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PubMed2000

Fundamentals of DNA hybridization arrays for gene expression analysis.

DNA hybridization arrays [also known as macroarrays, microarrays and/or high-density oligonucleotide arrays (Gene Chips)] bring gene expression analysis to a genomic scale by permitting investigators to simultaneously examine changes in the expression of literally thousands of genes. For hybridization arrays, the general approach is to immobilize gene-specific sequences (probes) on a solid state matrix (nylon membranes, glass microscope slides, silicon/ceramic chips). These sequences are then queried with labeled copies of nucleic acids from biological samples (targets). The underlying theory is that the greater the expression of a gene, the greater the amount of labeled target, and hence, the greater output signal. In spite of the simplicity of the experimental design, there are at least four different platforms and several different approaches to processing and labeling the biological samples. Moreover, investigators must also determine whether they will utilize commercially available arrays or generate their own. This review will cover the status of the hybridization array field with an eye toward underlying principles and available technologies. Future developments and technological trends will also be evaluated.

Cluster Analysis↗
PubMed2005

Feasibility of RNA collection for micro-array gene expression analysis in the treatment of cervical carcinoma: a scientific correlate of RTOG C-0128.

PURPOSE: To determine the feasibility of RNA collection in a multi-institutional cooperative group setting to be utilized for micro-array gene expression analysis, and to describe the methodology. METHODS: RTOG C0128, a phase I-II, protocol was designed to look at the safety and efficacy of external beam radiation therapy to 45 Gy with concomitant 5-FU and cisplatin chemotherapy, brachytherapy to deliver 85 Gy to point A, and Celecoxib at 400 mg twice daily for 1 year. Patients had the option of participating in a tissue collection portion of the protocol to be utilized for micro-array gene expression analysis before treatment and at the time of the first implant. RNA quality was determined by two parameters: the absorbance ratio at 260 nm/280 nm, and by the ratio of the integrated peak of 28S RNA to 18S RNA after gel electrophoresis. RESULTS: From August 2001 to March 2004, 84 patients were accrued to the trial, and tissue was obtained prior to initiation of therapy on 34 patients (40%). FIGO stages for the patients who provided tissue were IB (23%), II (57%), and IIIA-IVA (20%). Additionally, biopsies were obtained at the time of the first implant from 22 of the accrued patients making paired samples available on 26% for RNA extraction and micro-array gene expression analysis. The mean +/- SEM amount of tissue obtained pretreatment was 97 +/- 13 mg compared with 51 +/- 8 mg for tissue obtained at the time of the first implant (P = 0.009). The mean total RNA extracted from the samples prior to treatment was 119 +/- 19 microg versus 35 +/- 6 microg at the time of the first procedure (P = 0.001). The RNA quality was assessed via the absorbance ratio at 260 nm divided by 280 nm. The mean values pretreatment and at first implant were 1.87 +/- 0.07 versus 1.66 +/- 0.11, respectively (P = 0.002); however, the integrated peak of 28S RNA to 18S RNA after gel electrophoresis was not significantly different (P = 0.26). CONCLUSIONS: RNA extraction for gene expression analysis can be successfully performed in the multi-institutional cooperative group setting. Fresh tissue samples were obtained on 40% of accrued patients prior to treatment. The amount of biopsy material and the quantity of RNA extracted were greater prior to treatment compared with the first implant. The quality of RNA was superior prior to treatment as measured by the ratio of absorbance at 260/280 nm. These results indicate that gene expression analysis is feasible in the cooperative group setting utilizing amplification techniques for the RNA. Hopefully, this will allow for improvement in prognosis, therapeutic development, and correlation with acute and late toxicities in patients with cancer.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed2004

An mRNA amplification procedure with directional cDNA cloning and strand-specific cRNA synthesis for comprehensive gene expression analysis.

We developed an integrated system suitable for comprehensive gene expression studies including construction and analysis of cDNA microarrays starting from a small amount of mRNA. We amplified total mRNA first as cDNA mixtures by polymerase chain reaction and then as strand-specific cRNA mixtures by in vitro transcription. These amplified cDNA and cRNA enabled determination of mRNA levels by hybridization analyses such as Southern, Northern, reverse-Northern macroarray, and cDNA microarray analyses, as well as construction of the cDNA library with a unidirectional cDNA insert. By using strand-specific cRNA derived from rat primary-cultured hepatocytes, we detected putative antisense transcripts for the metallothionein gene. cDNA microarray analysis for genes regulated by glucocorticoids and glucagon in the hepatocytes revealed that a number of genes involved in signal transduction and transcriptional regulation were up- or down-regulated. The present system is widely applicable to gene expression analysis with limited amounts of RNA samples.

