A simple methodology for RNA isolation from bacteria by integration of formamide extraction and chitosan-modified silica purification

RNA isolation from bacteria is technically difficult due to the RNA characteristic of labile and vulnerable degradation. Many reagents were explored for cellular lysis and complete inhibition of RNase. However, the available methods for RNA isolation are either of low efficiency or time-consuming. H...

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Bibliographic Details
Published in:Analytical and bioanalytical chemistry 2021-11, Vol.413 (26), p.6469-6477
Main Authors: Zhao, Xiaoli, Li, Yong, Duan, Yake, Amin, Amr, Xie, Yingqiu, Shi, Chao, Ma, Cuiping
Format: Article
Language:English
Subjects:
Amides
Analytical Chemistry
Bacteria
Biochemistry
Biodegradation
Characterization and Evaluation of Materials
Chemical properties
Chemistry
Chemistry and Materials Science
Chitin
Chitosan
Chitosan - analogs & derivatives
Escherichia coli - chemistry
Escherichia coli - genetics
Extraction (Chemistry)
Food Science
Formamides - chemistry
Gene expression
Genetic aspects
Laboratory Medicine
Lysis
Membranes, Artificial
Methods
Monitoring/Environmental Analysis
Nucleic acids
Paper in Forefront
Purification
Reagents
Reverse transcription
Ribonuclease
Ribonucleic acid
RNA
RNA, Bacterial - genetics
RNA, Bacterial - isolation & purification
Separation
Silica
Silicon dioxide
Silicon Dioxide - chemistry
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Summary:RNA isolation from bacteria is technically difficult due to the RNA characteristic of labile and vulnerable degradation. Many reagents were explored for cellular lysis and complete inhibition of RNase. However, the available methods for RNA isolation are either of low efficiency or time-consuming. Here, we developed a rapid and accessible protocol for RNA isolation that combined a simplified cell lysis and RNA release by formamide-based solution and RNA purification by chitosan-modified silica membrane for the first time. With this method, we obtained about ~ 28 μg of total RNA from 10 8 Escherichia coli cells. The entire procedure can be done within 15 min without redundant pipetting steps. The purity of extracted RNA was comparable to that of commercial kits, but the cost was much lower. Furthermore, the yielded RNA was successfully used in downstream enzymatic reactions, such as reverse transcription and quantitative real-time PCR. This new method would be of benefit for an extensive range of gene expression analyses in bacterial organisms. Graphical abstract
ISSN:1618-2642
1618-2650
DOI:10.1007/s00216-021-03644-6