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PubMed2006

Gene expression analysis of mouse chromosome substitution strains.

An analysis of transcriptional variation in the liver using a panel of B.A chromosome substitution strains identified 4209 transcripts that are differentially expressed relative to the C57BL/6J background and 1010 transcripts that are differentially expressed between C57BL/6J and A/J strains. A subset of these strains (substituting Chromosomes 1, 6, and 15) was used to identify 386 additional differentially expressed transcripts in the kidney. Approximately 15% of differentially expressed transcripts are located on the substituted chromosome. These cis-QTL are codirectionally expressed with the donor strain A/J. By comparison, trans-regulated loci comprise 85% of differentially expressed transcripts, often show opposite direction of change compared with A/J, and can be regulated by multiple chromosome substitutions. Gene expression differences in this study provide evidence for transgressive segregation: Only 438 of 4209 QTL in liver were inside the parental range. By combining QTL data with known biological functions, we were able to identify physiologic pathways altered in multiple strains. In many cases the same pathways were altered by multiple distinct chromosome substitutions. Taken together, these results suggest that widespread epistatic background effects may result in complex and overlapping transcriptional relationships among different chromosome substitution strains. Transcriptional profiling of chromosome substitution strains reveals a complex genetic architecture of transcriptional regulation.

Animals↗
PubMed2026

Optimized Hot Phenol-Based RNA Extraction from Mycobacteria: A Robust Approach for Reliable Gene Expression Analysis.

Mycobacterium tuberculosis (Mtb) remains a major global health threat, underscoring the need for reliable transcriptomic studies to understand its biology and drug resistance mechanisms. Such analyses depend on obtaining high-quality, high-yield RNA. Although several RNA extraction methods are available, many require expensive reagents, large culture volumes, or specialized equipment, limiting their suitability for large-scale studies, particularly in resource-constrained settings. Here, an optimized Hot Phenol based RNA extraction method specifically tailored for mycobacteria is presented. The method uses minimal culture volume and commonly available reagents to consistently yield high-quality RNA suitable for high-throughput transcriptomic applications. RNA quantity and integrity were assessed by gel electrophoresis and RNA integrity analysis (RIN), and its suitability for downstream applications was confirmed by qPCR and Qubit 4. To benchmark the performance of the optimized method, a parallel RNA extraction using TRIzol and RNeasy under identical experimental conditions was carried out, including the same Mycobacterium species, culture volume, growth phase (logarithmic and stationary), and lysis conditions. This allowed a direct comparison of yield, quality, feasibility, and cost. The optimized Hot Phenol method demonstrated comparable or improved RNA yield and quality while significantly reducing reagent cost and dependence on specialized equipment. Owing to its efficiency, reproducibility, and affordability, this protocol provides a practical alternative for large-scale gene expression and transcriptomic studies in Mtb and other mycobacterial species.

RNA, Bacterial↗
PubMed2005

Large-scale gene expression analysis of cholesterol dependence in NS0 cells.

NS0, a nonsecreting mouse myeloma cell, is a major host line used for recombinant antibody production. These cells have a cholesterol-dependent phenotype and rely on an exogenous supply of cholesterol for their survival and growth. To better understand the physiology underlying cholesterol dependence, we compared NS0 cells, cultivated under standard cholesterol-dependent growth conditions (NS0), to cells adapted to cholesterol-independent conditions (NS0 revertant, NS0_r). Large-scale transcriptional analyses were done using the Affymetrix GeneChip array, MG-U74Av2. The transcripts expressed differentially across the two cell lines were identified. Additionally, proteomic tools were employed to analyze cell lysates from these two cell lines. Cellular proteins from both NS0 and NS0_r were subjected to 2D gel electrophoresis. MALDI-TOF mass spectrometry was performed to determine the identity of the differentially expressed spots. We examined the expression level of mouse genes directly involved in cholesterol biosynthesis, lipid metabolism, and central energy metabolism. Most of these genes were downregulated in the revertant cell type, NS0_r, compared to NS0. Overall, a large number of genes are expressed differentially, indicating that the reversal of cholesterol dependency has a profound effect on cell physiology. It is probable that a single gene mutation, activation, or inactivation is responsible for cholesterol auxotrophy. However, the wide-ranging changes in gene expression point to the distinct possibility of a regulatory event affecting the reversibility of auxotrophy, either directly or indirectly.

Animals↗
PubMed2004

Gene expression analysis in cartilage by in situ hybridization.

