title: Identification of biomarkers that distinguish chemical contaminants using a gradient feature selection method

status: Public on Jan 05 2010

summary:
There are many toxic chemicals to contaminate the world and cause harm to human and other organisms. How to quickly discriminate these compounds and characterize their potential molecular mechanism and toxicity is essential. High through put transcriptomics profiles such as microarray have been proven useful to identify biomarkers for different classification and toxicity prediction purposes. Here we aim to investigate how to use microarray to predict chemical contaminants and their possible mechanisms.
In this study, we divided 105 compounds plus vehicle control into 14 compound classes. On the basis of gene expression profiles of in vitro primary cultured hepatocytes, we comprehensively compared various normalization, feature selection and classification algorithms for the classification of these 14 class compounds. We found that normalization had little effect on the averaged classification accuracy. Two support vector machine methods LibSVM and SMO had better classification performance. When feature sizes were smaller, LibSVM outperformed other classification methods. Simple logistic algorithm also performed well. At the training stage, usually the feature selection method SVM-RFE performed the best, and PCA was the poorest feature selection algorithm. But overall, SVM-RFE had the highest overfitting rate when an independent dataset used for a prediction in this case. Therefore, we developed a new feature selection algorithm called gradient method which had a pretty high training classification as well as prediction accuracy with the lowest over-fitting rate. Through the analysis of biomarkers that distinguished 14 class compounds, we found a goup of genes that mainly invovled in cell cylce were significanly downregulated by the metal and inflammatory compounds, but were induced by anti-microbial, cancer related drugs, pesticides, and PXR mediators.

overall_design: For in vitro experiment, primary cultured rat hepatocytes were treated one of 105 compounds  with relative controls. At least three biological replicates were used for each unique condition. In total 531 arrays were used.

first: David

middle: R

last: Johnson

first: Choo

middle: Y

last: Ang


title: Whole-genome gene expression profiling of F11 dorsal root ganglion (DRG)-like cells stimulated with forskolin

status: Public on Jul 01 2010

city: Amsterdam

first: Harold

middle: D

last: MacGillavry

address: De Boelelaan 1087

department: Center for neurogenomics and cognitive research

country: Netherlands

institute: Vrije Universiteit

summary: We generated whole-genome gene expression profiles of F11 dorsal root ganglion (DRG)-like neuroblastoma cells stimulated with the adenylate cyclase activator forskolin for 0, 2, 4, 24 and 48 hours.

overall_design: F11 neuroblastoma cells were incubated in low-serum medium (DMEM with 0.5% FCS and antibiotics) for three hours and then stimulated with 10 µM forskolin for 2, 4, 24 and 48 hours. All samples are in triplicate. F11 cells incubated in low-serum medium for three hours, but not stimulated with forskolin, were used as a common reference.

first: Jochem

last: Cornelis

first: Joost

last: Verhaagen

first: August

middle: B

last: Smit

first: Ronald

middle: E

last: van Kesteren


title: Gene expression in colon tissue of rats fed digestion resistant carbohydrates

status: Public on Dec 18 2010

city: Palmerston North

first: Wayne

last: Young

state: Manawatu

address: Tennent Drive

department: Food Nutrition Genomics Team

country: New Zealand

institute: AgResearch Ltd

summary: Conventionally raised and germ-free newly weaned male Sprague-Dawley rats were fed a basal diet or a diet supplemented with digestion resistant carbohydrates in the form of inulin, resistant starch or konjac flour.  Gene expression in colon tissue was measured to characterise interaction between food, microbes and host.

overall_design: 2 colour microarray, reference design.  Biological replicates: 6 for all groups except for conventionally raised rats fed inulin, which consisted of 5 biological replicates


title: RNA profiles of rat olfactory epithelia : individual and age related variations (gender/supplier experiment)

status: Public on Nov 01 2009

city: RENNES

first: Francis

last: Galibert

address: 2 av du Pr Léon Bernard

department: Génétique et Developpement

country: France

institute: CNRS UMR6061/Université Rennes1

summary:
Background: Mammalian genomes contain a large number (~1000) of olfactory receptor (OR) genes, many of which (20 to 50 %) are pseudogenes. OR gene transcription is not restricted to the olfactory epithelium, but is found in numerous tissues. Using microarray hybridization and RTqPCR, we analyzed the mRNA profiles of the olfactory epithelium of male and female Brown Norway rats of different origins and ages (newborn, adult and old).