Animals↗
PubMed2002

Gene expression analysis of 1,25(OH)2D3-dependent differentiation of HL-60 cells: a cDNA array study.

The alterations in gene expression associated with 1,25(OH)2D3-induced differentiation of HL-60 cells were studied in order to identify potential targets for further investigation of the genetic basis of acute myeloid leukaemia. Atlas human haematology filters, including 406 genes (Clontech), were used to study gene expression in response to 1,25(OH)2D3 (concentration, 5 x 10-8 mol/l) for 24 and 72 h. Compared with untreated cells, expression differences were found in 43 genes. Downregulated genes at both time-points were: IL2RA, CMYC, NPM, DEK, AF4, FLI1, htlf, MNDA, BCR, IKAROS, BPI and NFAT4. Upregulated genes at both time-points were IL1B, CD14 and MCL1. CD55, CD58, IRF2, CREB1, ATF4, RAC1, TIAR, KIAA0053, BAT2, BTK, RCK, EV12B and EDN were downregulated at 24 h, while SPI1, MKK3, BTG1 and IL8 were upregulated. At 72 h the upregulated genes were IL1RA, IL2RG, CXCR4, SCYA1, SCYA3, SCYA4, SCYA5, SCYA22, ANX2, CD83 and UPAR. cDNA array results were confirmed on randomly selected genes using quantitative real-time polymerase chain reaction for three upregulated (CXCR4, IL1B and CD14) and three downregulated (DEK, AF4 and FLI1) genes. Gene expression analysis after differentiation induction may provide a tool to study the roles of DEK, AF4 and FLI1 in cell proliferation and differentiation. To demonstrate the genes that initiate differentiation, sequential gene expression analysis has to be performed during the first 24 h of the differentiation process.

Calcitriol↗
PubMed2000

Large scale gene expression analysis of cholesterol-loaded macrophages.

We conducted large scale gene expression analysis of the response of macrophages to exposure to oxidized low density lipoprotein (Ox-LDL). Much of the vessel wall lesion of atherosclerosis is composed of macrophages that have become engorged with cholesterol. These resulting "foam cells" contribute to the progression of vascular disease through several pathways. As a potential model of foam cell formation, we treated THP-1 cells with 12-O-tetradecanoylphorbol 13-acetate to differentiate them into a macrophage-like phenotype and subsequently treated them with oxidized low density lipoprotein for various time periods. RNA from Ox-LDL treated and time-matched control untreated cells was hybridized to microarrays containing 9808 human genes. 268 genes were found to be at least 2-fold regulated at one or more time points. These regulation patterns were classified into seven clusters of expression profiles. The data is discussed in terms of the overall pattern of gene expression, the thematic classification of the responding genes, and the clustering of functional groups in distinct expression patterns. The magnitude and the temporal patterns of gene expression identified known and novel molecular components of the cellular response that are implicated in the growth, survival, migratory, inflammatory, and matrix remodeling activity of vessel wall macrophages. In particular, the role of nuclear receptors in mediating the gene expression modulation by Ox-LDL is highlighted.

Cell Line↗
PubMed2005

Microarray gene expression analysis free of reverse transcription and dye labeling.

A new microarray system has been developed for gene expression analysis using cationic gold nanoparticles with diameters of 250 nm as a target detection reagent. The approach utilizes nonlabeled target molecules hybridizing with complementary probes on the array, followed by incubation in a colloidal gold solution. The hybridization signal results from the precipitation of nanogold particles on the hybridized spots due to the electrostatic attraction of the cationic gold particles and the anionic phosphate groups in the target DNA backbone. In contrast to conventional fluorescent detection, this nanoparticle-based detection system eliminates the target labeling procedure. The visualization of hybridization signals can be accomplished with a flatbed scanner instead of a confocal laser scanner, which greatly simplifies the process and reduces the cost. The sensitivity is estimated to be less than 2 pg of DNA molecules captured on the array surface. The signal from hybridized spots quantitatively represents the amount of captured target DNA and therefore permits quantitative gene expression analysis. Cross-array reproducibility is adequate for detecting twofold or less signal changes across two microarray experiments.

Bacteriophages↗
PubMed2005

[A modern method for differential gene expression analysis and its significance for cancer diagnostics and therapy].

