title: Evaluation of microbial RNA extractions from Streptococcus pneumoniae.
abstract: The mechanisms that control Streptococcus pneumoniae's ability to colonize the nasopharynx or to invade the middle ear and cause acute otitis media are not understood. Focused study of these mechanisms requires efficient methods for the extraction of microbial RNA from minute clinical samples. Several lysis/extraction methods were tested and compared to determine the optimal conditions for isolating intact total RNA from pneumococcal cells. The sensitivity and efficiency of the extractions were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Compared to other methods, mechanical homogenization in TRIZOL was the most efficient for releasing microbial RNA, and addition of polyinosinic acid (Poly I) as an RNA carrier increased the assay sensitivity to 10(2) colony forming units when detected by RT-PCR amplification of 16S ribosomal RNA or messenger RNA for penicillin binding protein 2b. Quantitative results were confirmed using a ribonuclease protection assay. Penicillin binding protein 2b was also detected in rat middle ear mucosa recovered 5 weeks after middle ear challenge with S. pneumoniae. This study describes a useful core methodology for use in identifying pneumococcal virulence genes from small titer samples and has promising applications in clinical studies of pneumococcal nasopharyngeal colonization and otitis media pathogenesis.
authors.affiliation: Division of Pediatric Otolaryngology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, 15213, USA. Ha-Sheng.Li@chp.edu
authors.forename: Ha-Sheng
authors.initials: HS
authors.lastname: Li-Korotky
authors.forename: Lori A
authors.initials: LA
authors.lastname: Kelly
authors.forename: Otavio
authors.initials: O
authors.lastname: Piltcher
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Hebda
authors.forename: William J
authors.initials: WJ
authors.lastname: Doyle
mesh_terms: D000818:Animals; D019286:Endopeptidase K / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D010033:Otitis Media / Q000382:microbiology*; D010636:Phenols / Q000494:pharmacology; D011008:Pneumococcal Infections / Q000382:microbiology*; D011069:Poly I / Q000494:pharmacology; D012329:RNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013296:Streptococcus pneumoniae / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D006146:Guanidines; D010636:Phenols; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; C411644:trizol; D011069:Poly I; D012967:Sodium Dodecyl Sulfate; D010450:Endopeptidases; D019286:Endopeptidase K; C021816:mutanolysin