Results: (1) We observed very little difference between males and females and between rats from two different suppliers. (2) Different OR genes were expressed at varying levels, rather than uniformly across the four endoturbinates. (3) A large proportion of the gene transcripts (2/3 of all probes) were detected in all three age groups. Adult and older rats expressed similar numbers of OR genes, both expressing more OR genes than newborns. (4) Comparisons of whole transcriptomes or transcription profiles of expressed OR genes only showed a clear clustering of the samples as a function of age. (5) Most OR genes were expressed at lower levels at birth than in older animals, but a small number of OR genes were expressed specifically or were overexpressed in newborns.

Conclusions: Not all OR genes are expressed at a detectable level. Pups expressed fewer OR genes than adult rats, and generally at a lower level; however, a small subset of OR genes were more strongly expressed in these newborn rats. The reasons for these differences are not understood. However, the specific expression of some OR genes in newborn olfactory epithelia may be related to the blindness and deafness of pups at birth, when these pups are heavily reliant on olfaction and their mother.

overall_design: We purchased four Brown Norway rats (two males and two females, 6 weeks-old) from Elevage Janvier and four rats (two males and two females, 6 weeks-old) from Charles River laboratory. Following reception, the animals were kept for one week in the animal house and sacrificed. Total RNA from left and right olfactory epithelia were extracted, labeled and used for hybridization on Agilent Whole Rat Genome 44K microarrays.

first: Maud

last: Rimbault

first: Stephanie

last: Robin

first: Amaury

last: Vaysse


title: Diet induced gene expression in rat peripheral blood mononuclear cells

status: Public on Dec 01 2009

city: Wageningen

first: Jaap

last: Keijer

state: gld

address: Marijkeweg 40

department: Human and Animal Physiology

institute: Wageningen University

summary:
Gene expression of rat peripheral blood mononuclear cells was analyzed by microarray analysis in normoweight and in diet-induced obese rats (cafeteria rats). The aim of this study was to identify genes involved in energy homeostasis that are altered in the obese state.

Keywords: Dietary treatment, obese-state analysis

overall_design: This study analyzed the gene expression of PBMC in normoweight and in diet-induced obese (cafeteria-fed) Wistar rats submitted to ad libitum feed conditions. Two-month-old male Wistar rats (n=10) were assigned into two dietary groups for 4 months: the control group (n=5) was fed with a standard chow diet, whereas the second group (cafeteria group, n=5) was fed with a fat-rich hypercaloric cafeteria diet in addition to the standard chow. At 6 months of age, blood samples were collected in feeding conditions from the safena vein. Peripheral blood mononuclear cells were isolated by Ficoll gradient separation, and RNA was extracted. One sample in the control group was excluded because of a low amount of RNA. Gene expression changes were assessed using an Agilent rat whole genome microarray (G4131F Agilent Technologies).

first: A

last: Caimari

first: P

last: Oliver

first: W

last: Rodenburg

first: J

last: Palou


title: Identification of genes which are responsible for induction of the initial differentiation process of PC12 cells

status: Public on Feb 13 2010

city: Bunkyo-ku

first: Jaehoon

last: Chung

state: Tokyo

address: 7-3-1 Hongo

department: Department of Biophysics and Biochemistry

country: Japan

institute: Tokyo university

summary: Continuous NGF stimulation induces PC12 cell differentiation; however, it is unclear whether NGF is continuously required for differentiation. We found that discontinuous NGF stimulation, consisting of the first transient stimulation followed by an interval and the second sustained stimulation, similarly induces differentiation. The first stimulation did not induce neurite extension, whereas the second stimulation induced fast neurite extension; therefore, the first stimulation is required as prerequisite conditions. This indicates that the action of NGF can be divided into two processes: the first stimulation-driven latent process and the second stimulation-driven extension process. The latent process appears to require ERK and transcription, but not PI3K, activities, whereas the extension-process requires ERK and PI3K, but not transcription, activities. We also found that NGF in the first stimulation can be replaced by PACAP, but not by insulin, EGF, bFGF or forskolin, whereas NGF in the second stimulation cannot be replaced by any of these stimulants. These findings allowed us to identify potential genes specifically involved in the latent process, rather than in other processes. These results demonstrate that NGF induces differentiation of PC12 cells via mechanically distinct processes: the ERK-driven and transcription-dependent latent process; and the ERK- and PI3K-driven, and transcription-independent extension process.

