Phetcharat Jaiaue1, Piroonporn Srimongkol2, Sitanan Thitiprasert3, Somboon Tanasupawat4, Benjamas Cheirsilp5, Suttichai Assabumrungrat6, Nuttha Thongchul7. 1. Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand. 2. Research Unit in Bioconversion and Bioseparation for Value-Added Chemical Production, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, Thailand. 3. Research Unit in Bioconversion and Bioseparation for Value-Added Chemical Production, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, Thailand. Sitanan.T@chula.ac.th. 4. Research Unit in Bioconversion and Bioseparation for Value-Added Chemical Production, Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand. 5. Department of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Songkla, Thailand. 6. Department of Chemical Engineering, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand. 7. Research Unit in Bioconversion and Bioseparation for Value-Added Chemical Production, Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, Thailand. Nuttha.T@chula.ac.th.
Abstract
BACKGROUND: High quality RNA is required for the molecular study. Sample preparation of the spore-forming, Gram-positive bacteria like Bacillus sp., remains challenging although several methods have been proposed. Those techniques were simply developed using cell samples at certain growth stages despite some molecular studies like transcriptomic analyses require RNA samples from different physiological stages. METHODS AND RESULTS: We developed the rapid, simple yet effective cell-lysis technique with limit use of harsh reagents by modifying the kit-based protocols. Appropriate lysozyme loading (20 mg/mL), incubation time (30 min), and temperature (37 °C) enabled cell lysis and enhanced RNA extraction from both vegetative cells and endospores of Bacillus subtilis TL7-3. High RNA Integrity Numbers and ratios of A260/A280 and A260/A230 of all RNA products collected during the batch cultivation confirmed that invert mixing with absolute ethanol prevented RNA damage during protein denaturation. With the process modification of the major steps in cell lysis and RNA extraction compared with the kit-based protocols that are typically used in laboratory work, interestingly, our modified protocol, simple-yet-effective, yielded higher concentration, purity, and integrity of RNA products from all cell samples collected at different physiological stages. While the kit-based protocols either failed to provide high RNA concentration or RNA purity and integrity for all cell samples particularly during the late-log, stationary, or sporulation. CONCLUSIONS: Therefore, we can claim the significance of this modified protocol to be applicable for RNA extraction to those spore-forming Gram-positive bacteria not limited to B. subtilis growing at varied physiological stages.
BACKGROUND: High quality RNA is required for the molecular study. Sample preparation of the spore-forming, Gram-positive bacteria like Bacillus sp., remains challenging although several methods have been proposed. Those techniques were simply developed using cell samples at certain growth stages despite some molecular studies like transcriptomic analyses require RNA samples from different physiological stages. METHODS AND RESULTS: We developed the rapid, simple yet effective cell-lysis technique with limit use of harsh reagents by modifying the kit-based protocols. Appropriate lysozyme loading (20 mg/mL), incubation time (30 min), and temperature (37 °C) enabled cell lysis and enhanced RNA extraction from both vegetative cells and endospores of Bacillus subtilis TL7-3. High RNA Integrity Numbers and ratios of A260/A280 and A260/A230 of all RNA products collected during the batch cultivation confirmed that invert mixing with absolute ethanol prevented RNA damage during protein denaturation. With the process modification of the major steps in cell lysis and RNA extraction compared with the kit-based protocols that are typically used in laboratory work, interestingly, our modified protocol, simple-yet-effective, yielded higher concentration, purity, and integrity of RNA products from all cell samples collected at different physiological stages. While the kit-based protocols either failed to provide high RNA concentration or RNA purity and integrity for all cell samples particularly during the late-log, stationary, or sporulation. CONCLUSIONS: Therefore, we can claim the significance of this modified protocol to be applicable for RNA extraction to those spore-forming Gram-positive bacteria not limited to B. subtilis growing at varied physiological stages.
Authors: Alexander J F Egan; Robert M Cleverley; Katharina Peters; Richard J Lewis; Waldemar Vollmer Journal: FEBS J Date: 2016-11-23 Impact factor: 5.542
Authors: Yongki Choi; Issa S Moody; Patrick C Sims; Steven R Hunt; Brad L Corso; David E Seitz; Larry C Blaszczak; Larry C Blaszcazk; Philip G Collins; Gregory A Weiss Journal: J Am Chem Soc Date: 2012-01-24 Impact factor: 15.419