Small RNA Library Preparation

Overview of Small RNA and Small RNA Library Preparation

Small RNA molecules, which typically range in size from 20 to 30 nucleotides, play a vital role in the regulation of genes across a wide range of organisms. These molecules participate in various biological processes, including development, cellular homeostasis, and response to external stimuli. By modulating gene expression at the post-transcriptional level, small RNAs act as crucial regulators through mechanisms such as RNA interference (RNAi) and microRNA (miRNA)-mediated gene silencing. To fully understand the complexities of gene regulation, it is imperative to comprehend the functions and mechanisms of small RNAs.

CD Genomics offers Small RNA Sequencing for small RNA profiling and discovery, such as microRNAs (miRNAs), siRNAs, piRNAs, etc. Refer to our RNA Sequencing Sample Submission and Preparation Guidelines for your next small RNA sequencing.

The preparation of a small RNA library is a foundational step in small RNA research that facilitates comprehensive profiling and analysis of small RNA populations within a given sample. This process involves the transformation of small RNA molecules into a library consisting of fragments that are ready for high-throughput sequencing. Small RNA libraries offer valuable insights into the expression patterns, diversity, and functional roles of small RNAs. By examining small RNA populations on a global scale, researchers can discover novel small RNA species, detect isoforms, explore differential expression patterns, and gain a deeper understanding of their regulatory functions.

Guideline to Small RNA Library Preparation

Small RNA library preparation involves a series of intricate steps aimed at extracting and enriching specific populations of small RNA molecules from the larger pool of total RNA. This process encompasses meticulous isolation techniques, size selection procedures, adapter ligation strategies, reverse transcription, library amplification, and ultimate sequencing using high-throughput platforms. The following sections will delve into the various aspects of small RNA library preparation, including RNA extraction methods, size selection techniques, traditional adapter ligation-based methods, and recent advancements in this field.

Schematic of protocol to prepare miRNA libraries for sequencing.Schematic of protocol to prepare miRNA libraries for sequencing. (Belair et al., 2019)

RNA Extraction Methods for Small RNA Isolation

The efficient and reliable isolation of small RNAs holds paramount importance in generating high-quality libraries. Numerous methods have been devised for small RNA extraction, each offering distinct advantages and considerations depending on the sample type, quantity, and downstream applications. Organic extraction methods utilize organic solvents to isolate small RNAs, while column-based purification techniques exploit the affinity of small RNAs to specialized matrices for selective isolation. Additionally, precipitation-based techniques utilize various precipitants to separate small RNAs from the bulk of total RNA. The selection of an appropriate method must be carefully determined based on the specific requirements of the experiment.

Size Selection Techniques for Small RNA Enrichment

Size selection represents a critical step in small RNA library preparation as it aims to enrich the desired population of small RNAs while eliminating contaminants. Several techniques are commonly employed for size selection, including gel electrophoresis, size-exclusion chromatography, and the utilization of commercially available kits. Gel electrophoresis allows for precise separation based on size, while size-exclusion chromatography employs specialized columns to selectively retain small RNAs of interest. Commercially available kits offer convenience and efficiency by providing pre-designed reagents and protocols. The choice of size selection method depends on the desired size range of small RNAs and the sensitivity required for downstream applications.

  • Adapter ligation-based methods
    Traditional adapter ligation-based methods, such as the small RNA-Seq library preparation protocol, have long been utilized in small RNA library preparation. In these methods, RNA adapters are ligated to the ends of small RNA molecules, allowing subsequent reverse transcription into complementary DNA (cDNA). The resulting cDNA serves as a template for library amplification, enabling downstream sequencing of the targeted small RNA population.
  • Modifications and improvements in adapter ligation-based methods
    To overcome limitations associated with traditional adapter ligation-based methods, researchers have introduced various modifications and improvements. One such enhancement involves the use of chemically modified adapters, which minimizes ligation biases and enhances the overall accuracy of library preparation. Another notable advancement is the incorporation of unique molecular identifiers (UMIs), short DNA sequences unique to each individual RNA molecule, which serve to mitigate the biases introduced during PCR amplification. Additionally, strand-specific library preparation protocols have been developed, enabling the identification of the RNA strand from which the small RNA originated.
  • PCR-based methods
    PCR-based methods for small RNA library preparation involve the utilization of PCR amplification to generate an ample quantity of material suitable for sequencing. This technique entails the introduction of specific primers during the reverse transcription process, enabling subsequent amplification of cDNA fragments containing small RNA sequences.
    Incorporating PCR amplification strategies, such as barcoding and multiplexing, allows for the simultaneous handling and sequencing of multiple samples. This approach enhances throughput and cost-efficiency by facilitating the pooling of libraries. However, it is crucial to exercise caution to prevent the occurrence of PCR biases and cross-contamination between samples.
  • Template-switching methods
    Template-switching methods facilitate the synthesis of cDNA libraries by incorporating an artificial template-switching oligonucleotide during reverse transcription. This oligonucleotide serves as a template for the addition of a universal primer-binding site, allowing subsequent PCR amplification and sequencing.
    Template-switching methods offer advantages such as increased sensitivity, reduced bias, and the ability to capture a broader range of small RNA species. However, these methods may be prone to the generation of chimeric sequences and require careful optimization to minimize technical artifacts.

