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Evaluation of commercially available small RNA methods for plant samples

ABSTRACT: Plant small RNAs are a diverse and complex set of molecules, ranging in length from 21 to 24 nt, involved in a wide range of essential biological processes. Nowadays, high-throughput sequencing is the most commonly used method for the discovery and quantification of small RNAs. However, it is known that several biases can occur during the preparation of small RNA libraries, especially using low input RNA. We used two types of plant biological samples to evaluate the performance of seven commercially available methods for small RNA library construction, using different RNA input amounts. We show that when working with plant material, library construction methods have differing capabilities to capture small RNAs, and that different library construction methods provide better results when applied to the detection of microRNAs, phased small RNAs, or tRNA-derived fragments.

ORGANISM(S): Zea mays

PROVIDER: GSE156973 | GEO | 2021/08/01

REPOSITORIES: GEO

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Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Amborella trichopoda leaves. The high depth of the resulting datasets enabled us to examine in detail critical small RNA features such as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study. A small RNA library was derived from leaves of Amborella trichopoda. The tissue represented a mixture of developmental stages, with a bias towards those from early, actively differentiating cells. Total RNA was isolated using the TriReagent (Molecular Research Center) and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA library was sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Doug Soltis and AndrÃ© Chanderbali for providing the plant material, as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.

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Evaluation of commercially available small RNA methods for plant samples

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