Small RNA Sequencing: Introduction, Workflow, and Applications

Introduction to Small RNA Sequencing

Gene silencing and post-transcriptional gene regulation are both aided by small non-coding RNAs. Small RNA sequencing (RNA-Seq) is a method for isolating and sequencing small RNA species such as microRNAs (miRNAs). With unparalleled sensitivity and versatile range, Small RNA-Seq can analyze dozens of small RNA especially miRNA sequences.

Small RNA-Seq can be used to discover new miRNAs and other small non-coding RNAs, as well as evaluate the expression of all small RNAs in a given sample. It is feasible to classify variations such as isomiRs with single-base resolution without prior sequence or secondary structure information, as well as assess any small RNA or miRNA.

Advantages of Small RNA Sequencing

miRNA sequencing libraries derived directly from total RNA aid in understanding the role of non-coding RNA and how post-transcriptional regulation contributes to phenotype. It can also be used to find new biomarkers and collect the full range of small RNA and miRNA species.

Small RNA sequencing can also be used to (1) determine miRNA and other small RNA expression profiles, (2) learn more about how cells are enforced or misregulated under pathological circumstances, (3) small RNA clustering, (4) novel small RNA discovery, (5) small RNA prediction, and (6) differential expression of all small RNAs in any specimen.

Workflow for Small RNA Sequencing

Small RNA Sequencing: Introduction, Workflow, and ApplicationsFigure 1. Workflow for sequencing and analysis of miRNA changes. (Nassirpour, 2014)

Purification: This phase is necessary and essential for any molecular-based technique because it ensures that the small RNA fragments found in the samples to be analyzed are pure and of good quality. Depending on the goals of the experiment, different purification methods can be used: spin column chromatography and acid guanidinium thiocyanate-phenol-chloroform extraction It's critical to quantify small RNAs and assess the purification quality once they've been isolated.

Library Preparation and Amplification: Many NGS sequencing protocols require the creation of a genomic library containing thousands of fragments of the target nucleic acids, which are then sequenced using appropriate technology. Retrotranscription happens after the adapters are ligated to both ends of the small RNAs, producing complementary DNA molecules (cDNAs) that will be amplified using various amplification techniques depending on the sequencing protocol in order to obtain billions of amplicons to be sequenced. Masking oligonucleotides targeting 5.8s rRNA are incorporated into the regular PCR mix to enhance sensitivity to small RNA targets and strengthen amplification results.

Sequencing: RNA-seq can be done in a variety of ways, depending on the purpose of the analysis:

(1) Ion Torrent sequencing: NGS technology is based on the use of a semiconductor chip with a sample incorporated with an ion-sensitive field-effect transistor that can pinpoint pH changes due to the release of one or more protons after the incorporation of one or more dNTPs during synthesis sequencing.
(2) Illumina sequencing: It is the most frequently used approach for small RNA sequencing and offers a good method.

Data Analysis and Storage: The last step is to analyze and store the data: UMI and index sequences are automatically removed from sequencing reads, and PHRED analyzes their quality. Reads can then be mapped or aligned to a reference genome to extract information about their similarity: Reads with the same length, sequence, and UMI are eliminated as equals from the hit list. The number of different UMIs for a given small RNA sequence does, in fact, reflect the number of copies. Finally, the small RNAs are quantified by matching molecules with transcript annotations from various databases.


  1. Srinivasan S, Yeri A, Cheah PS, et al. Small RNA sequencing across diverse biofluids identifies optimal methods for exRNA isolation. Cell. 2019 Apr 4;177(2).
  2. Nassirpour R, Mathur S, Gosink MM, et al. Identification of tubular injury microRNA biomarkers in urine: comparison of next-generation sequencing and qPCR-based profiling platforms. BMC genomics. 2014 Dec;15(1).
  3. t Hoen PA, Friedlander MR, Almlof J, et al. Reproducibility of high-throughput mRNA and small RNA sequencing across laboratories. Nat Biotechnol. 2013;(11).
* For Research Use Only. Not for use in diagnostic procedures.


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