Development of a method for recovering rickettsial RNA from infected cells to analyze gene expression profiling of obligate intracellular bacteria.
The Rickettsia genus is composed of Gram-negative bacteria responsible for Typhus and spotted fevers. Because of the limitations imposed by their obligate intracellular location, the molecular mechanisms responsible for their pathogenicity remain poorly understood. Several rickettsial genomes are now available, thus providing the foundation for a new era of post-genomic research. Here, using Rickettsia conorii as model, we developed a suitable method for microarray-based transcriptome analysis of rickettsiae. Total RNA was extracted from infected Vero cells using a protocol preserving its integrity, as observed by Bioanalyzer (Agilent) profiles. By a subtractive hybridization method, the samples were subsequently depleted of eukaryotic RNA that represents up to 90% of the whole extract and that hampers fluorochrome labeling of rickettsial nucleic acids. To obtain the amount of material required for microarray hybridization, the bacterial RNA was then amplified using random primers. Hybridizations were carried out on microarrays specific for R. conorii but containing a limited number of selected targets. Our results show that this method yielded reproducible signals. Transcriptional changes observed following exposure of R. conorii to a nutrient stress were verified by real-time quantitative PCR and by quantitative reverse transcription PCR starting from amplified cDNA and total RNA as templates, respectively. We conclude that this approach has great potential for the study of mechanisms behind the virulence and intracellular survival of members of the genus Rickettsia.
La
My-Van
MV
Unité des Rickettsies, CNRS-UMR6020, IFR48, Faculté de Médecine, 27, Bd Jean Moulin, Marseille, France.
François
Patrice
P
Rovery
Clarisse
C
Robineau
Sylvianne
S
Barbry
Pascal
P
Schrenzel
Jacques
J
Raoult
Didier
D
Renesto
Patricia
P
D000818:Animals; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020114:Rickettsia conorii / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014709:Vero Cells
D017931:DNA Primers; D012333:RNA, Messenger