Contemporary approaches to diagnostics and therapy in oncology are nowadays tightly coupled to novel findings of biomedical science. One of the main trends in molecular medicine is the development of methodologies enabling parallel monitoring of expression of large quantities of genes or proteins--so called functional genomics and proteomics. These techniques allow determination of differential gene expression, i.e. evaluation of differences in gene expression between two or more cell or tissue samples of different types (e.g. normal or cancer cells) or coming from different culture conditions. These approaches help in elucidating causes of malignant transformation and can serve as a base for development of targeted anticancer gene therapy, monitoring of patient response to treatment and prediction of further disease development. Genomic approaches have undergone rapid development in the last few years--from differential and subtractive hybridisation through differential display all the way to serial analysis of gene expression and DNA microarrays. Besides that, tissue and protein arrays and other proteomic approaches have been also used. Currently DNA microarrays covering expression of the whole human genome, having significant potential in oncological research and clinical praxis, have been used more and more frequently. Many new tumor growth and progression markers were found using such approaches. Some of these markers have been already successfully used in clinical practice (e.g. in breast cancer) for therapy optimisation and minimisation of patient discomfort.

Gene Expression Profiling↗
PubMedDate not supplied

House keeping genes and gene expression analysis in transplant recipients: a note of caution.

BACKGROUND: House keeping genes are often used as a means of standardising results obtained in gene expression investigations. This study was performed to investigate whether beta-actin, beta2-microglobulin (two genes frequently quoted as house keeping genes) and/or transferrin receptor would be suitable house keeping genes for use in gene expression analysis of renal transplant recipients. METHODS: Sequential expression of all three genes was measured in the peripheral blood mononuclear cells of 13 living donors and 45 renal transplant recipients, pre-operatively and then daily for up to 2 weeks. Fifteen of the recipients experienced an episode of biopsy proven acute rejection. Gene expression measurement was performed using quantitative real time 'TaqMan' PCR technology. RESULTS: Gene expression of all three genes was unchanged in the living donor cohort. However, in the transplant recipients there were significant increases in expression following transplantation in the non-rejectors, and preceding the diagnosis of acute rejection. In the latter group, levels returned to pre-transplant values after the commencement of anti-rejection therapy. CONCLUSIONS: Beta-actin, beta2-microglobulin and transferrin receptor gene expression, although not influenced by surgery, is influenced by transplantation, acute rejection and anti-rejection therapy making these genes unsuitable as house keeping genes following renal transplantation. These findings may cast doubt on the results of some studies that used these genes for the purposes of standardisation when looking at cDNA measurement. We suggest that any group wishing to use a house keeping gene ensure that its expression is independent of study parameters prior to the start of the study.

Actins↗
PubMed2000

Large-scale screen for genes controlling mammalian embryogenesis, using high-throughput gene expression analysis in mouse embryos.

We have adapted the whole-mount in situ hybridization technique to perform high-throughput gene expression analysis in mouse embryos. A large-scale screen for genes showing specific expression patterns in the mid-gestation embryo was carried out, and a large number of genes controlling development were isolated. From 35760 clones of a 9.5 d.p.c. cDNA library, a total of 5348 cDNAs, enriched for rare transcripts, were selected and analyzed by whole-mount in situ hybridization. Four hundred and twenty-eight clones revealed specific expression patterns in the 9.5 d.p.c. embryo. Of 361 tag-sequenced clones, 198 (55%) represent 154 known mouse genes. Thirty-nine (25%) of the known genes are involved in transcriptional regulation and 33 (21%) in inter- or intracellular signaling. A large number of these genes have been shown to play an important role in embryogenesis. Furthermore, 24 (16%) of the known genes are implicated in human disorders and three others altered in classical mouse mutations. Similar proportions of regulators of embryonic development and candidates for human disorders or mouse mutations are expected among the 163 new mouse genes isolated. Thus, high-throughput gene expression analysis is suitable for isolating regulators of embryonic development on a large-scale, and in the long term, for determining the molecular anatomy of the mouse embryo. This knowledge will provide a basis for the systematic investigation of pattern formation, tissue differentiation and organogenesis in mammals.

Animals↗
PubMed2006

Gene expression analysis in blood cells in response to unmodified and 2'-modified siRNAs reveals TLR-dependent and independent effects.