In situ hybridization allows detection and localization of specific nucleic acid sequences directly within a cell or tissue. We present an in situ hybridization protocol using double-stranded DNA or single-stranded RNA probes labeled with [32P] to localize and visualize the temporal and spatial distribution of cartilage-characteristic mRNAs. Probes labeled with this high-energy isotope provide good resolution at the tissue level with relatively low background; as a result of the probes that can be obtained that have a higher specificity to emulsion activity, very short exposure times are required.

Animals↗
PubMed2003

Differential gene expression analysis by micro-preparative capillary gel electrophoresis.

Differential display analysis by cDNA fractionation, collection of differentially expressed fractions of interests and their downstream characterization is demonstrated. cDNA pools from two strains of Cochliobolus heterostrophus fungus were generated by specific restriction digestion and selective ligation. Micropreparative separation and isolation of differentially expressed transcript representatives were accomplished by high-performance capillary gel electrophoresis. The collected individual DNA molecules were polymerase chain reaction amplified and sequenced to create expressed sequence tags for the genes of interests. High resolving power and sensitivity of capillary gel electrophoresis enabled fast and automated processing of minute amounts of cDNA samples with high precision.

Base Sequence↗
PubMed2004

Gene expression analysis by real-time reverse transcription polymerase chain reaction: influence of tissue handling.

Factors such as warm ischemia and time at room temperature before tissue treatment may influence the results of mRNA expression analyses on tissue specimens obtained during surgery. We evaluated the effect of these factors on RNA integrity and mRNA expression levels by incubating freshly obtained mouse liver tissue at 25 or 37 degrees C for periods of 0-4 h. Changes in the mRNA expression levels of seven genes, Tbp, Eef1a, Fos, Junb, Myc, Vegf, and Glut2, were determined by real-time reverse transcription-polymerase chain reaction. Incubation at 25 degrees C for up to 4 h only slightly altered (by a factor of less than 2) levels of mRNA for Tbp, Eef1a, Junb, Myc, Vegf, and Glut2. This result is consistent with limited RNA degradation at this temperature. Incubation at 37 degrees C strongly affected the levels of these mRNAs. Four hours of incubation at this temperature resulted in extensive RNA degradation, with mRNA levels falling to 1/10th those before incubation. When relative quantification was performed, i.e., quantification of the target gene transcripts in comparison to an endogenous housekeeping transcript (Tbp or Eef1a), the changes in mRNA levels were reduced to less than 2.5-fold. Fos behaved very differently from the other genes tested on incubation, with Fos mRNA levels increasing considerably following incubation at either 25 or 37 degrees C. Our data suggest that, with the exception of certain genes induced by tissue injury, relative quantification of mRNA, even on degraded RNA samples, can provide a reliable estimate of in vivo mRNA levels.

Animals↗
PubMedDate not supplied

Functional and gene expression analysis of the p53 signaling pathway in clear cell sarcoma of the kidney and congenital mesoblastic nephroma.

Mutation of p53 has been implicated in progression of classical Wilms tumor (WT) into the anaplastic variant (AWT), drug resistance, and poor prognosis. Because of prognostic similarities, clear cell sarcoma of the kidney (CCSK) has been classified with AWT and other aggressive pediatric renal tumors, apart from congenital mesoblastic nephroma (CMN), which is instead a relatively benign tumor of neonates. Initially, CCSK and CMN were assumed to be ontologically related, but the role of p53 in the pathogenesis of either disease has not been sufficiently evaluated as in AWT. We examined the status of p53 in CMN and CCSK in comparison to AWT by immunohistochemistry and mRNA analysis of p53, the downstream effector p21(WAF-1/CIP-1) ( p21), the multidrug resistance gene MDR-1, a putative target of p53, and the p53-antagonist Mdm-2. Surprisingly, strong p53 nuclear immunoreactivity was found in cultures from two CMN specimens, but not in frozen or fixed tumor tissue from five other CMN specimens, nor in cell lines or tumor tissue from CCSK. Sequence analysis excluded p53 mutations. The size of the p53 mRNA in CMN and CCSK primary tumors excluded gross deletions or rearrangements. Low levels of Mdm-2 mRNA in CCSK and CMN primary tumors and cultures did not support a role for Mdm-2. Absence of MDR-1 mRNA excluded MDR-1 in the drug-resistant phenotype of CCSK. Cisplatin-induced p21 transactivation assays and G(1) cell cycle arrest analyses showed that p21 transactivation and G(1) arrest occurred in both CCSK and CMN cultures, demonstrating integrity of the p53 signal transduction pathway. Absence of p53 functional abnormalities excluded relationships between CCSK and CMN as in AWT, supporting the association of cellular CMN with congenital fibrosarcomas as more recently proposed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Microarray gene expression analysis of murine tumor heterogeneity defined by dynamic contrast-enhanced MRI.

