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
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| Custom small RNA Blockers | NEXTFLEX Custom Small RNA blockers
NEXTFLEX Custom Small RNA Blockers
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| Spike-in controls | miND® Spike-in Controls | |
| Small RNA library prep | NEXTFLEX Small RNA-seq Kit v4
NEXTFLEX Small RNA Sequencing Kit V4
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