overall_design: Gene expression in PC12 cells was measured at 3 hours after following stimulations, complete medium (n = 4), continuous NGF (n = 2), 1 hour of NGF (n = 4), 1 hour of PACAP (n = 2), 1 hour of Insulin (n = 2), 1 hour of NGF in presence of U0126 (n = 2) or 1 hour of NGF in presence of LY294002 (n = 2).

first: Hiroyuki

last: Kubota

first: Yu-ichi

last: Ozaki

first: Shinsuke

last: Uda

first: Shinya

last: Kuroda


title: RNA profiles of rat olfactory epithelia : individual and age related variations (age experiment)

overall_design: Four female rats with their newborn pups (n=19; 3 to 5 days old) were purchased from Charles River laboratory. RNA was extracted from one side only of the nasal epithelium of each newborn rat. RNA was also extracted from the left and right olfactory epithelium of four 22 months-old male rats, kept in the animal house since they were 3 weeks-old, and from four 9 weeks-old male rats. Total RNA from olfactory epithelia were extracted, labeled and used for hybridization on Agilent Whole Rat Genome 44K microarrays.


title: Proteomics and transcriptomics analysis of rat retinal ganglion cells exposed to chronic ocular hypertension.

status: Public on Aug 20 2009

city: Miami

first: Dmitry

last: Ivanov

state: FL

address: 1638 NW 10th Ave

country: USA

institute: University of Miami

summary: In this work, we tested feasibility of high content screening of acutely isolated RGCs to generate systems biology knowledge of intrinsic cellular pathways associated with the onset of glaucomatous RGC loss in the retina. We performed differential profiling of these neurons using two complementary techniques: proteomics and microarray-based transcriptomics. The analysis of these RGC-specific proteomic and transcriptomic data using pathway informatics databases and biological network tools have revealed complex metabolic and regulatory changes in the COH-challenged neurons. We used the iTRAQ reagents that allow for the identification and quantitation of up to four different samples simultaneously, to assess the COH-induced changes in protein content of adult rat RGCs. Similar principle is utilized in the two-color Agilent arrays that we used for our transcriptomic analysis of the same RGC samples.

overall_design: We performed high-content analyses of molecular changes in retinal ganglion cells associated with chronic ocular hypertension (COH). Gene expression profiling was performed using two-color microarrays. To assess differences in the gene expression levels in COH-challenged vs. normotensive RGCs, the “glaucomatous RGC/ reference RNA” ratios were divided by the ratios of “normotensive RGCs /reference RNA” obtained from the same group of experimental animals. Equivalent amounts of reference and experimental aRNAs were hybridized with the Agilent Rat Oligo Microarrays (Agilent, USA) according to the manufacturer’s instructions. Each microarray was scanned and normalized for signal intensities across the whole array and locally, using the Lowess normalization. We analyzed only those genes that passed Agilent quality control criteria. To reveal the genes with significant changes in glaucoma, we first calculated ratios of expression levels in experimental vs. fellow control eyes for each of four independent groups, and then selected genes with consistent changes using ANOVA and the cut-off p-value <0.05. Within each group, experimental and control RNA was extracted from RGCs purified from eye pairs originating from the same animal.  Within each pair, as well as within each pooled group, total RNA abundances  were, therefore, normalized for individual differences, so as were the resultant gene expression ratios. A fold change difference ≥1.5 was used to determine differentially expressed genes.

first: Valery

middle: I

last: Shestopalov


title: Characterization of the transcriptome of the low oil 1 (loo1) mutant

status: Public on Apr 12 2009

city: Urbana

first: Yun

last: Lin

state: IL

address: 183 NSRC, 1101 W. Peabody Dr.

department: Crop Sciences

institute: University of Illinois

summary:
Gene expression profiles in 15 day old low oil 1 (loo1) mutant and the wild type A15 seedlings were compared.
Keywords: Comparison and loo1 and wild type seedling transcriptomes

overall_design: The loo1 mutant and the wild type seedlings transcriptomes were compared using 70-mer oligoarrays. Two experimental repeats were conducted, each with two dye-swap hybridizations.