Ribonucleic nucleic acid recognition by Toll-like receptors (TLRs) induces innate immune responses. However, no comprehensive analysis of gene expression in human blood cells in response to unmodified and 2'-modified immunostimulatory RNAs has been reported. Using oligonucleotide microarrays, we show that around 400 genes were significantly (P<0.001) altered in peripheral blood mononuclear cells (PBMC) in response to either single-stranded (ss) or double-stranded (ds) small interfering RNAs (siRNAs). Most of the upregulated genes encode proteins involved in innate and adaptive immune responses, including proinflammatory cytokines, interferons, chemokines and chemokine receptors. Genes encoding proteins involved in lymphocyte activation (e.g. CD80, CD40, and CD69) and in regulation of the immune responses (e.g. SOCS proteins) were upregulated. Also, genes encoding for antiviral proteins (Mx1, Mx2, TRIM proteins), and interferon regulatory factors (e.g. IRF7) were upregulated. Around 90% of the genes (140 out of 160) affected by R-848, a specific ligand for TLR7 and TLR8, were also affected by ss siRNAs or ds siRNAs, indicating that the signaling pathways activated by R-848 are also activated by immunostimulatory siRNAs. In addition to immunoactivation via TLRs, ss siRNAs and ds siRNAs induced TLR-independent gene alterations. Surprisingly, replacement of only uridine bases with either 2'-fluoro or 2'-O-methyl modified counterparts abrogated all the observed bystander effects. Collectively, these microarray data offer for the first time an insight into human PMBC response to immunostimulatory RNAs such as ss siRNAs and ds siRNAs. The data should help to define strategies to either enhance or avoid the non-specific effects of siRNAs in order to develop safe therapeutics.

Gene Expression Profiling↗
PubMed2004

Reliability of t7-based mRNA linear amplification validated by gene expression analysis of human kidney cells using cDNA microarrays.

Genome wide gene expression analysis by cDNA microarrays is often limited by minute amounts of starting RNA. We therefore tested an optimized linear RNA amplification protocol using the RiboAmp amplification kit in the setting of cDNA microarrays. We isolated mRNA from a human kidney cell line (HK-2; ATCC) and from Universal Human Reference RNA (STR; Stratagene). After performing one and two rounds of linear RNA amplification, respectively, the amplified RNAs were co-hybridized to cDNA microarrays. Linearity and reproducibility of the individual experiments were then assessed by calculating the Pearson correlation. The intra-amplification consistency showed a correlation of 0.968 for the first round, 0.907 for the second round and 0.912 for two successive rounds of amplification. If the first round was compared to unamplified material, r was 0.925. The second round amplification yielded a correlation of 0.897 if compared to unamplified mRNA. Two rounds of amplification starting from 200 pg of mRNA compared to unamplified material resulted in a correlation of 0.868. These results indicate that linear amplification using RiboAmp kit yields amplified RNA with a high degree of linearity and reproducibility.

Cell Line↗
PubMed2005

Experimental comparison and cross-validation of the Affymetrix and Illumina gene expression analysis platforms.

The growth in popularity of RNA expression microarrays has been accompanied by concerns about the reliability of the data especially when comparing between different platforms. Here, we present an evaluation of the reproducibility of microarray results using two platforms, Affymetrix GeneChips and Illumina BeadArrays. The study design is based on a dilution series of two human tissues (blood and placenta), tested in duplicate on each platform. The results of a comparison between the platforms indicate very high agreement, particularly for genes which are predicted to be differentially expressed between the two tissues. Agreement was strongly correlated with the level of expression of a gene. Concordance was also improved when probes on the two platforms could be identified as being likely to target the same set of transcripts of a given gene. These results shed light on the causes or failures of agreement across microarray platforms. The set of probes we found to be most highly reproducible can be used by others to help increase confidence in analyses of other data sets using these platforms.

Adult↗
PubMed2005

Scalable transcriptional analysis routine--multiplexed quantitative real-time polymerase chain reaction platform for gene expression analysis and molecular diagnostics.

We report the development of a new technology for simultaneous quantitative detection of multiple targets in a single sample. Scalable transcriptional analysis routine (STAR) represents a novel integration of reverse transcriptase-polymerase chain reaction and capillary electrophoresis that allows detection of dozens of gene transcripts in a multiplexed format using amplicon size as an identifier for each target. STAR demonstrated similar or better sensitivity and precision compared to two commonly used methods, SYBR Green-based and TaqMan probe-based real-time reverse transcriptase-polymerase chain reaction. STAR can be used as a flexible platform for building a variety of applications to monitor gene expression, from single gene assays to assays analyzing the expression level of multiple genes. Using severe acute respiratory syndrome (SARS) corona virus as a model system, STAR technology detected single copies of the viral genome in a two-gene multiplex. Blinded studies using RNA extracted from various tissues of a SARS-infected individual showed that STAR correctly identified all samples containing SARS virus and yielded negative results for non-SARS control samples. Using alternate priming strategies, STAR technology can be adapted to transcriptional profiling studies without requiring a priori sequence information. Thus, STAR technology offers a flexible platform for development of highly multiplexed assays in gene expression analysis and molecular diagnostics.

