Comparative analysis and innovation of a simple and rapid method for high-quality RNA and DNA extraction of kiwifruit

Seyyed Fatemeh Fallah,Mansour Afshar-Mohammadian,Mohammad Hossein Rezadoost

doi:10.1016/j.mex.2018.03.008

Seyyed Fatemeh Fallah, Mansour Afshar-Mohammadian + Show 1 more

Open Access

https://doi.org/10.1016/j.mex.2018.03.008

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Journal: MethodsX	Publication Date: Jan 1, 2018
Citations: 4	License type: cc-by

Affiliation: University of Guilan

Abstract

RNA and DNA extraction is a requirement for the study of gene expression and has an increasingly important role in genetic studies of all fleshy fruits. RNA and DNA extraction is difficult in kiwifruit due to the significant amount of polysaccharides and polyphenols compounds. So far, no commercial kit has been developed specifically for high-quality RNA and DNA extraction in kiwifruit and the common protocols for RNA extraction have poor yields. This study developed a new protocol for high quality RNA extraction in Actinidia deliciosa. According to the results, the average yield of RNA extraction of fruit and leaf of A. deliciosa was ~2180.7?ng/µl (~545.175?µg/g?FW) and ~3424.9?ng/µl (~856.225?µg/g?FW), respectively with A260/A280 between 1.95 to 2.07 and A260/A230 higher than 2 indicating high RNA purity. While the averages yield of RNA extraction using previous methods from kiwifruit and leaf was 23?µg/g?FW and 527?µg/g?FW, respectively. Also, the average yields of genomic DNA from kiwifruit ranged from 52 to 98?ng/µl with A260/A230 between 0.60 to 1.64 and A260/A280 between 1.40 to 1.48. To our knowledge, this is the first report of a highly efficient and rapid method of RNA and DNA extraction in kiwifruit which can be used for a broad spectrum of the all fleshy fruits.

Highlights

Hayward kiwifruit samples from the cultivar of A. deliciosa with a mean soluble solid content of 6.1– 6.95% and firmness of 80–100 N were collected from the research center gardens close to the city of Tonekabon at the north of Iran, at early November 2016
The amplified products were separated on a 1% agarose/TBE gel electrophoresis and imagined after staining with Loading Dye ((Buffer) 6x Cinnagen [2,3]. Some protocols such as those described by Smart and Roden [1], Minguzzi et al [4], Ma et al [5], and Yockteng et al [6] which are used for RNA extraction from plant tissues had low yield because of the high amount of polyphenols, polysaccharides and other secondary metabolites in the plant tissues
The results showed that the average yields of total RNA from fruit and leaf of A. deliciosa

Summary

Method details

Hayward kiwifruit samples from the cultivar of A. deliciosa with a mean soluble solid content of 6.1– 6.95% and firmness of 80–100 N were collected from the research center gardens close to the city of Tonekabon at the north of Iran, at early November 2016. Reagents 2 M Sodium acetate (2 M sodium acetate preparation for RNA extraction: add 16.42 g sodium acetate (anhydrous) to 40 ml water and 35 ml glacial acetic acid. Adjust to a pH of 4 with glacial acetic acid and bring to a final volume of 100 ml with DEPC-treated water), 2 M LiCl, chloroform– isoamylalcohol (24:1, v/v), phenol-chloroform–isoamylalchol (25:24:1, v/v), 4 M NaCl, 2 M guanidinethiocyanate, polyvinylpolypyrrolidone (PVPP), isopropanol, 70% (v/v) ethanol (EtOH). 10 Add 300 ml DEPC-treated water, add 500 ml phenol-chloroform-isoamylalchol (25:24:1, v/v) and centrifuge at 13,000 rpm for 10 min at 4 C. 13 pellet should be dried and dissolved in 50 ml DEPC-treated water and stored at À80 C. 11 pellet was dried and dissolved in 50 ml nuclease-free water and stored at À80 C

Modified procedure

Findings

Method validation