Current methods of studying angiogenesis are limited in their ability to serially evaluate in vivo function throughout a target tissue. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and pharmacokinetic modeling provide a useful method for evaluating tissue vasculature based on contrast accumulation and washout. While it is often assumed that areas of high contrast enhancement and washout comprise areas of increased angiogenesis and tumor activity, the actual molecular pathways that are active in such areas are poorly understood. Using DCE-MRI in a murine subcutaneous tumor model, we were able to perform pharmacokinetic functional analysis of a tumor, coregistration of MRI images with histological cross-sections, immunohistochemistry, laser capture microdissection, and genetic profiling of tumor heterogeneity based on pharmacokinetic parameters. Using imaging as a template for biologic investigation, we have not found evidence of increased expression of proangiogenic modulators at the transcriptional level in either distinct pharmacokinetic region. Furthermore, these regions show no difference on histology and CD31 immunohistochemistry. However, the expression of ribosomal proteins was greatly increased in high enhancement and washout regions, implying increased protein translation and consequent increased cellular activity. Together, these findings point to the potential importance of posttranscriptional regulation in angiogenesis and the need for the development of angiogenesis-specific contrast agents to evaluate in vivo angiogenesis at a molecular level.

Animals↗
PubMed2003

Characterization of fluoxetine plus olanzapine treatment in rats: a behavior, endocrine, and immediate-early gene expression analysis.

A large number of individuals afflicted with psychiatric disorders, particularly depression with psychotic features, do not respond to conventional drug therapy. An option for this phenomenon is to augment a standard selective serotonin (5-HT) reuptake inhibitor with an atypical antipsychotic agent. In this regard, fluoxetine and olanzapine have been used concomitantly for treatment-resistant depression and bipolar depression. Although highly efficacious in terms of producing superior improvement of symptoms across a variety of psychological measures, the motor patterns, endocrine profiles, and intracellular signaling pathways affected by drug augmentation have not been determined. Here we show that fluoxetine (10 mg/kg) plus olanzapine (5 mg/kg) given to rats for 7 consecutive days (i.e., subchronic treatment) alters motor activity and diminishes spontaneous behaviors as measured by spatial position and angular path analyses. In addition, the same drug combination pattern sensitizes peak adrenal corticosterone secretion without altering serum glucose levels. We also show that subchronic fluoxetine and olanzapine exposure suppresses the induction of two immediate-early gene transcription factors (e.g., pCREB and FOS) that are associated with long-lasting changes in synaptic efficacy and structural modifications in the prefrontal cortex, piriform cortex, and hippocampus. These results suggest that fluoxetine plus olanzapine can interact in a fashion not predicted by the currently accepted model of fluoxetine monotherapy and provide insight into the synergistic actions of drug augmentation in patients with treatment-resistant depression.

Animals↗
PubMed2001

Herbivore-induced volatile production by Arabidopsis thaliana leads to attraction of the parasitoid Cotesia rubecula: chemical, behavioral, and gene-expression analysis.

Many plant species defend themselves against herbivorous insects indirectly by producing volatiles in response to herbivory. These volatiles attract carnivorous enemies of the herbivores. Research on the model plant Arabidopsis thaliana (L.) Heynh. has contributed considerably to the unraveling of signal transduction pathways involved in direct plant defense mechanisms against pathogens. Here, we demonstrate that Arabidopsis is also a good candidate for studying signal transduction pathways involved in indirect defense mechanisms by showing that: (1) Adult females of Cotesia rubecula, a specialist parasitic wasp of Pieris rapae caterpillars, are attracted to P. rapae-infested Arabidopsis plants. (2) Arabidopsis infested by P. rapae emits volatiles from several major biosynthetic pathways, including terpenoids and green leaf volatiles. The blends from herbivore-infested and artificially damaged plants are similar. However, differences can be found with respect to a few components of the blend, such as two nitriles and the monoterpene myrcene, that were produced exclusively by caterpillar-infested plants, and methyl salicylate, that was produced in larger amounts by caterpillar-infested plants. (3) Genes from major biosynthetic pathways involved in volatile production are induced by caterpillar feeding. These include AtTPS10, encoding a terpene synthase involved in myrcene production, AtPAL1, encoding phenylalanine ammonia-lyase involved in methyl salicylate production, and AtLOX2 and AtHPL, encoding lipoxygenase and hydroperoxide lyase, respectively, both involved in the production of green leaf volatiles. AtAOS, encoding allene oxide synthase, involved in the production of jasmonic acid, also was induced by herbivory.

