TY - JOUR

T1 - A Phenol-Free Method for the Robust Isolation of the Double-Stranded RNA Produced in the E. coli HT115 Strain

AU - Ivanov, A. A.

AU - Golubeva, T. S.

N1 - The work was supported by the state assignment of the Ministry of Science and Higher Education of the Russian Federation FWNR-2022-0017.

PY - 2025/8

Y1 - 2025/8

N2 - Objective: Different variants of phenol-chloroform nucleic acid extraction are widely used for double-stranded RNA isolation following its production in E. coli. All existing protocols require phenol, which is hazardous to the environment, and further clean-up steps to concentrate and purify the target molecule before it can be used in experiments. Aiming to develop an economical phenol-free method with high target dsRNA yield and no need for additional clean-up steps, we substituted phenol with methanol and incorporated DNA hydrolysis into the isolation pipeline. Methods: DNA hydrolysis following thermal lysis of bacteria was performed prior to the main extraction step. Methanol-chloroform phase separation was used to separate the target RNA from proteins. A PEG-8000 precipitation step allowed precipitation of high molecular weight RNA only. Results and Discussion: We found that methanol could be used instead of phenol and that some traditional purification techniques could be incorporated into the cell lysis and nucleic acid precipitation steps, allowing extraction of RNA with up to 25% target dsRNA content. Given the high and reproducible target molecule content, the dsRNA thus obtained may be used directly or further processed to remove unwanted RNA. However, no published method suggests a purification approach for achieving 100% target molecule content. Conclusions: The proposed method facilitates double-stranded RNA isolation from E. coli culture without requiring phenol or expensive reagents. Consistently high content of the target molecule can be achieved, in contrast to phenol-chloroform extraction where additional clean-up steps are required. The application of this methodology will be beneficial in laboratories engaged in fundamental or applied research on RNA interference. However, scaling the technology for agricultural use may require adjustments to the protocol described in this work.

AB - Objective: Different variants of phenol-chloroform nucleic acid extraction are widely used for double-stranded RNA isolation following its production in E. coli. All existing protocols require phenol, which is hazardous to the environment, and further clean-up steps to concentrate and purify the target molecule before it can be used in experiments. Aiming to develop an economical phenol-free method with high target dsRNA yield and no need for additional clean-up steps, we substituted phenol with methanol and incorporated DNA hydrolysis into the isolation pipeline. Methods: DNA hydrolysis following thermal lysis of bacteria was performed prior to the main extraction step. Methanol-chloroform phase separation was used to separate the target RNA from proteins. A PEG-8000 precipitation step allowed precipitation of high molecular weight RNA only. Results and Discussion: We found that methanol could be used instead of phenol and that some traditional purification techniques could be incorporated into the cell lysis and nucleic acid precipitation steps, allowing extraction of RNA with up to 25% target dsRNA content. Given the high and reproducible target molecule content, the dsRNA thus obtained may be used directly or further processed to remove unwanted RNA. However, no published method suggests a purification approach for achieving 100% target molecule content. Conclusions: The proposed method facilitates double-stranded RNA isolation from E. coli culture without requiring phenol or expensive reagents. Consistently high content of the target molecule can be achieved, in contrast to phenol-chloroform extraction where additional clean-up steps are required. The application of this methodology will be beneficial in laboratories engaged in fundamental or applied research on RNA interference. However, scaling the technology for agricultural use may require adjustments to the protocol described in this work.

KW - RNA interference

KW - RNA isolation

KW - bacterial dsRNA production

KW - double-stranded RNA

KW - spray-induced gene silencing

UR - https://www.scopus.com/pages/publications/105011953854

UR - https://www.elibrary.ru/item.asp?id=82684577

UR - https://www.mendeley.com/catalogue/ebbf4826-6d2f-35a7-965c-56f23b88bfc3/

U2 - 10.1134/S1068162024607067

DO - 10.1134/S1068162024607067

M3 - Article

VL - 51

SP - 1521

EP - 1528

JO - Russian Journal of Bioorganic Chemistry

JF - Russian Journal of Bioorganic Chemistry

SN - 1068-1620

IS - 4

M1 - 8

ER -