RT Journal Article
T1 Protocol: low cost fast and efficient generation of molecular tools for small RNA analysis.
A1 López-Márquez, Diego
A1 Del-Espino, Ángel
A1 Rodríguez-Bejarano, Eduardo
A1 Beuzón-López, Carmen del Rosario
A1 Ruiz-Albert, Francisco Javier
K1 Genética vegetal
K1 Genómica
K1 Clonación molecular
K1 Acido ribonucleico
AB Small RNAs are sequence-dependent negative regulators of gene expression involved in many relevant plant processes such as development, genome stability, or stress response. Functional characterization of sRNAs in plants typically relies on the modification of the steady state levels of these molecules. State-of-the-art strategies to reduce plant sRNA levels include molecular tools such as Target Mimics, Short Tandem Target Mimic, or molecular SPONGES. Construction of these tools routinely involve many different molecular biology techniques, steps, and reagents rendering such processes expensive, time consuming, and difficult to implement, particularly high-throughput approaches. We have developed a vector and a cloning strategy that significantly reduces the number of steps requiredfor the generation of MIMs against any given small RNA (sRNA). Our pGREEN-based binary expression vector (pGREENDLM100) contains the IPS1 gene from A. thaliana bisected by a ccdB cassette itself flanked by restriction sites for a type IIS endonuclease. Using a single digestion plus a sticky-end ligation step, the ccdB cassette that functions as a negative (counter) selection system is replaced by a pair of 28 nt self-annealing primers that provide specificity against the selected target miRNA/siRNA. The method considerably reduces the number of steps and the time required to generate the construct, minimizes the errors derived from long-range PCRs, bypasses bottlenecks derived from subcloning steps, and eliminates the need for any additional cloning technics and reagents, overall saving time and reagents. Our streamlined system guarantees a low cost, fast and efficient cloning process that it can be easily implemented into high-throughput strategies, since the same digested plasmid can be used for any given sRNA. We believe this method represents a significant technical improvement on state-of-the-art methods to facilitate the characterization of functional aspects of sRNA biology.
PB Springer Nature
YR 2020
FD 2020-03-20
LK https://hdl.handle.net/10630/35707
UL https://hdl.handle.net/10630/35707
LA eng
NO López-Márquez, D., Del-Espino, Á., Bejarano, E.R. et al. Protocol: low cost fast and efficient generation of molecular tools for small RNA analysis. Plant Methods 16, 41 (2020). https://doi.org/10.1186/s13007-020-00581-w
NO Programa Operativo FEDER Andalucía (UMA18-FEDERJA-070)Predoctoral Fellowship from the Spanish Ministerio de Educación, Cultura y Deporte: (FPU14/04233) (FPU17/03520)Plan Propio Universidad de Málaga (UMA)Project Grant MINECO (RTI2018-095069-B-100)
DS RIUMA. Repositorio Institucional de la Universidad de Málaga
RD 20 ene 2026