• Robustness across diverse sample types, from tissues to biofluids
• Streamlined workflow without gel cuts
• Fully automated - easy scalability from small to large studies
• Protocols available to select for small RNA species other than miRNA

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 sequencing kit v4 uses patent-pending technology to provide a completely gel-free small RNA library preparation solution for Illumina sequencing platforms. Our approach to reducing bias involves optimization of the ligation reaction and the use of dimer reduction oligonucleotides. These changes result in decreased bias in comparison to standard protocols.

NEXTFLEX Small RNA-Seq Kit v4 allows robust preparation of libraries from diverse sample types in approximately 6 hours, with as little input as 1 ng of total RNA. Libraries prepared with this kit have a higher proportion of mapping reads and unique miRNAs than leading competitors. The kit contains tRNA and YRNA blockers to efficiently remove these species in biofluid samples (Figure 1 and 2).

Figure 1: Gel-free miRNA library prep from various biofluids

Figure 2: Gel-free miRNA library prep from exosomes

This convenient gel-free workflow incorporated in the NEXTFLEX Small RNA-Seq Kit v4 enables the construction of libraries from exoRNA. Using this workflow, researchers can achieve a high number of reads aligning to mature miRNA and low adapter dimers, even with the very low miRNA inputs characteristic of exosome samples (Figure 3).

Figure 3: Average top 10 miRNA observed with exosomes from bone marrow and pooled serum samples.

Improved miRNA discovery

The performance of small RNA sequencing is heavily dependent on the workflow used. The NEXTFLEX® Small RNA Sequencing Kit v4 can identify a significantly higher number of unique miRNA species compared to other available solutions, even at low inputs (Figure 4). This comprehensive profiling is essential for understanding the full spectrum of miRNA expression in each sample. Detecting even the low-expressed miRNA present in the sample provides deeper insights into regulatory mechanisms and facilitates biomarker discovery.

Figure 4. Venn diagram showing that 45% more unique miRNA species were identified when using the NEXTFLEX^® Small RNA-Seq Kit v4 than Competitor A kit. Input was 1 ng of RNA extracted from serum.

Ribosome Profiling (Ribo-Seq) Application

The NEXTFLEX Small RNA-Seq v4 Kit can also be used for ribosome profiling (Ribo-Seq), which analyzes ribosome-protected mRNA fragments to study translation. By adapting small RNA library preparation methods, researchers can gain insight into gene regulation and translation dynamics, making Ribo-Seq valuable in fields like gene expression, disease mechanisms, and drug 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

Small RNA sequencing library prep optimized for automation

By eliminating all gel-based steps even for low input samples, the NEXTFLEX Small RNA-Seq Kit V4 is optimized for easy migration onto automated liquid handling platforms. This workflow is automated on the Sciclone G3 NGSx, Sciclone G3 NGSx iQ, and the Zephyr G3 NGS workstations

Illumina® and Element® small RNA sequencing multiplexing with UDI barcodes

Up to 384 UDI barcoded primers are included in each kit for multiplexing. These UDIs allow libraries to be run on any Illumina and Element Biosciences sequencing platform. These include the Illumina NextSeq® and Novaseq® sequencers and only require standard Illumina sequencing primers.

Maximize miRNA mapped reads with NEXTFLEX blood miRNA blockers

The NEXTFLEX blood miRNA blockers deplete common miRNA in blood and plasma such as miR-486-5p, miR-92a-3p and miR-451a. These constitute 50-70% of the small RNA present in those samples and usually are not of interest for researchers.