Understanding biological complexity arising from patterns of gene expression requires accurate and precise measurement of RNA levels across large numbers of genes simultaneously. Real time PCR (RT-PCR) in a microtiter plate is the preferred method for quantitative transcriptional analysis but scaling RT-PCR to higher throughputs in this fluidic format is intrinsically limited by cost and logistic considerations. Hybridization microarrays measure the transcription of many thousands of genes simultaneously yet are limited by low sensitivity, dynamic range, accuracy and sample throughput. The hybrid approach described here combines the superior accuracy, precision and dynamic range of RT-PCR with the parallelism of a microarray in an array of 3072 real time, 33 nl polymerase chain reactions (RT-PCRs) the size of a microscope slide. RT-PCR is demonstrated with an accuracy and precision equivalent to the same assay in a 384-well microplate but in a 64-fold smaller reaction volume, a 24-fold higher analytical throughput and a workflow compatible with standard microplate protocols.

No abstract

No abstract

No abstract

Interleukin (IL)-1 beta, IL-8, and tumor necrosis factor-alpha (TNF-alpha) induce anorexia when administered intracerebroventricularly at doses that yield estimated pathophysiological concentrations reported in the cerebrospinal fluid (CSF). Our hypothesis is that pivotal cytokines released during pathological processes interact to induce anorexia during disease. In the present study, we investigated the effects of the intracerebroventricular microinfusion of individual or multiple combinations (8 dyads and 5 triads) of IL-1 beta, IL-8, and TNF-alpha on feeding and the microstructure of eating in rats maintained ad libitum. Estimated pathophysiological concentrations of cytokine combinations exhibited additive or synergistic activities in inducing anorexia. Computerized analysis of behavioral patterns demonstrated that the most effective treatment (triad of 1.0 ng IL-1 beta + 20 ng IL-8 + 20 ng TNF-alpha/rat, n = 11) decreased nighttime meal size by 42% and feeding rate (meal size/meal duration) by 26%, whereas it increased the satiety ratio (postprandial intermeal intervals/meal size) by 80%; meal duration and meal frequency were not significantly affected. Analysis of meal parameters in 4-h intervals revealed a maximum effect during the first 4-h interval after the intracerebroventricular administration. The time course during this initial 4-h interval analyzed in 10-min periods was similar for IL-8 (n = 10) and TNF-alpha (n = 11), and both were significantly different from the time course induced by IL-1 beta (n = 10) and the most effective triad (n = 11). Intracerebroventricular microinfusion of heat-inactivated triad or intraperitoneal administration of the most effective triad, in doses-equivalent to those administered centrally, had no effect on feeding. The results suggest that estimated pathophysiological concentrations of cytokines in the CSF act centrally and additively or synergistically to decrease feeding, and this effect may participate in the anorexia frequently accompanying pathological processes.