J. K. Wickiser Lab

Posts Tagged ‘microarray’

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Reconstructing Directed Signed Gene Regulatory Network from Microarray Data.

Monday, September 5, 2011

The analysis of networks dominates the field of Network Science, but it might well be argued that the construction of the network is the difficult first step that involves the real science: Gene Networks and Microarrays

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The Reactivity of the Cellular Transcriptome to Xenobiotic Compound Perturbation

Tuesday, August 23, 2011

This looks like it’ll be a very cool meeting at NYAS.

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High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay

Friday, August 19, 2011

Here’s an evaluation of from the Faculty of 1000.

“Here is a method for determining, in a single experiment, rates of synthesis and decay of mRNAs, individually or genome-wide.

Modifying previously published procedures, the authors use 4-thiouridine (4-thio-U) for pulse-labeling newly synthesized RNA in mammalian cells. After RNA isolation, RNA containing 4-thio-U is selectively biotinylated. This newly synthesized RNA is separated from ‘old’ RNA, and all three fractions — total, new and old — can be analyzed, e.g. by microarrays. Rates of synthesis and decay are obtained from simple equations. The method can potentially replace separate determinations of synthesis and decay rates. Nuclear run-on experiments to determine rates of synthesis are tedious and based on assumptions. Actinomycin-D time courses for analysis of RNA decay run the risk of interfering with the process that is to be measured and cannot measure long half-lives reliably. Actinomycin-D can be avoided through the use of regulated promoters, but this almost invariably requires a separate construct to be made for every transcript to be examined. 4-thio-U labeling looks like an attractive alternative.

For a recent application of the procedure and its adaptation to Saccharomyces cerevisiae, please see ref. {1}.

References:
{1} Miller et al. Mol Syst Biol 2011, 7:458 [PMID:21206491].
Competing interests: None declared”

Citation:
Wahle E: “Here is a method for determining, in a single experiment, rates of synthesis and decay…” Evaluation of: [Dölken L et al. High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. RNA. 2008 Sep; 14(9):1959-72; doi: 10.1261/rna.1136108]. Faculty of 1000, 18 Apr 2011. F1000.com/9679960

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Oxidative stress response and gene expression with atrazine exposure in adult female zebrafish

Friday, August 12, 2011

From Hannah Lachance as she was perusing the literature: Atrazine (ATZ) is a herbicide that runs off from agricultural facilities into the ground and surface water sources. ATZ is suspected to be a cause of oxidative stress in fish because it interferes with reactive oxygen species (ROS) production and different endpoints. Fish species, such as Zebrafish have an antioxidant defense system that helps them limit damage from oxidative stress. Therefore, measuring antioxidant enzyme activity was used to measure oxidative stress levels. This study analyzed the liver and ovaries of adult female zebra fish to determine oxidative stress levels caused by ATZ. The oxidative stress levels we determined by the MDA and GSH content as well as by the activities of SOD and CAT. Oxidation stress levels were also measured by analyzing the gene expression of Cu/Zn-Sod, Mn-Sod, Cat, and Gpx which are antioxidant proteins. The ROS production, the respiratory chain, and ATP synthase in the liver were also monitored through analyzing the transcription of mitochondrial inner membrane genes related to these processes. The subjects of this experiment were 5 month-old, healthy, female zebrafish weighing between 0.08g and 0.52g that were randomly selected for the different experimental and control groups . All the fish were kept on a 14h light/10h dark cycle and feed brine shrimp two times a day. The experimental groups based on the different concentrations of ATZ; 1,10, 100, and 1000ug L-1 of ATZ were in water containing 0.2% acetone. The control group was in water with 0.2% acetone and no ATZ. Each experimental group consisted of 10 fish in 3L of the water ATZ mixture. The zebrafish were exposed to the solutions for 14 days after which the livers and ovaries were harvested and divided into pooled samples; 4 pooled samples for enzyme extraction and biochemical analysis, 4 pooled samples for RNA extraction and mRNA transcription analysis. RNA was isolated from the liver and ovaries using TRIzol. In order to verify the quality of the RNA, gels containing 1% agarose formaldehyde gel and the 260nm/280nm ratio were used. cDNA was then synthesized through a reverse transcriptase kit. PCR and Q-PCR (using syber green and ∆∆Ct) were preformed and oligonucleotide primers were used to detect the gene expression of the oxidative stress indicators. The results showed that the SOD and CAT activity increased sequentially (low concentration to high concentration) in the liver but not so much in the ovaries. GSH levels decreased and MDA levels increased in a dose dependent manner in the liver but on the GSH levels changed in the ovaries. mRNA levels of Mn -Sod and Cu/Zn- Sod increased significantly in the liver but neither changed in the ovaries. Cat and Gpx increased at the 100ug L-1 level in the liver but no change in the ovaries. This suggest that the ovaries are less susceptible to oxidative stress caused by ATZ. Bcl-2 decreased and Ucp-2 increased yet Ndi, Coxl, and ATPo6 showed no signs of change. All of these results show that the liver is actively reacting to the ATZ trying to prevent oxidative stress.

Zebrafish

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Bioinformatical tool used in our lab

Wednesday, January 12, 2011

Here is the bioinformatical tool we use in the lab to analyze our gene expression microarray data:
http://www.chem.agilent.com/en-US/PRODUCTS/SOFTWARE/LIFESCIENCESINFORMATICS/Pages/default.aspx

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