Efficient and High-Quality RNA Isolation from Metabolite-Rich Tissues of Stevia rebaudiana, an Important Commercial Crop
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Abstract
Stevia rebaudiana, a perennial herb native to northeastern Paraguay, has gained immense attention globally over the recent decades due to the natural sweetness of its leaves. Like in most plants, this particular species contains high amount of secondary metabolites, thus rendering the isolation of high quality and quantity RNA extract for molecular applications rather challenging. An effective, high-yield and high-quality RNA isolation protocol for this economically important plant species was devised here based on the cetyltrimethylammonium bromide (CTAB) extraction method, with an additional genomic DNA (gDNA) removal step. DNA and other contaminants that may affect downstream applications were effectively removed. Our results exhibited that RNA samples isolated from the leaves and stems of Stevia rebaudiana using this improvised method are high in integrity and quality with RNA integrity number (RIN) of more than 8 and low in contaminants.
Kajian ini melaporkan biodiversiti strain bakteria selulosa termofilik yang terdapaStevia rebaudiana, satu tumbuhan herba saka yang berasal dari timur laut Paraguay, telah mendapat perhatian yang banyak untuk beberapa dekad kebelakangan ini kerana kemanisan semula jadi daun-daunnya. Seperti kebanyakan tumbuhan, spesies ini mengandungi kandungan metabolit sekunder yang tinggi, dengan itu menyebabkan pengasingan RNA yang berkualiti dan berkuantiti tinggi untuk aplikasi-aplikasi hiliran menjadi agak mencabar. Sebuah protokol pengasingan RNA yang efektif, dapat memberikan hasil yang banyak dan berkualiti tinggi telah dirumuskan berdasarkan kaedah pengekstrakan setiltrimetilamonium bromida (CTAB), dengan penambahan langkah pembuangan DNA genom (gDNA). DNA dan bahan kontaminasi lain yang berkemungkinan mempengaruhi aplikasi hiliran juga disingkirkan secara efektif. Hasil kajian ini menunjukkan bahawa sampelsampel RNA yang diasingkan daripada daun-daun dan batang-batang Stevia rebaudiana menggunakan kaedah yang direka ubah ini adalah dalam kualiti dan integriti yang tinggi dengan nombor integriti RNA (RIN) yang melebihi 8 dan tahap pencemaran yang rendah.
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