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Utilizing Dichloromethane as an Extremely Proficient Substitute for Phenol/Chloroform in Extracting RNA with Exceptional Purity from Woody Tissues of Coconut

Procuring high-grade RNA from mature coconut tissues is a tricky and labor-intensive process due to the intricate scaffold of polysaccharides, polyphenols, lipids, and proteins that form firm complexes with nucleic acids. However, we have effectively developed a novel method for the first time, lett...

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Autores principales: Iqbal, Amjad, Yang, Yaodong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10514881/
https://www.ncbi.nlm.nih.gov/pubmed/37736958
http://dx.doi.org/10.3390/mps6050075
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author Iqbal, Amjad
Yang, Yaodong
author_facet Iqbal, Amjad
Yang, Yaodong
author_sort Iqbal, Amjad
collection PubMed
description Procuring high-grade RNA from mature coconut tissues is a tricky and labor-intensive process due to the intricate scaffold of polysaccharides, polyphenols, lipids, and proteins that form firm complexes with nucleic acids. However, we have effectively developed a novel method for the first time, letting the retrieval of high-grade RNA from the roots, endosperm, and mesocarp of mature coconut trees take place. In this method, we exploited dichloromethane as a replacement to phenol/chloroform for RNA recovery from mature coconut tissues. The amount of high-grade RNA acquired from the roots of mature coconut trees was 120.7 µg/g, with an A260/280 ratio of 1.95. Similarly, the mature coconut mesocarp yielded 134.6 µg/g FW of quality RNA with A260/280 ratio of 1.98, whereas the mature coconut endosperm produced 120.4 µg/g FW of quality RNA with A260/280 ratio of 2.01. Furthermore, the RNA isolation using the dichloromethane method exhibited excellent performance in downstream experiments, particularly in RT-PCR for cDNA production and amplification. On the contrary, the RNA plant kit, TRIZOL, and Cetyl Trimethyl Ammonium Bromide (CTAB) methods were unsuccessful in isolating substantial quantities of RNA with exceptional purities from the mentioned coconut tissues. In view of these findings, we conclude that the newly developed method will be pivotal in effectively extracting RNA with high purity from mature coconut tissues.
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spelling pubmed-105148812023-09-23 Utilizing Dichloromethane as an Extremely Proficient Substitute for Phenol/Chloroform in Extracting RNA with Exceptional Purity from Woody Tissues of Coconut Iqbal, Amjad Yang, Yaodong Methods Protoc Protocol Procuring high-grade RNA from mature coconut tissues is a tricky and labor-intensive process due to the intricate scaffold of polysaccharides, polyphenols, lipids, and proteins that form firm complexes with nucleic acids. However, we have effectively developed a novel method for the first time, letting the retrieval of high-grade RNA from the roots, endosperm, and mesocarp of mature coconut trees take place. In this method, we exploited dichloromethane as a replacement to phenol/chloroform for RNA recovery from mature coconut tissues. The amount of high-grade RNA acquired from the roots of mature coconut trees was 120.7 µg/g, with an A260/280 ratio of 1.95. Similarly, the mature coconut mesocarp yielded 134.6 µg/g FW of quality RNA with A260/280 ratio of 1.98, whereas the mature coconut endosperm produced 120.4 µg/g FW of quality RNA with A260/280 ratio of 2.01. Furthermore, the RNA isolation using the dichloromethane method exhibited excellent performance in downstream experiments, particularly in RT-PCR for cDNA production and amplification. On the contrary, the RNA plant kit, TRIZOL, and Cetyl Trimethyl Ammonium Bromide (CTAB) methods were unsuccessful in isolating substantial quantities of RNA with exceptional purities from the mentioned coconut tissues. In view of these findings, we conclude that the newly developed method will be pivotal in effectively extracting RNA with high purity from mature coconut tissues. MDPI 2023-08-26 /pmc/articles/PMC10514881/ /pubmed/37736958 http://dx.doi.org/10.3390/mps6050075 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Protocol
Iqbal, Amjad
Yang, Yaodong
Utilizing Dichloromethane as an Extremely Proficient Substitute for Phenol/Chloroform in Extracting RNA with Exceptional Purity from Woody Tissues of Coconut
title Utilizing Dichloromethane as an Extremely Proficient Substitute for Phenol/Chloroform in Extracting RNA with Exceptional Purity from Woody Tissues of Coconut
title_full Utilizing Dichloromethane as an Extremely Proficient Substitute for Phenol/Chloroform in Extracting RNA with Exceptional Purity from Woody Tissues of Coconut
title_fullStr Utilizing Dichloromethane as an Extremely Proficient Substitute for Phenol/Chloroform in Extracting RNA with Exceptional Purity from Woody Tissues of Coconut
title_full_unstemmed Utilizing Dichloromethane as an Extremely Proficient Substitute for Phenol/Chloroform in Extracting RNA with Exceptional Purity from Woody Tissues of Coconut
title_short Utilizing Dichloromethane as an Extremely Proficient Substitute for Phenol/Chloroform in Extracting RNA with Exceptional Purity from Woody Tissues of Coconut
title_sort utilizing dichloromethane as an extremely proficient substitute for phenol/chloroform in extracting rna with exceptional purity from woody tissues of coconut
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10514881/
https://www.ncbi.nlm.nih.gov/pubmed/37736958
http://dx.doi.org/10.3390/mps6050075
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