In this article, we review the use of these methods in the bioanalysis of 15 antimalarial drugs, based on papers published since 1988, with emphasis on sensitivity, sample-preparation procedures and detection techniques used. (C) 2012 Elsevier Ltd. All rights reserved.”
“Objective: To ascertain a viral vector-based short hairpin RNA (shRNA) capable of reducing the interleukin-1 beta (IL-1 beta) transcript in osteoarthritis (OA)-prone chondrocytes and detect corresponding
changes in the expression patterns AZD1208 of several critical disease mediators.
Methods: Cultured chondrocytes from 2-month-old Hartley guinea pigs were screened for reduction of the IL-1 beta transcript following plasmid-based delivery of U6-driven shRNA sequences. A successful plasmid/shRNA knockdown combination was identified and used to construct an adeno-associated virus serotype 5 (AAV5) vector for further evaluation. Relative real-time reverse transcription polymerase chain reaction (RTPCR) was used to quantify in vitro transcript changes of IL-1 beta and an additional nine genes following transduction with this targeting knockdown vector. To validate in vitro findings, this ATPase inhibitor AAV5 vector was injected
into one knee, while either an equivalent volume of saline vehicle (three animals) or non-targeting control vector (three animals) were injected into opposite knees. Fold differences and subsequent percent gene expression levels relative to control groups were
calculated using the comparative CT (2(-Delta Delta CT)) method.
Results: Statistically significant decreases in IL-1 beta expression were achieved by the targeting knockdown vector relative to both the mock-transduced control and non-targeting vector control groups in vitro. Transcript levels of anabolic transforming growth factor-beta (TGF-beta) Selleck I-BET151 were significantly increased by use of this targeting knockdown vector. Transduction with this targeting AAV5 vector also significantly decreased the transcript levels of key inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha), IL-2, IL-8, and IL-12] and catabolic agents [matrix metalloproteinase (MMP)13, MMP2, interferon-gamma (IFN-gamma), and inducible nitrous oxide synthase (iNOS)] relative to both mock-transduced and non-targeting vector control groups. In vivo application of this targeting knockdown vector resulted in a >50% reduction (P = 0.0045) or >90% (P = 0.0001) of the IL-1 beta transcript relative to vehicle-only or non-targeting vector control exposed cartilage, respectively.
Conclusions: Successful reduction of the IL-1 beta transcript was achieved via RNA interference (RNAi) techniques. Importantly, this alteration significantly influenced the transcript levels of several major players involved in OA pathogenesis in the direction of disease modification.