NEXTFLEX Small RNA Sequencing Kit V4

• Ready-to-run Sciclone/Zephyr automation scripts with deck maps, tip-usage estimates, and 96-/384-well timing
• No PAGE excision – standard magnetic bead clean-ups eliminate laborious gel slices while preserving low-input yield
• Wide sample compatibility: plasma, serum, urine, CSF, exosomal RNA, etc
• Captures diverse < 200 nt species – miRNA, siRNA, piRNA, tRNA-derived fragments in a single ligation-based workflow
• Scalable study design – one workflow supports pilot studies (8 samples) through 384-plex UDI projects without protocol changes

We compared different vendors, and we found that the small RNA sequencing kit from Revvity was the one that performed best.

- Dr. Sören Franzenburg, Head of NGS platform, Kiel University 

Gel-free small RNA library prep with reduced bias

The NEXTFLEX Small RNA-Seq Kit v4 offers a completely gel-free library prep workflow for Illumina^® and Element^® sequencers. Optimized 3′ and 5′ adapter-ligation chemistry, together with proprietary dimer-reduction oligonucleotides, minimizes adapter-dimer formation and sequence-dependent ligation bias without the need for PAGE cleanup.

The six-hour protocol reliably produces high-complexity libraries from as little as 1 ng of total RNA derived from plasma, serum, urine, CSF, cultured cells or tissue lysates. Internal benchmarking shows higher on-target mapping rates and a larger repertoire of unique miRNA species than standard small RNA workflows.

To further enhance sensitivity in biofluid studies, the kit includes tRNA and YRNA blocking oligos, reducing over-represented fragments and enriching true miRNA reads (Figures 1 & 2). 

Figure 1. NEXTFLEX Small RNA-Seq Kit v4 increases miRNA reads while reducing tRNA/YRNA carry-over. Libraries from 1 ng plasma RNA were sequenced and aligned to miRNA, tRNA, and YRNA reference sets. Compared with a competitor workflow, the NEXTFLEX Small RNA kit aligns with more miRNAs while, while minimizing reads associated with t/YRNA and RNA, improving effective sequencing depth for downstream small-RNA analysis.

Figure 2: NEXTFLEX Small RNA-Seq Kit v4 detects higher number of unique miRNA species across different sample types

Exosomal RNA (exoRNA) Library Prep for Liquid Biopsy miRNA Profiling

The gel-free NEXTFLEX Small RNA-Seq Kit v4 workflow is equally effective for exosomal RNA (exoRNA). Even when starting with the picogram-level miRNA inputs typical of exosome preparations, the protocol generates libraries with high mature-miRNA alignment rates and negligible adapter-dimer carry-over (Figure 3). This enables reliable profiling of miRNAs derived from extracellular vesicles for liquid-biopsy biomarker discovery and other low-input applications.

Improved miRNA discovery

Internal benchmarking shows that the NEXTFLEX Small RNA-Seq Kit v4 recovers up to 45% more unique miRNA species than a competitor small RNA workflow when starting with as little as 1 ng of total RNA (Figure 4). This comprehensive profiling is essential for understanding the full spectrum of miRNA expression in each sample enabling a more complete view of post-transcriptional regulation and strengthens downstream differential-expression and biomarker-discovery analyses. The kit incorporates a highly processive rRT enzyme able to read through some of the common covalent modifications appearing in miRNA.

Figure 3. NEXTFLEX Small RNA v4 identifies 45% more unique miRNA species than competitor workflows. Venn diagram of libraries prepared from 1 ng serum RNA shows 81 miRNAs detected with both kits, an additional 53 miRNAs unique to NEXTFLEX, and 11 unique to the comparator.

Ribosome profiling (Ribo-Seq) and nascent-RNA transcriptomics

The gel-free NEXTFLEX Small RNA-Seq Kit v4 readily converts ribosome-protected fragments (28–30 nt RPFs) and nascent transcripts into sequencing libraries for Ribo-Seq and nascent transcriptomics application. By adapting small RNA library preparation methods, researchers can quantify translational efficiency, map ribosome-stall sites, and reveal upstream open-reading frames, critical insights for gene-expression control, disease-mechanism studies, and drug-target discovery.

“We frequently use Revvity small RNA sequencing kits for Ribosome- and Disome-profiling. It offers a very streamlined library generation and allows for easy customization to adapt to experimental needs. With this, it consistently delivers good results. Additionally, the technical support from Revvity’s team is excellent and very helpful which further enhanced our very positive experience with this Revvity product.”

- Dr. Tobias Schmidt, CRUK Scotland Institute

Automation & 384-Plex Multiplexing

Validated scripts make the NEXTFLEX workflow easy to setup on Sciclone G3 NGSx / iQ and Zephyr G3 workstation. It is also readily adapted to other liquid handlers. Up to 384 Unique Dual Index (UDI) barcodes are available, enabling high-throughput multiplexing on Illumina® and Element® sequencers while minimizing index hopping.

