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Optimization of RNA extraction from pecan (Carya illinoinensis) leaves with high phenolic content

agricultural-science
molecular-biology
plant-science

Abstract

Efficient RNA extraction from pecan (Carya illinoinensis) leaves is challenging due to high polyphenol content, which compromises the RNA yield and quality. In this study, we evaluated nine commercially available RNA extraction kits and tested modifications to three selected protocols to optimize RNA isolation from pecan leaves. We also used Satsuma mandarin (Citrus unshu) leaf tissues as a control. The commonly used kits from Qiagen, Thermo Scientific, Macherey-Nagel, and Omega did not yield high-quality RNA from Pecan leaves, whereas Tiangen and Vazyme kits performed better. The Trizol method produced the highest RNA yield in young pecan leaves, but with low purity. Incorporating polyvinylpyrrolidone and β-mercaptoethanol into the Trizol protocol significantly improved RNA purity with consistent total yield. Similar improvements were achieved with the Quick-RNA Miniprep Kit after modification. The optimized methods consistently extracted high-quality RNA from both young and mature pecan leaves. Downstream validation through cDNA synthesis and quantitative real time PCR (qRT-PCR) analysis confirmed the suitability of the extracted RNA for gene expression studies. We developed a reliable RNA extraction protocol for pecan leaves, which will facilitate a broad range of molecular studies, including qRT-PCR, microarray analysis, cDNA library preparation, and transcriptomic studies using next-generation sequencing in polyphenol-rich pecan.
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