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Comparison of three different genomic RNA extraction methods from peel and pulp of Thomson navel orange fruit (Citrus sinensis L.)

Document Type : Research Paper - Plant Breeding

Authors

1 PhD Student, Department of Horticultural Science and Engineering, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran.

2 Associate Professor, Department of Horticultural Science and Landscape Engineering, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran

3 Associate Professor, Department of Plant Molecular Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

4 Assistant Professor, Department of Horticulture Crops Research, Mazandaran Agricultural and Natural Resources Research and Education Center, AREEO, Sari, Iran.

5 Associate Professor, Department of Horticultural Science and Landscape Engineering, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran.

6 Assistant Professor, Department of Plant Breeding & Biotechnology, Faculty of Plant Production, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Golestan, Iran.

Abstract

Introduction
Efficient genomic RNA extraction is a basic requirement of molecular biology and the primary step in the study of gene expression. Woody plants are problematic due to the presence of polyphenolics, polysaccharides and other compounds that bind or co-precipitate with the RNA. The present study is to compare different methods of extracting high-quality total RNA in Thomson navel orange. Citrus is one of the most strategic fruits of Iran, finding an efficient and low-cost method is important for executive and molecular works.
 
Materials and Methods
Plant materials used to extract total RNA were collected from two different parts of Thomson navel orange peel (consisting of both flavedo and albedo) and pulp from Ghaemshahr Horticultural Research Station, Mazandaran in 2020. Fruits were frozen in liquid nitrogen after sampling and were stored at -80°C until used. These samples were transferred to the laboratory department of horticultural science and engineering, Gorgan where the samples were applied to analyze the molecular expriment. For this study three methods including of: Trizol, Non-column Denazist and RNX-PlusTM were evaluated for total RNA extraction. In order to show the quality and quantity of RNA, were assessed on 1% agarose gel and using a nano­drop spectrophotometer with 230, 260 and 280 wavelength. Results were analyzed using a completely randomized design with three replicates, Duncan test with a significance level of 0.05 using SAS 9.0 software.
 
Results and Discussion
The result showed a distinct 28S and 18S rRNA bands visible on an agarose gel. The RNA integrity was assessed by the sharpness of ribosomal RNA bands. Also by using this protocols, the absorbance ratio at 260/280 was ranged between 1.80 – 1.93 which indicated there were no protein in the extracted RNA. A ratio A260/ A280 ratio from 2.0 to 2.14 indicated a lack of polysaccharides and phenol contamination. Trizol method, which was significantly different than compared to other methods. This protocol was successfully applied to isolate total RNA from the organ of two section fruit. Generally, the samples with a ratio in the range of ~ 1.9 to 2 revealed high purity of the extracted RNA using Trizol and the average concentration of RNA ranged from 379.90 (ng/µl) of orange peel and 352.15 (ng/µl) of orange pulp, respectively.  And concentration was observed in Non-column Denazist method which treated with the lowest concentration (263.10 ng/µl) of orange pulp.
 
Conclusion
The Trizol method was the best choice for extraction of high-quality total RNA. In this respect method represented the highest RNA yield and purity from various samples (peel and pulp) of citrus. However, generally peel samples (flavedo and albedo) produced high concentration total RNA.

Keywords

Main Subjects

References
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Plant Productions
Volume 46, Issue 1
June 2023
Pages 1-10
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