Maximize miRNA mapped reads with NEXTFLEX blood miRNA blockers

Boost usable miRNA reads in blood and plasma with NEXTFLEX Blood miRNA Blockers. These oligos selectively deplete highly abundant miR-486-5p, miR-92a-3p, and miR-451a—species that can consume 50–70% of reads so more sequencing depth is devoted to biologically informative miRNAs.

We have been using Revvity small RNA sequencing kits for a number of years, and we are satisfied with the results and the support received.

- Dr. Marcel van Herwijnen, Researcher/Lab Manager, Maastricht University 
 

Boost Functional Insights with Dharmacon™ miRNA Modulators

Pair the NEXTFLEX® Small RNA-Seq Kit v4 with Dharmacon™ miRNA mimics and inhibitors to quickly elevate or silence target miRNAs, confirm their biological impact in any cell system, and move seamlessly from expression profiling to functional validation in a single streamlined workflow.

Profiling from extracellular vesicles

• Apostolov, A., Mladenović, D., Tilk, K. (2025) Multi-omics analysis of uterine fluid extracellular vesicles reveals a resemblance with endometrial tissue across the menstrual cycle: biological and translational insights, Human Reproduction Open, https://doi.org/10.1093/hropen/hoaf010.
• Marocco. F. (2025) The RNA binding protein PCBP2 is a regulator of miRNAs partition between cell and extracellular vesicles. [Doctoral dissertation, Sapienza University of Rome]. IRIS Uniroma1. https://iris.uniroma1.it/retrieve/6d5c8022-59e2-44ec-b8b6-eb7a6ea8cc6e/Tesi_dottorato_Marocco.pdf.
• Pan, Y., Meng, L., Yoshida, K., … & Asakawa, S. (2025). Comparative and functional analysis of exosomal microRNAs during semelparous reproduction in ayu fish. Journal of Extracellular Biology, 4(3), e70038. https://doi.org/10.1002/jex2.70038
• García-Concejo, A., Schwager, A., … & Blanco, B. (2025). Exosome-derived miRNAs distinguish septic shock from non-septic shock in postoperative patients. Journal of Translational Medicine, 23, XXX. (PMID: 40033446)

Biomarker discovery/validation

• Lill C. M., et al. (2025) EPIC4ND: European Prospective Investigation into Cancer and Nutrition follow-up for neurodegenerative diseases. medRxiv 2025.01.29.25321340; doi: https://doi.org/10.1101/2025.01.29.25321340.
• Chu, C. P., Nabity, M. B. (2025) Technical considerations and review of urinary microRNAs as biomarkers for chronic kidney disease in dogs and cats. Vet Clin Pathol. 2025; 00: 1-19. doi:10.1111/vcp.13412.
• Karere, G. M., Hsu, F.-C. Hepple, R. T. et al. (2025) MicroRNA signatures of VO2peak in older adult participants of the Study of Muscle, Mobility and Aging. bioRxiv 2025.01.08.631999; doi: https://doi.org/10.1101/2025.01.08.631999.
• Love, C. G., Coombs, L., & Van Laar, R. (2024). RNA-seq validation of microRNA expression signatures for precision melanoma diagnosis and prognostic stratification. BMC Medical Genomics, 17, 256. https://doi.org/10.1186/s12920-024-02028-w

Other

• Almeida, M.V., Blumer, M., Yuan, C.U. et al. Dynamic co-evolution of transposable elements and the piRNA pathway in African cichlid fishes. Genome Biol 26, 14 (2025). https://doi.org/10.1186/s13059-025-03475-z.
• Rossner, p., Javorkova, E., Sima, M. et al. Skin wound healing: the impact of treatment with antimicrobial nanoparticles and mesenchymal stem cells. (2025) Authorea. February 17, 2025. DOI: 10.22541/au.173977945.54128573/v1.
• Tahiri, I., Llana, S.R., Díaz-Castro, F. et al. AgRP neurons shape the sperm small RNA payload. Sci Rep 15, 7206 (2025). https://doi.org/10.1038/s41598-025-91391-4.

Ribo-Seq

• Ting MKY, Gao Y, Barahimipour R, et al. Optimization of ribosome profiling in plants including structural analysis of rRNA fragments. Plant Methods. 2024;20(1):143. doi: 10.1186/s13007-024-01267-3.
• Bohlen J, Fenzl K, Kramer G, Bukau B, Teleman AA. Selective 40S footprinting reveals cap-tethered ribosome scanning in human cells. Molecular Cell. 2020;79(4):561-574.e5. doi: 10.1016/j.molcel.2020.06.005.
• Hoerth K, Reitter S, Schott J. Normalized Ribo-Seq for quantifying absolute global and specific changes in translation. Bio-Protocol. 2022;12(4):e4323. doi: 10.21769/BioProtoc.4323