Adaptation, Physiological↗
PubMed2004

Biochemical characterization and quantitative gene expression analysis of the multi-stress inducible metallothionein from Tetrahymena thermophila.

A cadmium-binding protein with biochemical features of a metallothionein (MT) has been isolated and purified to homogeneity from the ciliate Tetrahymena thermophila. N-terminal sequencing revealed the posttranslational cleavage of the first two amino acids and, in general, a high degree of identity with known MTs from other ciliates. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) analysis of the apothionein revealed a molecular mass (16,763 Da) higher to those of mammals and of other protozoa. Finally, quantitative real-time PCR has been used to investigate the susceptibility of this ciliate MT to gene activation in response to heavy metals and to other stressors. Our data indicate that while zinc is not effective at all and cadmium is the best inducer, other stress factors, such as mercury, copper, heat and hydrogen peroxide, also activated gene transcription. As in vertebrate cells, interleukin-6 (IL-6) that stimulates ciliate growth, was able to enhance MT gene synthesis. This complex of data seems to indicate a general role of this protein in stress response.

Animals↗
PubMed2004

Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E).

The erythrocyte colony-forming unit (CFU-E) is a rare bone marrow (BM) progenitor that generates erythrocyte colonies in 48 hours. The existence of CFU-Es is based on these colonies, but CFU-Es have not been purified prospectively by phenotype. We have separated the "nonstem," "nonlymphoid" compartment (lineage marker [lin]-c-Kit+Sca-1-IL-7Ralpha-) into interleukin 3 receptor alpha negative (IL-3Ralpha-) and IL-3Ralpha+ subsets. Within IL-3Ralpha- but not IL-3Ralpha+ cells we have identified TER119-CD41-CD71+ erythrocyte-committed progenitors (EPs). EPs generate CFU-E colonies at about 70% efficiency and generate reticulocytes in vivo. Depletion of EPs from BM strongly reduces CFU-E frequencies. EPs lack potential for erythrocyte burst-forming unit, megakaryocyte, granulocyte (G), and monocyte (M) colonies, and for spleen colony-forming units. Chronically suppressed erythropoiesis in interferon consensus sequence-binding protein (ICSBP)-deficient BM is associated with reduced frequencies of both the EP population and CFU-E colonies. During phenylhydrazine-induced acute anemia, numbers of both the EP population and CFU-E colonies increase. Collectively, EPs (lin-c-Kit+Sca-1-IL-7Ralpha-IL-3Ralpha-CD41-CD71+) account for most, if not all, CFU-E activity in BM. As a first molecular characterization, we have compared global gene expression in EPs and nonerythroid GM progenitors. These analyses define an erythroid progenitor-specific gene expression pattern. The prospective isolation of EPs is an important step to analyze physiologic and pathologic erythropoiesis.

Anemia↗
PubMed1993

Feline immunodeficiency virus gene expression: analysis of the RNA splicing pattern and the monocistronic rev mRNA.

The transcription pattern of the feline immunodeficiency virus (FIV) genome in a feline CD4+ cell line was examined. In addition to the genomic RNA (9.2 kb), at least five FIV-specific transcripts [5.2, 4.4 (doublet), 1.7 and 1.4 kb] were detected by using subgenomic restriction enzyme fragments of an FIV molecular clone or FIV-specific oligonucleotides as probes. Among these transcripts, the 9.2, 5.2 and 4.4 (doublet) kb mRNAs were not expressed in the cytoplasm of cells transfected with a rev- mutant. To determine the location of splice junctions in the FIV genome, we used PCR to amplify and clone cDNAs corresponding to the viral mRNAs from infected cells. The region between pol and env was found to contain at least two splice donor and three splice acceptor sites. Two splice acceptor sites were detected in the 3' region of env. By hybridization analysis and sequencing of cDNA clones, it was revealed that the medium sized mRNAs are derived from a single splice event, with different splice acceptor sites, and that the two smaller transcripts are doubly or triply spliced mRNAs. Our results demonstrate a complex pattern of alternative splicing of FIV mRNAs. Furthermore, we identified monocistronic rev mRNA species that employ a unique splice acceptor site.

