NEXTFLEX Small RNA Sequencing Kit V4

• 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.

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

Listen to the Experts:

Listen to our webinar, "Exploring Circulating Extracellular Vesicles in Liquid Biopsies for Neurological Disorders," where Bessie Meechoovet, a Senior Research Associate from the Jensen lab at the Translational Genomics Research Institute (TGen), explains how cell-free biomarkers are used to enhance our understanding of neurodegenerative diseases.

The Jensen lab utilizes long-read single-cell and RNA transcriptomics data to identify minimally invasive biomarkers in biofluids. Ms. Meechoovet describes a method they've developed to enrich brain extracellular vesicles, aiding in the discovery of potential biomarkers.

FAQ

Q. What types of RNAs are included in the Small RNA library?

The NEXTFLEX Small RNA Sequencing kit v4 is designed to capture short RNAs (17-65 nucleotides) with 5´ monophosphorylated and 3´ hydroxylated ends. The kit also allows for the capture of larger molecules (<200 bp) by using the no size selection protocol. MicroRNAs typically have a 5’ monophosphate group after cleavage by the enzyme Dicer. Other molecules, such as tRNA and yRNA, also have a 5’ monophosphate at their end and they will be incorporated in the libraries if present in the sample, such as serum and plasma. The kit includes blocking oligos to prevent unwanted tRNAs and yRNA from being incorporated into the library. For removal of other types of RNA, please contact our technical support team.

Q. Do you have any recommendations for extraction of RNA for Small RNA sequencing?

Any extraction method that generates a high-purity RNA preparation while preserving the fraction of RNA molecules with size <200 nt is compatible with small RNA sequencing. Popular extractions method used together with our NEXTFLEX® Small RNA Sequencing kit v4 include, but are not limited to, chemagic™ miRNA kits (Revvity), miRNeasy Kits (QIAGEN), MagMAX™ mirVana™ (Thermofisher) and MicroRNA Purification kit (Norgen Biotek).

Q. Are the libraries produced by NEXTFLEX Small RNA Sequencing Kit directional?

Yes, the NEXTFLEX Small RNA libraries are directional. During the library preparation 5’ and 3’ adapters are ligated to the RNA molecule. This ensures that the resulting cDNA retains the original RNA strand’s orientation.

Q. Can I use total RNA, or do I have to isolate or enrich the sample for small RNA?

Small RNA libraries can be prepared using total RNA. Enriched small RNA samples can be used but are not necessary. Total RNA with a RIN (RNA Integrity Number) higher than 7 is recommended.

Q. What happens if the quality of my RNA is very low?

The NEXTFLEX Small RNA v4 is a gel-free workflow designed to remove adapter dimers (libraries with no insert). However, if the input sample is heavily degraded, it will contain a significant proportion of fragments with size <16 nt. Those fragments will appear in the final library preparation and will decrease the percentage of reads mapping small RNA. To improve mapping rate there are two options: one option is to perform gel electrophoresis to separate the band of intact small RNA from other fragments, followed by gel band excision and purification. The second option is to use an upstream kit to remove degraded RNAs before entering library preparation. Please contact Revvity technical support team to discuss if this is needed and the best approach for your project.

Q. How should my NEXTFLEX Small RNA libraries be trimmed?

Please download the trimming instructions provided.

Q. What should I do if my Small RNA may not have 5´ Phosphate and 3´ OH groups?

Please contact Revvity technical support for recommendations on treatment of the RNA using T4 Polynucleotide Kinase (T4 PNK).

Q. How are the barcodes introduced in the NEXTFLEX Small RNA libraries?

Two unique 8 bp indices are introduced during the PCR step (Step F).

Q. What are the recommended settings for sequencing?

Due to the size of small RNAs, a read of 50 bp is sufficient to cover the entire insert and includes enough of the adapter to be identified and trimmed during data analysis. Single or paired-ended is possible, although paired-end generally does not add additional information. Care should be taken to ensure the number of cycles does not exceed the total length of the library molecule (insert + adapter). This will cause low quality sequencing cycles which can result in lower quality data or no data generation.

Q. What is the number of reads required for small RNA sequencing?

The number of reads required for small RNA sequencing typically ranges from 1 to 5 million reads per sample. This range can vary depending on the specific goals of your experiment, for example differential gene expression or microRNA discovery typically require greater numbers of reads. In addition, the complexity of the input tissue and amount of starting microRNAs (or other target RNAs) may require higher number of sequencing reads. If in doubt, please contact Revvity technical support team for additional information on your particular project.

Q. Do I have to use custom Illumina® sequencing primers for the libraries generated with NEXTFLEX Small RNA Sequencing Kit v4?

No. NEXTFLEX Small RNA v4 libraries are compatible with Illumina sequencing reagents, therefore designing custom sequencing primers is not necessary.

Q. What do I need to do to run the NEXTFLEX Small RNA libraries on the AVITI™ sequencer from Element® Biosciences?

NEXTFLEX Small RNA v4 libraries can run native on Element Cloudbreak™ Freestyle Sequencing Kits, without any extra step.

Q. Are the Monarch™ Spin RNA Cleanup Kits (NEB) compatible with NEXTFLEX reagents for RNA library prep?

Yes. RNA purified with this kit can be used for small RNA library preparation using NEXTFLEX Small RNA Sequencing Kit v4. This kit has a 15 nt cut off, which means that eluates from this kit will increase significantly the proportion of mapped reads for small RNA and is a convenient option to improve the percentage of small RNA mapped reads in very degraded samples.

Q. Do you have recommendations for analysis of small RNA sequencing data?

General recommendations for analysis of small RNA data are provided in our blog “Navigating the complexities of miRNA sequencing data analysis”. For specific questions, or if you are new to this field, please contact our technical support team.