A simple and efficient Triton X-100 boiling and chloroform extraction method of RNA isolation from Gram-positive and Gram-negative bacteria.
A fast, reliable, and inexpensive Triton X-100 boiling procedure for RNA isolation from both the Gram-positive and Gram-negative bacteria was developed. The yield of RNA was 0.2-2 mg per 10 ml bacterial culture. The method was tested on Gram-positive and Gram-negative bacteria of eight genera and nine species and yielded reproducible results. In parallel experiments, the Qiagen and hot phenol extraction methods both yielded RNA that contained contaminating 16S and 23S rRNA. The Triton X-100 boiling method reported here yielded RNA that was free from 16S and 23S rRNA, contained full-length transcripts and did not require additional purification. The presence of specific mRNA in one of the RNA samples obtained by this procedure was demonstrated by partial amplification of a 732 bp vancomycin resistance gene, vanA, by reverse transcription-polymerase chain reaction (RT-PCR). The presence of a full-length transcript (1031 bases) of the vanA gene was verified by Northern hybridization and probing with a digoxigenin (DIG)-labeled vanA PCR partial product. The method provides a rapid, reliable, and simple tool for the isolation of good quality RNA suitable for various molecular biology experiments.
Sung
Kidon
K
US Food and Drug Administration, National Center for Toxicological Research, Division of Microbiology, 3900 NCTR Road, Jefferson, AR 72079, USA.
Khan
Saeed A
SA
Nawaz
Mohamed S
MS
Khan
Ashraf A
AA
D002725:Chloroform; D003902:Detergents; D006090:Gram-Negative Bacteria / Q000235:genetics*; D006094:Gram-Positive Bacteria / Q000235:genetics*; D008967:Molecular Biology / Q000379:methods; D017830:Octoxynol; D012329:RNA, Bacterial / Q000302:isolation & purification*; D012997:Solvents
D003902:Detergents; D012329:RNA, Bacterial; D012997:Solvents; D002725:Chloroform; D017830:Octoxynol