Isolation and Purification of High-quality RNA from Pistachio (Pistacia vera L.)
Malihe Rajabi
1
(Department of Horticulture, Damghan Branch, Islamic Azad University, Damghan, Iran)
Bahareh Kashefi
2
(Department of Horticulture, Damghan Branch, Islamic Azad University, Damghan, Iran)
Hossein Afshari
3
(Department of Horticulture, Damghan Branch, Islamic Azad University, Damghan, Iran)
Zarrin Taj Alipour
4
(Department of Soil Science, Damghan Branch, Islamic Azad University, Damghan, Iran)
الکلمات المفتاحية: Isolation, Mini kit, Pistachio, Real time, RNA, TRIzol,
ملخص المقالة :
Pistachios (Pistacia vera L.) are known as one of the nuts with high nutritional value, and an important commercial product worldwide. Pistachio is a tree that is compatible with dry climate regions and has the largest cultivated area in Iran. Pistachio yields have declined in recent years due to the rise in soil salinity caused by climate change (Mohit Rabari et al., 2023; Behzadi Rad et al., 2021). Nevertheless, the presence of phenolic compounds and other secondary metabolites may impede the isolation of high-quality RNA from pistachio leaves in numerous instances. In the present study, several methods were used to purify the total RNA of Shahpasand variety. These methods included Trizol kit; mini kit and modified method (TRIzol & mini kit) were used. The obtained results showed the superiority of modified method (TRIzol & kit) compared to TRIzol and Kit methods. In this method, the A260/A280 ratio of RNA sample was 2.12. In the next step, cDNA was synthesized and used to analyze SOS1 gene expression via Real Time PCR. Gene-specific amplification was confirmed by a single peak in the melt-curve analysis. Furthermore, Ct values showed that SOS1 gene extracted by the (TRIZOLE & Kit) was the most abundant transcript with a mean Ct value of 20 whereas in two other ways was the least abundantly transcribed. In conclusion, the results of this investigation demonstrated that the optimized extraction method is capable of isolating high-quality RNA from pistachios. The extracted RNA can be used for further molecular studies, such as real-time PCR and other downstream applications.
Amaranatha RV, Somayanda I, Doherty CJ, Jagadish Sk (2020) A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber. Scientific Reports. https://doi.org/10.1038/s41598-020-73958-5.
Arnold ME, Slomka MJ, Coward VJ, Mahmood S, Raleigh PJ, Brown IH (2013) Evaluation of the pooling of swabs for real-time PCR detection of low titre shedding of low pathogenicity avian influenza in turkeys. Epidemiology and Infection. 141, 1286-1297. https://doi.org/ 10.1017/ S0950268812001811.
Babaei L, Sharifani MM, Darvishzadeh R, Abbaspour N, Henareh M (2021) Impact of drought stress on photosynthetic response of some pear species. International Journal of Horticultural. Science and Technology. 8, 353-69. https://doi.org// 10.22059/ijhst. 2020.309629.394.
Baltasar Z, Verdonk JC (2022) RNA Extraction from Plant Tissue with Homemade Acid Guanidinium Thiocyanate Phenol Chloroform (AGPC). Current Protocols. 2, e351. doi: 10.1002/cpz1.351.
Behzadi Rad P, Roozban MR, Karimi S, Ghahremani R, Vahdati K (2021) Osmolyte accumulation and sodium compartmentation has a key role in salinity tolerance of pistachios rootstocks. Agriculture. 11(8), 708.
Chang E, Zhao Y, Wei Q, Shi S, Jiang Z (2016) Isolation of high-quality RNA from Platycladus orientalis and other
Cupressaceae plants. Electronic Journal of Biotechnology 23, 21–27. https://doi.org//10.1016/j.ejbt.2016.08.003
F Faostat (2016) FAO web page. http://www.fao.org/faostat.
Gangwar M, Alka Sh, Virendra K, Pradyot P (2021) Assessment of Successful qRT-PCR of SARS-CoV-2 Assay in Pool Screening Using Isopropyl Alcohol Purification Step in RNA Extraction. BioMed Research International. https://doi.org//10.1155/2021/6653950.
Hosseini N, Rezanejad F, Zamani Bahramabadi E (2022) Effects of Soil Texture, Irrigation Intervals, and Cultivar on some Nut Qualities and Different Types of Fruit Blankness in Pistachio (Pistacia vera L.). International Journal of Horticultural Science and Technology. 9, 41-53. https://doi.org//10.22059/ijhst.2020.303725.374.
Honaas L, Kahn E (2017) A practical examination of RNA isolation methods for European pear (Pyrus communis). BMC Research Notes 10:237. https://doi.org/10.1186/s13104-017-2564-2.
Honeywooda MJ, Milesb SJ, Wonga K, Harringtonb ChB, Cara C, Obersteb MS, Bowenb Michael D, Vegab Everardo (2022) Use of guanidine thiocyanate-based nucleic acid extraction buffers to inactivate poliovirus in potentially infectious materials. Journal of Virological Methods. 297, 114262. https://doi.org/10.1016/j.jviromet.