Animals↗
PubMed2004

Microarray gene expression analysis of the Fob3b obesity QTL identifies positional candidate gene Sqle and perturbed cholesterol and glycolysis pathways.

Obesity-related diseases are poised to become the primary cause of death in developed nations. While a number of monogenic causes of obesity have recently been identified, these are responsible for only a small proportion of human cases of obesity. Quantitative trait locus (QTL) studies using animal models have revealed hundreds of potential loci that affect obesity; however, few have been further analyzed beyond the original QTL scan. We previously mapped four QTL in an F(2) between divergently selected Fat (F) and Lean (L) lines. A QTL of large effect on chromosome 15 (Fob3) was subsequently mapped to a higher resolution into two smaller-effect QTL (Fob3a and Fob3b) using crosses between the F-line and a congenic line containing L-line alleles at the Fob3 QTL region. Here we report the gene expression characterization of Fob3b. Microarray expression analysis using the NIA-NIH 15K cDNA array set containing 14,938 mouse ESTs was employed to identify candidate genes and pathways that are differentially expressed between the F-line and a congenic line containing only the Fob3b QTL (Fob3b-line). Our study suggests squalene epoxidase (Sqle), a cholesterol biosynthesis enzyme, as a strong positional candidate gene for Fob3b. Several other cholesterol biosynthesis pathway genes unlinked to Fob3b were found to be differentially expressed, suggesting that a perturbation of this pathway could be in part responsible for the phenotypic difference between the F-line and Fob3b-line mice.

Animals↗
PubMed2005

Gene expression analysis of Tek/Tie2 signaling.

The elaboration of the vasculature during embryonic development involves restructuring of the early vessels into a more complex vascular network. Of particular importance to this vascular remodeling process is the requirement of the Tek/Tie2 receptor tyrosine kinase. Mouse gene-targeting studies have shown that the Tie2-deficient embryos succumb to embryonic death at midgestation due to insufficient sprouting and remodeling of the primary capillary plexus. To identify the underlying genetic mechanisms regulating the process of vascular remodeling, transcriptomes modulated by Tie2 signaling were analyzed utilizing serial analysis of gene expression (SAGE). Two libraries were constructed and sequenced using embryonic day 8.5 yolk sac tissues from Tie2 wild-type and the Tie2-null littermates. After tag extraction, 45,689 and 45,275 SAGE tags were obtained for the Tie2 wild-type and Tie2-null libraries, respectively, yielding a total of 21,376 distinct tags. Close to 62% of the tags were uniquely annotated, whereas 10% of the total tags were unknown. Using semiquantitative PCR, the differential expression of eight genes was confirmed that included Elk3, an important angiogenic switch gene which was upregulated in the absence of Tie2 signaling. The results of this study provide valuable insight into the potential association between Tie2 signaling and other known angiogenic pathways as well as genes that might have novel functions in vascular remodeling.

Animals↗
PubMed2003

Gene expression analysis of plant host-pathogen interactions by SuperSAGE.

The type III restriction endonuclease EcoP15I was used in isolating fragments of 26 bp from defined positions of cDNAs. We call this substantially improved variant to the conventional serial analysis of gene expression (SAGE) procedure "SuperSAGE." By applying SuperSAGE to Magnaporthe grisea (blast)-infected rice leaves, gene expression profiles of both the rice host and blast fungus were simultaneously monitored by making use of the fully sequenced genomes of both organisms, revealing that the hydrophobin gene is the most actively transcribed M. grisea gene in blast-infected rice leaves. Moreover, SuperSAGE was applied to study gene expression changes before the so-called hypersensitive response in INF1 elicitor-treated Nicotiana benthamiana, a "nonmodel" organism for which no DNA database is available. Again, SuperSAGE allowed rapid identification of genes up- or down-regulated by the elicitor. Surprisingly, many of the down-regulated genes coded for proteins involved in photosynthesis. SuperSAGE will be especially useful for transcriptome profiling of two or more interacting organisms like hosts and pathogens, and of organisms, for which no DNA database is available.

Base Sequence↗