Application of real-time polymerase chain reaction for the quantitation of interleukin-1β mRNA upregulation in brain ischemic tolerance

Abstract

Differential gene expression plays an important role in normal development and pathophysiological conditions. The accurate quantitation of mRNA expression is critical to assess the differential gene expression. While a number of techniques, such as Northern analysis, (semi-)quantitative reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization, are available to measure the levels of mRNA expression, certain limitations exist, including the insensitive and inaccurate quantitation of mRNA expressed at low abundance. In the present study, we describe the application of a recently developed TaqMan real-time quantitative RT-PCR for the detection of interleukin-1β (IL-1β) mRNA expression in rat cortical tissue after a short duration of ischemia (i.e., ischemic preconditioning). The principle of the TaqMan real-time detection is based on the fluorogenic 5′ nuclease assay that allows simple and rapid quantitation of a target sequence during the extension phase of PCR amplification. Using a cloned plasmid DNA as a standard and normalizing RNA samples with a housekeeping gene for the TaqMan real-time PCR, we detected the significant induction in absolute copy numbers of IL-1β mRNA in the ipsilateral cortex after preconditioning, suggesting a potential role of this inflammatory cytokine in ischemic brain tolerance. Themes: Disorders of the nervous system Topics: Ischemia