Liao X, Zhao Y, Li H, Hou W, Tang X, Zhou R (2023) A simple and rapid method for isolating high-quality RNA from kenaf with high polysaccharide and polyphenol contents. BioTechniques 75, 218–226. https://doi.org/10.2144/btn-2023-0077.
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25 p. 402-8. ,https://doi.org/10.1006/meth.2001.1262.
Moazzzam Jazi M, Seyedi SM, Ebrahimie E, Ebrahimi M, De Moro G, Botanga Ch (2017) A genome-wide transcriptome map of pistachio (Pistacia vera L.) provides novel insights into salinity-related genes and marker discovery. BMC Genomics 18:627. https://doi.org/10.1186/s12864-017-3989-7.
Moazzam Jazi M, Rajaei S, Seyedi SM (2015) Isolation of high-quality RNA from pistachio (Pistacia vera L.) and other woody plants high in secondary metabolites. Physiology and Molecular Biology of Plants. 21, 597–603. https://doi. org/ 10.1007/s 12298-015-0319.
Majidi M, Bahmani Y (2018) Isolation of high-quality RNA from a wide range of woody plants. Journal of Plant Molecular Breeding. 5, 50-59. https://doi.org/10.22058/JPMB.2018.82001.1157.
Mohit Rabari K, Roozban MR, Souri MK, Sadeghi-Majd R, Hamedpour-Darabi M, Vahdati K (2023) Exogenous calcium improves growth and physiological responses of pistachio rootstocks against excess boron under salinity. Journal of Plant Nutrition, 46(17), 4252-4266.
Morantel CJ, Marchart SS, Márquez MA, Martínez EM, Luque I (2014) RNA isolation from loquat and other recalcitrant woody plants with high quality and yield. Analytical Biochemistry. 452, 46–53. https://doi.org /10.1016/j.ab.2014.02.010.
Nadiya F, Anjali N, Gangaprasad A, Sabu KK (2015) High-quality RNA extraction from small cardamom tissues rich in polysaccharides and polyphenols. Analytical Biochemistry 15:25–7. https://doi.org/10.1016/j.ab.2015.05.017.
Nazoori F, ZamaniBahramabadi E, Mirdehghan SH, Yousefi M (2022) Preharvest application of sulfur as Pesticide on Fresh Hull and Kernel of Pistachio (Pistacia vera L.). International Journal of Horticultural Science and Technology 9, 117-29. doi. org/10. 22059 / ijhst. 2021.313206.408.
Nazoori F, ZamaniBahramabadi E, Rezaei H (2024) Effect of sun-drying and roasting on pistachio quality and health benefits. International Journal of Horticultural Science and Technology. 11, 299-308. https://doi.org/10.22059/ijhst.2023.358161.635
Norozi M, ValizadehKaji B, Karimi R, Nikoogoftar Sedghi M (2019) Effects of foliar application of potassium and zinc on pistachio (Pistacia vera L.) fruit yield. International Journal of Horticultural Science and Technology 6, 113-23. https://doi.org/10.22059/ijhst.2021.313206.408.
Seçgin Z, Gökhan G, Elif SA, Kızıldoğan KA, Kavas M (2020) Development of new total RNA isolation method for tissues with rich phenolic compounds. Turkish Journal of Biochemistry 45, 343–350. https://doi.org/ 10.1515/tjb-2019-0375.
Roozban MR, Mohamadi N and Vahdati K (2006) Fat content and fatty acid composition of four Iranian pistachio varieties grown in Iran. Acta Horticulturae. 726, 573-577.
Sharifkhah M, Bakhshi D, Pourghayoumi M, Abdi S, Hokmabadi H (2020) Effect of pollination time on yield and antioxidant properties of some pistachio cultivars. International Journal of Horticultural Science and Technology. 7, 51-58. https://doi. org/10.22059/ijhst.2020.285527.306
Tang D, Wei F, Kashif MH, Munsif F, Zhou R (2019) Identification and analysis of RNA editing sites in chloroplast transcripts of kenaf (Hibiscus cannabinus L.). 3 Biotech. 9(10), 361. https://doi.org/10.1007/s13205-019-1893-3.
Toni LS, Garcia AM, Jefrey DA, Jiang X, Stauffer B (2018) Optimization of phenol-chloroform RNA extraction, MethodsX 5, pp. 599–608. https://doi.org/10.1016/j.mex.2018.05.011.
Xiao Y, Yang Y, Cao H, Fan H, Ma Z, Lei x, Mason A.S, Xia Z, Huang X (2012) Efficient isolation of high quality RNA from tropical palms for RNA-seq analysis. Plant Omics. 5, 584–558.
Yee W, Kumar JN, Muthusamy PD (2018) Inclusion of 2-Mercaptoethanol in Lysis Buffer Could Interfere with Isolation of High Molecular Weight DNA from Freshwater Microalgae. Indian Journal Microbiology. 58,109–113. https://doi.org/ 10.1007/s12088-017-0698-5.
