ABSTRACT
Background: Nucleic acid amplification assays (NAAs), such as polymerase chain reaction or loop-mediated isothermal amplification (LAMP), are used for disease diagnosis. Current nucleic acid isolation kits require several hours for completion of protocol including the complicated handling steps. Objective: In this study, a simple and cost-effective nucleic acid preparation method was developed, and its performance was compared with those of commercial kits. Materials and Methods: RNA was prepared using our method and three commercial RNA isolation kits. The RNA quantity and quality were evaluated using the NanoDrop spectrophotometer and Agilent 2100 bioanalyser. Reverse transcription LAMP (RT-LAMP) reactions were performed to determine the usability of the RNA preparation methods. Results: The concentrations of RNA extracted from blood samples by four different methods were sufficient for use in NAAs. The RNA integrity number was >7.0 when RNA was isolated using other RNA isolation kits but lower when prepared using our method. The RT-LAMP reaction was successfully performed when RNA was prepared using any of the methods. Conclusions: These results demonstrate that despite the lower purity and integrity of RNA, our RNA preparation protocol is simple and rapid and shows reasonable performance in RT-LAMP.
ABSTRACT
Biofilm formed by Staphylococcus aureus is considered an important virulence trait in the pathogenesis of infections associated with implantable medical devices. Gene expression analyses are important strategies for determining the mechanisms involved in production and regulation of biofilm. Obtaining intact RNA preparations is the first and most critical step for these studies. In this article, we describe an optimized protocol for obtaining total RNA from sessile cells of S. aureus using the RNeasy Mini Kit. This method essentially consists of a few steps, as follows: 1) addition of acetone-ethanol to sessile cells, 2) lysis with lysostaphin at 37°C/10 min, 3) vigorous mixing, 4) three cycles of freezing and thawing, and 5) purification of the lysate in the RNeasy column. This simple pre-kit procedure yields high-quality total RNA from planktonic and sessile cells of S. aureus.