Quality Control and Validation of Small RNA Libraries

Assessment of Library Quality Using Bioanalyzer or qPCR

The initial step in quality control is the assessment of small RNA library quality. This involves evaluating the integrity and purity of the library to ensure that it meets the required standards. Two commonly employed techniques for library quality assessment are the Bioanalyzer and quantitative polymerase chain reaction (qPCR).

  • Bioanalyzer: The Bioanalyzer system utilizes microfluidic electrophoresis to analyze RNA samples, providing information about their size distribution and integrity. It generates electropherograms that display the peaks corresponding to the different RNA species present in the library. By analyzing the peaks, researchers can identify any anomalies such as degradation, adapter dimers, or contamination, which may affect downstream analyses.
  • qPCR: Quantitative PCR is a sensitive and accurate technique used to measure the abundance of specific RNA sequences. It can be utilized to assess the library quality by quantifying the presence of specific small RNA molecules or adapter sequences. By comparing the amplification curves and cycle threshold values, researchers can gauge the efficiency of library preparation and identify potential issues such as poor reverse transcription or amplification biases.

Size Distribution Analysis of Small RNA Libraries

Size distribution analysis provides crucial information about the enrichment and representation of specific small RNA populations within the library. It helps identify potential biases, assess the success of size selection, and verify the presence of specific small RNA species or isoforms.

The size distribution of small RNA molecules in the library is an important factor to consider, as it influences the efficiency of sequencing and downstream analyses. Deviations from the desired size range can indicate technical artifacts or biases that may affect the interpretation of results. Several methods are employed to assess the size distribution of small RNA libraries, including gel electrophoresis, capillary electrophoresis, and next-generation sequencing.

  • Gel Electrophoresis: Traditional gel electrophoresis provides a visual representation of the size distribution of small RNA molecules. By comparing the library bands with molecular weight markers, researchers can determine the presence of desired small RNA species and identify any unexpected fragments or contaminants.
  • Capillary Electrophoresis: Capillary electrophoresis is a high-resolution technique that enables precise size determination of small RNA molecules. It provides detailed information about the distribution of small RNA lengths within the library, ensuring the accuracy of downstream analyses.
  • Next-Generation Sequencing: While primarily used for sequencing, next-generation sequencing platforms also generate data regarding the size distribution of small RNA libraries. By examining the read length distribution, researchers can confirm the presence of the desired small RNA species and evaluate any biases or artifacts introduced during library preparation.

Quantification of Small RNA Libraries Using Fluorometric or qPCR-based Methods

Accurate quantification of small RNA libraries is crucial for normalizing sample inputs, pooling libraries for sequencing, and ensuring reproducible results across different experiments. Two commonly employed methods for quantifying small RNA libraries are fluorometric assays and qPCR-based methods.

  • Fluorometric Assays: Fluorometric assays, such as the Qubit system, utilize specific dyes that selectively bind to nucleic acids. By measuring the fluorescence emitted upon binding, researchers can quantify the total RNA concentration in the library. This method provides a rapid and sensitive means of assessing the library's nucleic acid content.
  • qPCR-Based Methods: Quantitative PCR can also be used to quantify small RNA libraries. It involves amplifying specific regions within the library, typically adapter sequences or small RNA targets, using qPCR primers. By comparing the amplification curves and cycle threshold values with known standards, researchers can determine the absolute or relative abundance of the small RNA library.

Validation of Library Diversity and Representation

Library diversity and representation are critical factors in small RNA sequencing experiments. It ensures that the library accurately represents the entire small RNA population and avoids biases introduced during library preparation. Several approaches can be employed to validate library diversity and representation.

  • Unique Molecular Identifiers (UMIs): UMIs are short DNA sequences incorporated during library preparation that uniquely tag each RNA molecule. By incorporating UMIs, researchers can assess the library's diversity by counting the number of unique tags present. A higher number of unique tags indicates a more diverse library. Access to our article Unique Molecular Identifiers (UMIs): Enhancing Accuracy and Precision in RNA Sequencing for more details.
  • Spike-in Controls: Incorporating spike-in RNA molecules with known sequences and concentrations into the library allows for the assessment of library representation. By comparing the observed abundance of spike-in controls with their expected values, researchers can determine if the library introduces biases or if specific small RNA species are underrepresented.
  • Sequencing Depth Analysis: Analyzing the sequencing depth, or the number of reads obtained for each small RNA species, provides insights into library representation. Uneven sequencing depths across different small RNA species may indicate biases introduced during library preparation.


  1. Belair, Cassandra D., et al. "High-throughput, efficient, and unbiased capture of small RNAs from low-input samples for sequencing." Scientific reports 9.1 (2019): 2262.
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