Browsing by Author "Pérez-Osorio, Ailyn C."
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Item Isolation of RNA and DNA from biofilm samples obtained by laser capture microdissection microscopy(2008-10) Pérez-Osorio, Ailyn C.; Franklin, Michael J.The metabolic activities of bacteria growing in biofilms result in spatial gradients of oxygen, nutrients, and waste products. Because bacteria respond to local environmental conditions through changes in gene expression, mRNA levels of individual genes may vary spatially among bacteria within the biofilm. This article describes an approach to isolate RNA for quantification from cells at localized sites within biofilms. Biofilm thin sections are generated by embedding biofilms in cryoembedding resin, freezing the embedded biofilms on dry ice, and cutting with a cryomicrotome. The sections are placed on membrane-coated microscope slides and maintained on dry ice. Laser capture microdissection microscopy (LCMM) is used to dissect small subsets of cells at different regions within the biofilms, and RNA is extracted from the samples using either hot phenol or TRI reagent. A TRI reagent-based DNA extraction method is also presented.Item qRT-PCR of microbial biofilms(2008-10) Pérez-Osorio, Ailyn C.; Franklin, Michael J.Bacteria growing in biofilms often express a different subset of genes compared to the same strains growing planktonically. Quantitative reverse transcriptase real time PCR (qRT-PCR) can be used effectively to quantify the number of RNA transcripts of specific genes from bacteria growing in biofilms. qRT-PCR has a large dynamic range and may be used to verify gene expression data obtained from microarrays. In addition, qRT-PCR is sensitive, and therefore may be used to quantify gene expression from biofilm samples where only a small amount of biological material is available, as in samples obtained by laser capture microdissection microscopy (LCMM). The most commonly used qRT-PCR methods are the SYBR Green and dual-labeled probe (Taqman) approaches. Both approaches use reverse transcription to convert mRNA to cDNA, followed by PCR amplification of the cDNA. This article describes steps involved in aspects of qRT-PCR including (1) primer design, (2) primer and probe performance testing, (3) qRT-PCR using the Corbett Rotor-Gene system, and (4) data export and analysis.