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Abstract Translation of mRNA into functional proteins is a fundamental process underlying many aspects of plant growth and development. Yet, the role of translational regulation in plants across diverse tissue types, including seeds, is not well known due to the lack of methods targeting these processes. Studying the seed translatome could unveil seed‐specific regulatory mechanisms, offering valuable insights for breeding efforts to enhance seed traits. Polysome profiling is a widely used technique for studying mRNAs being translated. However, the traditional method is time‐consuming and has a low polysome recovery rate; therefore, it requires substantial starting material. This is particularly challenging for species or mutants with limited seed quantities. Additionally, seed polysome fractions often yield low quality RNA due to the abundance of various compounds that interfere with conventional RNA extraction protocols. Here we present a robust polysome extraction method incorporating a size‐exclusion step for polysome concentration streamlined with a rapid RNA extraction method optimized for seeds. This protocol works across multiple plant species and offers increased speed and robustness, requiring less than half the amount of seed tissue and time compared to conventional methods while ensuring high polysome recovery and yield of high‐quality RNA for downstream experiments. These features make this protocol an ideal tool for studying seed translation efficiency and hold broad applicability across various plant species and tissues. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Robust polysome extraction for seeds Basic Protocol 2: Rapid fraction total RNA extraction
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An efficient and effective RNA extraction protocol for ferns
Abstract PremiseThe extraction of high‐quality RNA is the critical first step for the analysis of gene expression and gene space. This remains particularly challenging in plants, and especially in ferns, where the disruption of the cell wall and separation of organic compounds from nucleic acids is not trivial. MethodsWe developed a cetyltrimethylammonium bromide (CTAB)‐based RNA extraction protocol that consistently performs well across a large phylogenetic breadth of ferns—a lineage of plants high in secondary compounds—and in an array of tissue types. Two alternative options (precipitation vs. clean‐up without intermediate precipitation) are presented, both of which yield high‐quality RNA extracts with optical density (OD) ratios of OD 260/280 = 1.9–2.1 and OD 260/230 > 1.6, and RNA integrity numbers >7. ConclusionsThis study presents an efficient protocol for the extraction of high‐quality RNA from multiple tissues and across the fern phylogeny, a clade of plants that still lags behind other major lineages in the development of genomic resources. We hope that this method can be used to help facilitate the closing of this gap.
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- Award ID(s):
- 2310485
- PAR ID:
- 10623865
- Publisher / Repository:
- Applications in Plant Sciences
- Date Published:
- Journal Name:
- Applications in Plant Sciences
- Volume:
- 12
- Issue:
- 6
- ISSN:
- 2168-0450
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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