first: Shui

last: Wang


title: The genetics of wound healing in tympanic membranes in rats

status: Public on Aug 19 2009

city: Perth

first: Peter

middle: Luke

last: Santa Maria

state: WA

address: 2nd floor M Block, QE II Medical Centre, Nedlands

department: Ear Sciences

country: Australia

institute: University of Western Australia

summary: The aim of this study was to identify the genes differentially expressed between timepoints in the week following tympanic membrane perforation in rats. Tissue from 240 individual rats was used in this study following random allocation into timepoint groups to be sacrificed over 7 days. An Agilent one color microarray technique was performed and the results were analyzed using Genespring GX9 software. A total of 3262 genes were identified as significant (p<0.05) and differentially expressed above a two-fold threshold between the timepoints. This study provides a complete genetic review of rat tympanic membrane wound healing over 7 days. The results can be used as a model for other wound healing in other mammals and in different parts of the body. The information on differential gene expression can be used in research towards developing chronic tympanic membrane perforations and also in research to treat acute and chronic tympanic membrane perforations. The microarray was performed on animals in a disease free environment and the genetic information can be compared to future research in disease states of the TM including Otitis media, cholesteatoma, chronic perforation and tympanosclerosis.

overall_design: Rats were randomly selected as either controls or in the perforation group. Perforations were created unilaterally (left ear) in the upper outer quadrant of the pars tensa of rats’ tympanic membranes using sterile 23 gauge needles . Rats were then randomly allocated into timepoint groups to be sacrificed at either 12, 24, 36, day 2, 3, 4, 5, 6, 7.  At the point of microarray, there were 18 rats per timepoint group and 18 controls.

middle: L

last: SantaMaria

first: S

last: Redmond

first: R

last: McInnes

first: M

last: Atlas

last: Ghassemifar


title: Prostates of rats with induced partial bladder outlet obstruction and the effect of Eviprostat on those changes

status: Public on Jul 21 2009

city: Tsukuba

first: Mitsuhiro

last: Tagaya

state: Ibaraki

address: 3-14-1 Sakura

department: Discovery Research Laboratories

institute: Nippon Shinyaku co.,ltd.

summary: Prostatic inflammation plays a role in the progression of benign prostatic hyperplasia (BPH). Eviprostat is an antiinflammatory and antioxidant phytotherapeutic agent widely used to treat lower urinary tract symptoms in BPH. However, because Eviprostat is a mixture of compounds from multiple natural sources, its mechanism of action has been difficult to investigate. In this study, we used oligonucleotide microarrays to identify changes in gene expression that occur in the prostate of rats with surgically induced partial bladder outlet obstruction and the effect of Eviprostat on those changes.

overall_design: Male rats were divided into four groups of five or six animals each. The rats in groups 1 and 2 underwent a sham operation. Five days after the sham operation, group 1 was treated twice a day with vehicle (0.1% (w/v) Tween 80; sham/vehicle group) and group 2 was treated twice a day with 18 mg/kg Eviprostat (36 mg/kg daily; sham/Eviprostat group). The rats in groups 3 and 4 underwent surgical partial bladder outlet obstruction. Briefly, with the rat in the supine position, a midline suprapubic incision was made and the bilateral prostate was retracted to expose the neck of the bladder and the urethra, care being taken not to damage the bladder. The loose connective tissue at the base of the bladder was dissected away from the proximal urethra. A rubber ring was cut open and placed around the proximal urethra, and a 4-0 silk ligature was tied around the rubber ring. From the day after the operation, group 3 was treated twice a day for five days with vehicle (operated/vehicle group) and group 4 was treated twice a day for five days with 18 mg/kg Eviprostat (36 mg/kg daily; operated/Eviprostat group). On the sixth day, 2 h after the last administration, the rats were sacrificed and the prostate was rapidly removed.

