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)
کلید واژه: Isolation, Mini kit, Pistachio, Real time, RNA, TRIzol,
چکیده مقاله :
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.
چکیده انگلیسی :
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.
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