Optimized Small RNA Library Prep for Service Facilities

Exploring the use of circulating microRNAs as biomarkers for various pathogenic conditions is a rapidly expanding area of clinical
research. Small RNA library prep requires more attention to detail than most standard sequencing
applications. Labs may typically experience the following issues:

  • Inherent introduction of bias in the chemistry, which reduces data accuracy
  • Cumbersome protocol, which prevents it from being easy to use
  • Significant number of handling steps, which makes scaling up throughput difficult
  • Lack of multiplexing options, which increases sequencing cost

PerkinElmer has eliminated these problems, delivering a practical solution which simplifies miRNA library preparation workflows.


“The NEXTFLEX® Small RNA-Seq kit v3 “consistently performed well with respect to enrichment of miRNA mapping reads in biofluids and tissues” and “exhibited a relatively low quantification bias and have the capacity to be automated for high-throughput miRNA library preparation”

Coenen-Stass, A.M.L., et al. (2018) Evaluation of methodologies for microRNA biomarker detection by next generation sequencing. RNA Biology.,, 15:8, 1133-1145.

“The high reproducibility rate is one of the reasons why we chose protocol Z (NEXTFLEX® Small RNA-Seq Kit v3) for our small RNA sequencing service.”

CeGaT, global service provider, Small RNA Sequencing Tech Note

Adaptable & Scalable Solutions for Varying Throughput

The NEXTFLEX® Small RNA-Seq kit v4 uses patented and patent-pending technology to provide a reduced-bias small RNA library preparation solution for Illumina® sequencing platforms. Our approach to reducing ligation-associated bias involves the use of adapters with randomized bases at the ligation junctions, resulting in greatly decreased bias in comparison to standard protocols. This reduction in bias results in data which more accurately represents abundances of small RNAs in the starting material. In addition, reduction of bias allows more miRNAs to be detected with fewer total reads, increasing efficiency and reducing cost for small RNA sequencing.

Sequencing Depth Versus miRNAs Detected

Small RNA libraries were generated in triplicate from the indicated amount of human brain total RNA and sequenced on an Illumina® MiSeq® sequencer. The indicated number of reads were sampled from each library, and the average number of miRNA groups with ≥20 reads were determined.

“In summary, library preparation with NEXTFLEX® Small RNA-Seq Kit v3 (Bioo Scientific) leads to reproducible results even with very limited amounts of sample. Depending on the batch size, there are different flow cells available for small RNA sequencing on NovaSeq 6000.”

CeGaT, global service provider, Small RNA Sequencing Tech Note

See What Others are Saying About the NEXTFLEX® Chemistry

Six independent, peer-reviewed studies were published in 2018 and 2019 comparing small RNA sequencing data obtained using commercially available small RNA-seq library prep kits in human samples. The NEXTFLEX® Small RNA-Seq kit was recommended in every study because of the low bias and consistent results obtained using the kit.

Coenen-Stass, A.M.L., et al. (2018) Evaluation of methodologies for microRNA biomarker detection by next generation sequencing. RNA Biology. 15: 8. 15:8, 1133-1145. doi: 10.1080/15476286.2018.1514236.

Dard-Dascot, C., et al. (2018) Systematic comparison of small RNA library preparation protocols for next-generation sequencing. BMC Genomics 19(118), doi:10.1186/s12864-018-4491-6.

Giraldez, M. D., Spengler, R. M., Etheridge, A., Godoy, P. M., Barczak, A. J., Srinivasan, S., . . . Tewari, M. (2018). Comprehensive multi-center assessment of small RNA-seq methods for quantitative miRNA profiling. Nature Biotechnology. doi:10.1038/nbt.4183.

Ku, A., Ravi, N., Yang, M., Evander, M., Laurell, T., Lilja, H., & Ceder, Y. (2019). A urinary extracellular vesicle microRNA biomarker discovery pipeline; from automated extracellular vesicle enrichment by acoustic trapping to microRNA sequencing. Plos One, 14(10). doi: 10.1371/ journal.pone.0224604.

Wright, C., Rajpurohit, A., Burke, E. E., Williams, C., Collado-Torres, L., Kimos, M., . . . Shin, J. H. (2018). Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods. bioRxiv 445437. doi:10.1101/445437.

Yeri, A., et al. (2018) Evaluation of commercially available small RNASeq library preparation kits using low input RNA. BMC Genomics 201819:331. doi: 10.1186/s12864-018-4726-6.

Read why CeGaT, a CLIA certified and CAP accredited sequencing service provider, chose the NEXTFLEX® Small RNA-seq kit v3 for their miRNA library prep.

Small RNA-seq on the Illumina® NovaSeq® Platform

The NEXTFLEX® Small RNA-Seq kit v4 is supplied with up to 384 Unique Dual Index barcoded primers (UDIs) and is optimized to take advantage of the Illumina® NovaSeq® sequencer.

Scalable & Easy-to-use

Weary of limited lab resources for the anticipated number of samples in need of processing or the introduction of potential human errors for an important clinical research project or collaboration? We offer a fully walk-away automated protocol on the Sciclone® G3 NGSx iQ workstation.

Sciclone G3 NGS iQ Workstation

Maximize Reads Mapped to miRNAs

NEXTFLEX® tRNA/YRNA blockers seamlessly integrate into the library prep to block the formation of tRNA and YRNA products increasing the relative proportion of reads mapped to miRNA. This is particularly helpful for miRNA discovery from complex samples such as biofluids, which can have a high abundance of tRNA/YRNA fragments.

Potential Applications for Small RNAs

As more and more investigators are convinced of the utility of miRNAs as biomarkers for various complex disorders, labs can anticipate a growing demand for a reliable service partner. Don’t delay because the time is now to differentiate your labs’ capabilities to actively facilitate the progress of clinical research. The NEXTFLEX® Small RNA-Seq kit v3 has been used in various applications, not limited to the following:

Prieto-Fernández E, Aransay AM, Royo F, et al. (2019) A Comprehensive Study of Vesicular and Non-Vesicular miRNAs from a Volume of Cerebrospinal Fluid Compatible with Clinical Practice. Theranostics. 9(16):4567–4579. doi:10.7150/thno.31502

Zaragoza C, Saura M, Hernández I, et al. (2019) Differential expression of circulating miRNAs as a novel tool to assess BAG3-associated familial dilated cardiomyopathy. Biosci Rep. 39(3):BSR20180934. doi:10.1042/BSR20180934

Zhang J, Zhang Y, Shen W, Fu R, Ding Z, Zhen Y, Wan Y. (2019) Cytological effects of honokiol treatment and its potential mechanism of action in non-small cell lung cancer. Biomedicine & Pharmacotherapy. 9(117): 109058.

Kim K, Yoo DA, Lee HS, Lee KJ, Park SB, Kim C, Jo JH, Jung DE, Song SY. (2019) Identification of potential biomarkers for diagnosis of pancreatic and biliary tract cancers by sequencing of serum microRNAs. BMC Medical Genomics. 12:62.

Wei J, Blenkiron C, Tsai P, James JL, Chen QI, Stone PR, Chamley LW. (2017) Placental trophoblast debris mediated feto-maternal signaling via small RNA delivery: implications for preeclampsia. Scientific Reports. 7:14681.

Hicks SD, Johnson J, Carney MC, Bramley H, Olympia RP, Loeffert AC, Thomas NJ. (2019) Overlapping MicroRNA Expression in Saliva and Cerebrospinal Fluid Accurately Identifies Pediatric Traumatic Brain Injury. Journal of Neurotrauma 35:1.

For research use only. Not for use in diagnostic procedures.