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

Abstract

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.

Keywords

• dichloromethane
• RNA extraction
• cDNA library
• RT-PCR
• TRIZOL
• CTAB