative measurement of non-coding RNAs shorter than 200 nucleotides. Most widely studied small RNA species fall in the 15–90 nucleotide range and include microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), transfer RNAs and their derived fragments (tRNAs and tRFs), and Y-RNA fragments, alongside other classes of non-coding small RNA. These molecules play key roles in gene regulation, genome defense, and cellular stress responses. Small RNAs are detectable in tissues and biofluids, making them valuable for mechanistic studies and biomarker discovery. By converting small RNAs into sequencing-ready libraries, small RNA-seq supports systematic detection and quantification across samples, supporting applications from basic to translational research.
Facing a small RNA research challenge?
Our experts help you define the right small RNA-seq strategy for your samples.
Selection guide
| Item | Revvity solution |
|---|---|
| Blocker of abundant human miRNA found in blood | NEXTFLEX blood miRNA blocker |
| Custom small RNA Blockers | NEXTFLEX Custom Small RNA blockers |
| Spike-in controls | miND Spike-in Controls |
| Small RNA library prep | NEXTFLEX Small RNA-seq Kit v4 |
Main applications for small RNA-seq:
Main applications for small RNA-seq
Small RNA-seq supports both expression profiling and fragment-level analysis across diverse small RNA classes, from intracellular regulation to extracellular biomarker research.
miRNA expression profiling
Small RNA-seq enables detection and quantitative measurement of mature miRNAs and sequence variants (isomiRs). This supports comparisons across conditions and time points and helps connect miRNA shifts to pathways and biological targets.
tRNA and Y-RNA derived fragments
tRNAs and Y-RNAs generate short fragments with distinct regulatory roles. Sequencing resolves fragment classes, including common tRF subclasses and 5′ and 3′ Y-RNA fragments, helping clarify biogenesis, tissue specificity, and biological mechanisms.
piRNAs and other germline small RNAs
piRNAs are enriched in germline contexts and contribute to transposon silencing and genome defense. Sequencing characterizes piRNA repertoires, including sequence diversity and length distributions, to support functional interpretation.
Extracellular and extracellular vesicle-associated small RNAs
Small RNAs circulate in biofluids and can be carried in extracellular vesicles. Small RNA-seq enables detection and quantification across plasma, serum, and other matrices to support biomarker discovery and longitudinal studies.
For research use only. Not for use in diagnostic procedures.
Small RNA library prep and accessory products
Your small RNA-seq hub
Everything you need for small RNA‑seq, all in one place. Revvity offers a comprehensive solution for small RNA‑seq including library prep kits, targeted blockers designed to remove highly abundant species, and spike-in controls designed to support reproducible library construction across a wide dynamic range. Explore the products below to match workflow needs, sample type, throughput, and sequencing compatibility.
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 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 around 160 bp. 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, and a handful of highly expressed miRNAs), consuming most sequencing reads and masking low-abundance signals. To increase library diversity, we offer a depletion strategy based on targeted blockers. These blockers are short, sequence-specific oligos that hybridize to abundant small RNAs during the 3' adapter-ligation step of the NEXTFLEX Small RNA-Seq Kit v4 workflow, preventing adapter addition to the targeted species and reducing their carryover.
For RNA isolated from human whole blood or plasma, NEXTFLEX™ blood miRNA blockers reduce the abundance of common high-abundance miRNAs, including hsa-miR-451a, hsa-miR-486-5p, and hsa-miR-92a-3p, freeing read depth for study-relevant targets. For other sample types or organisms, NEXTFLEX™ Custom Small RNA Blockers can be designed to deplete user-specified high-abundance small RNAs, including tRNA or Y-RNA fragments and overexpressed miRNAs, with up to 10 targets per project.
Spike-in controls
Small RNA spike-in controls are a mix of synthetic small RNAs not present in natural samples. Added to total RNA before library preparation, they function as internal standards to quality-check assay performance and normalize results across samples and batches. miND® Spike-In Controls enable absolute normalization by converting sequencing read counts into molecules per microliter (copies/µL), improving comparability across experiments, cohorts, and laboratories, especially for low-input matrices such as biofluids.
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