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Optimization of Nucleic Acid Extraction from Cyclamen coum Miller
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Mohammad Sadegh Tamkin   , Marzieh Ghanbari Jahromi , Amir Mousavi |
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Abstract: (76 Views) |
Most molecular genetic tests begin by extracting DNA and RNA. To achieve this, obtaining a suitable, reliable, and repeatable extraction method is of great importance. In this regard, optimization of the extraction of nucleic acids from the Iranian cyclamen plant (Cyclamen coum Miller) and the introduction and expression of housekeeping genes was performed. After collecting plant samples from the three organs of leaf, petal and hypocotyl, modified CTAB and SDS methods were used for DNA isolation and modified Trizol, CTAB-LiCl and RNX-Plus methods were used for RNA isolation. The quality of isolated DNA and RNA was checked by electrophoresis and their concentration was measured with nanodrop. The presence of 18S ribosomal, actin (ACT), tubulin (TUB) and elongation factor (EF-1a) gene fragments was investigated using polymerase chain reaction (PCR). Based on the results, the lowest DNA concentration was related to the CTAB method. The DNA concentration and quality of Iranian cyclamen leaf, petal and hypocotyl samples were higher in the modified SDS method than in the CTAB method. The isolation of DNA using the modified SDS method not only had a high purity but also had relatively high performance. According to quality analysis of the RNA isolated from leaf, petal, and hypocotyl samples using Trizol, CTAB-LiCl, and RNX-Plus protocols, all three strategies may be used to isolate RNA from Iranian cyclamen tissues. However, the Trizol method is superior to other methods in terms of high band resolution and RNA concentration. The production of cDNA from RNA isolated from three Iranian cyclamen tissues was successfully performed only by Trizol and RNX-Plus methods. Based on the results, cDNA made from RNA isolated by Trizol method had better quality. To check the efficiency of RNA samples in RT-PCR reaction and the expression of housekeeping genes, RT-PCR test was performed. Based on the results, the presence of the bands related to the housekeeping genes fragment in the electrophoresis stage confirmed the good quality of the produced cDNA and also indicated the high quality of the isolated RNA from the Trizol and RNX-Plus methods. RT-PCR results showed that the expression of actin, EF-1a, 18S and tubulin gene was positive in most Iranian cyclamen organs. Among housekeeping genes, tubulin and 18S genes, had higher band resolution and higher quality in all samples and tissues. |
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| Keywords: Nucleic acid, RNA extraction, Phenol-Chloroform, RNX, Tarizol, CTAB |
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Full-Text [PDF 1492 kb]
(16 Downloads)
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Type of Study: Research |
Subject:
Biotechnology and Tissue culture
Received: 2020/07/24 | Accepted: 2023/07/8 | Published: 2024/03/16
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