first: Michiko

last: Oka

first: Makoto

last: Ueda

first: Kazuchika

last: Takagaki

first: Mitsushi

last: Tanaka

first: Tadaaki

last: Ohgi

first: Junichi

last: Yano


status: Public on Jul 17 2009

city: Ede

first: Marloes

last: Schepens

address: P.O. Box 20

institute: NIZO food research

summary:
Background and Aims: We have shown in several controlled rat and human infection studies that dietary calcium improves intestinal resistance and strengthens the mucosal barrier. Reinforcement of gut barrier function is also relevant for inflammatory bowel disease (IBD). Therefore, we investigated the effect of supplemental calcium on spontaneous colitis development in HLA-B27 transgenic rats, an experimental animal model of IBD.
Methods: HLA-B27 transgenic rats were fed a purified high-fat diet containing either a low or high calcium content (30 and 120 mmol CaHPO4/kg diet, respectively) for almost 7 weeks. Inert chromium ethylenediamine-tetraacetic acid (CrEDTA) was added to the diets to quantify intestinal permeability by measuring urinary CrEDTA excretion. Relative fecal dry-weight was determined to quantify diarrhea. Colonic inflammation was determined histologically, and by measuring mucosal interleukin-1β. In addition, colonic mucosal gene expression of individual rats was analyzed, using whole genome microarrays. Interesting results were verified by Q-PCR. Results: The high-calcium diet significantly prevented the increase in intestinal permeability and diarrhea with time in HLA-B27 rats developing colitis as compared to the low-calcium group. The histological colitis score and mucosal interleukin-1β levels were lower in high-calcium fed rats. Supplemental calcium prevented the colitis-induced increase in the expression of extracellular matrix remodeling genes (e.g. matrix metalloproteinases, procollagens and fibronectin), which was confirmed by Q-PCR.
Conclusions: Dietary calcium inhibits colitis development in HLA-B27 transgenic rats. Calcium prevents the colitis-related increase in intestinal permeability, diminishes diarrhea, and lowers the inflammatory response in the mucosa, resulting in less extracellular matrix breakdown.
Keywords: nutritional intervention

overall_design:
Female HLA-B27/β2-microglobulin transgenic rats on an inbred Fisher 344 background (n=8 in experimental group and n=9 in control group) (Taconic Farms, Inc, Germantown, NY), 8-10 weeks old and with a mean body weight of 128 g at the start of the experiment, were housed individually in metabolic cages. Animals were kept in a temperature- and humidity-controlled environment and in a 12-h light-dark cycle. Rats were fed a purified ‘humanized’ Western diet which contained in the control situation (per kg): 200 g acid casein, 326 g corn starch, 174 g glucose, 160 g palm oil, 40 g corn oil, 50 g cellulose and 5.16 g CaHPO4.2H2O (corresponding to 30 mmol calcium/kg diet; Sigma-Aldrich, St Louis, MO). Vitamins and minerals (other than calcium) were added to all diets according to the recommendations of the American Institute of Nutrition 1993.17 The experimental diet contained more calcium (120 mmol calcium/kg diet) at the expense of glucose.
All samples were individually labelled and hybridized (Cy5). Equal amounts of Cy3 cRNA of all animals were pooled to serve as standard reference pool.


title: Transcriptomic Analysis of Nephrotoxicity Induced by Cephaloridine, a Representative Cephalosporin Antibiotic

status: Public on Jun 23 2008

city: Osaka

first: Masatomo

last: Rokushima

state: Osaka

address: 12-4, Sagisu 5-chome, Fukushima-ku

institute: SHIONOGI & CO., LTD.

summary:
Cephaloridine (CER) is a classical beta-lactam antibiotic that has long served as a model drug for the study of cephalosporin antibiotic-induced acute tubular necrosis.  In the present study, we analyzed gene expression profiles in the kidney of rats given subtoxic and toxic doses of CER in order to identify gene expression alterations closely associated with CER-induced nephrotoxicity.  Male Fisher 344 rats were intravenously injected with three different doses (150, 300, and 600 mg/kg) of CER, and sacrificed after 24 h.  Only the high dose (600 mg/kg) caused mild proximal tubular necrosis and a slight renal dysfunction.  Microarray analysis identified hundreds of genes differentially expressed in the renal cortex following the exposure to CER, which could be classified into two main groups that were deregulated in dose-dependent and high dose-specific manners.  The genes upregulated dose-dependently mainly included those involved in detoxification and antioxidant defense, which was considered to be associated with CER-induced oxidative stress.  In contrast, the genes showing high dose-specific (lesion-specific) induction included a number of genes related to cell proliferation, which appeared to reflect a compensatory response to CER injury.  We also found a subset of G2/M phase genes that exhibited hormesis-like (U-Shape) biphasic dose response; namely, downregulation only at the low and/or middle (subtoxic) doses.  Furthermore, we could predict potential transcription regulators responsible for the observed gene expression alterations, such as Nrf2 and E2F family.  Among the candidate gene biomarkers, kidney injury molecule 1 was markedly upregulated at the mildly toxic dose, suggesting that this gene can be used as an early and sensitive indicator for cephalosporin nephrotoxicity.  In conclusion, our transcriptomic data revealed several characteristic expression patterns of genes associated with specific cellular processes, including oxidative stress response and proliferative response, upon exposure to CER, which may enhance our understandings of molecular mechanisms behind cephalosporin antibiotic-induced nephrotoxicity.
Keywords: compound treatment, dose response

overall_design: In the present study, we acquired gene expression profiles in the kidney of rats given subtoxic and toxic doses of cephaloridine (CER) using whole-genome oligonucleotide microarrays.  Male rats were injected with vehicle alone and three different doses (150, 300, and 600 mg/kg) of CER, and sacrificed after 24 h.  Each dose group contained 3 animals.

first: Kae

last: Fujisawa

first: Naoko

last: Furukawa

first: Fumio

last: Itoh

first: Toru

last: Yanagimoto

first: Ryou

last: Fukushima

first: Akiko

last: Araki

first: Manabu

last: Okada

first: Mikinori

last: Torii

first: Ikuo

last: Kato

first: Jun

last: Ishizaki

first: Kazuo

last: Omi


title: Sex-specific early growth hormone response genes in rat liver (Rattus norvegicus)

status: Public on May 24 2008

city: Boston

first: David

middle: J.

last: Waxman

state: MA

address: 5 Cummington Mall

department: Department of Biology and Bioinformatics Program

institute: Boston University

summary: A series of two color gene expression profiles obtained using Agilent 44K expression microarrays was used to examine sex-dependent and growth hormone-dependent differences in gene expression in rat liver.  This series is comprised of pools of RNA prepared from untreated male and female rat liver, hypophysectomized (‘Hypox’) male and female rat liver, and from livers of Hypox male rats treated with either a single injection of growth hormone and then killed 30, 60, or 90 min later, or from livers of Hypox male rats treated with two growth hormone injections spaced 3 or 4 hr apart and killed 30 min after the second injection.  The pools were paired to generate the following 6 direct microarray comparisons: 1) untreated male liver vs. untreated female liver; 2) Hypox male liver vs. untreated male liver; 3) Hypox female liver vs. untreated female liver; 4) Hypox male liver vs. Hypox female liver; 5) Hypox male liver + 1 growth hormone injection vs. Hypox male liver; and 6) Hypox male liver + 2 growth hormone injections vs. Hypox male liver.  A comparison of untreated male liver and untreated female liver liver gene expression profiles showed that of the genes that showed significant expression differences in at least one of the 6 data sets, 25% were sex-specific.  Moreover, sex specificity was lost for 88% of the male-specific genes and 94% of the female-specific genes following hypophysectomy. 25-31% of the sex-specific genes whose expression is altered by hypophysectomy responded to short-term growth hormone treatment in hypox male liver.  18-19% of the sex-specific genes whose expression decreased following hypophysectomy were up-regulated after either one or two growth hormone injections.  Finally, growth hormone suppressed 24-36% of the sex-specific genes whose expression was up-regulated following hypophysectomy, indicating that growth hormone acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression.  For full details, see V. Wauthier and D.J. Waxman, Molecular Endocrinology (2008)

overall_design: This series is comprised of pools of liver RNA prepared from untreated male, hypophysectomized (‘Hypox’) male, untreated female and Hypox female rats (3-4 livers/pool), as well as liver RNA prepared from Hypox male rats treated with a single growth hormone injection and killed either 30, 60, or 90 minutes later (pool of n = 4 livers) or from Hypox male rats treated with two growth hormone injections spaced 3 or 4 hr apart (pool of n = 5 livers).  The pools were paired to generate the following 6 direct microarray comparisons: 1) untreated male liver vs. untreated female liver; 2) Hypox male liver vs. untreated male liver; 3) Hypox female liver vs. untreated female liver; 4) Hypox male liver vs. Hypox female liver; 5) Hypox male liver + 1 growth hormone injection vs. Hypox male liver; and 6) Hypox male liver + 2 growth hormone injections vs. Hypox male liver.  Dye swapping experiments were carried out for each of the six hybridization experiments, as follows. The Alexa 555-labeled cDNA from one of the two untreated male pools was mixed with the Alexa 647-labeled cDNA from one of the two untreated female pools.  Similarly, Alexa 647-labeled cDNA from the second untreated male pool was mixed with the Alexa 555-labeled cDNA from the second untreated female pool.  Together, these two mixed cDNA samples comprise a fluorescent reverse pair (dye swap).  Dye swaps were similarly carried out for each of the five other competitive hybridization experiments, except that for experiments 5 and 6, a single pool of M-Hypox + GH liver cDNA, or a single pool of M-Hypox + 2GH liver cDNA, was used in each half of the fluorescent reverse pair.  Two microarrays, one for each mixed cDNA sample, were hybridized for each of the six fluorescent reverse pairs, giving a total of 12 microarrays.

first: Valerie

last: Wauthier

middle: J