Amino Acid Sequence↗
PubMed2006

Histidine decarboxylase, DOPA decarboxylase, and vesicular monoamine transporter 2 expression in neuroendocrine tumors: immunohistochemical study and gene expression analysis.

Histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (v-MAT2) are involved in the biosynthesis and storage of histamine. DOPA decarboxylase (DDC) is involved in the biosynthesis of a variety of amines and shares a high degree of homology with HDC. HDC and v-MAT2 immunoreactivities (IR) have recently been detected in well-differentiated neuroendocrine tumors (WDNETs) and poorly differentiated neuroendocrine carcinomas (PDNECs) of various sites and have been proposed as general endocrine markers. We evaluated HDC and v-MAT2 IR in a series of 117 WDNETs and PDNECs from different sites. Western blotting analysis was performed to verify the specificity of anti-DDC and anti-HDC antibodies. Real-time RT-PCR was performed using specific probes for HDC and DDC on 42 cases, examined also for DDC IR. HDC and v-MAT2 IR were observed in the majority of WDNETs and PDNECs of all sites and HDC-IR cases were always also DDC-IR. In contrast, high levels of HDC mRNA were detected only in the gastroenteropancreatic WDNETs, which did not show increased DDC mRNA levels. On the other hand, bronchial carcinoids and lung PDNECs showed high DDC mRNA levels, but nearly undetectable HDC mRNA levels. Western blotting analysis showed a cross-reaction between anti-HDC and anti-DDC antibodies. HDC should not be considered as a general endocrine marker and HDC IR in bronchial carcinoids and PDNECs of the lung can probably be attributed to a cross-reaction with DDC.

Antibody Specificity↗
PubMed2006

Gene expression analysis of a human lymphoblastoma cell line exposed in vitro to an intermittent 1.9 GHz pulse-modulated radiofrequency field.

This study was designed to determine whether radiofrequency (RF) fields of the type used for wireless communications could elicit a cellular stress response. As general indicators of a cellular stress response, we monitored changes in proto-oncogene and heat-shock protein expression. Exponentially growing human lymphoblastoma cells (TK6) were exposed to 1.9 GHz pulse-modulated RF fields at average specific absorption rates (SARs) of 1 and 10 W/kg. Perturbations in the expression levels of the proto-oncogenes FOS, JUN and MYC after exposure to sham and RF fields were assessed by real-time RT-PCR. In addition, the transcript levels of the cellular stress proteins HSP27 and inducible HSP70 were also monitored. We demonstrated that transcript levels of these genes in RF-field-exposed cells showed no significant difference in relation to the sham treatment group. However, concurrent positive (heat-shock) control samples displayed a significant elevation in the expression of HSP27, HSP70, FOS and JUN. Conversely, the levels of MYC mRNA were found to decline in the positive (heat-shock) control. In conclusion, our study found no evidence that the 1.9 GHz RF-field exposure caused a general stress response in TK6 cells under our experimental conditions.

Cell Line, Tumor↗
PubMed2001

Gene expression analysis of the catalytic subunit of human telomerase (hEST2) in the differential diagnosis of serous effusions.

Diagnostic accuracy in effusion cytology based on morphologic examination is not always satisfactory. Therefore, various diagnostic adjuncts such as immunocytochemistry or deoxyribonucleic acid cytometry are employed in this diagnostic field. Recently, demonstration of telomerase activity has been proposed as a possible marker for malignancy. In this study a seminested reverse transcription-polymerase chain reaction (RT-PCR) strategy for expression analysis of the catalytic subunit of human telomerase (hEST2) was used in 58 serous effusions. RT-PCR results correlated with cytologic diagnoses in 14 of 17 malignant effusions. In eight effusions cytologically suspicious for malignancy, PCR results were in accordance with the clinical follow-up. However, hEST2 RT-PCR was also positive in six of 15 cytologically benign effusions that consisted predominantly of inflammatory and mesothelial cells. Using the telomeric repeat amplification protocol, it could be demonstrated that cultured, proliferating benign mesothelial cells may present a weak telomerase activity, as is known in other benign cells including activated lymphocytes. In conclusion, the simple and rapid method of hEST2 RT-PCR serves to support the cytologic diagnosis of malignancy, but false-positive PCR results resulting from activated lymphocytes and proliferating mesothelial cells must be considered.

Ascitic Fluid↗
PubMed1998

Molecular characterization of tobacco sulfite reductase: enzyme purification, gene cloning, and gene expression analysis.

A cDNA clone, NtSiR1, that encodes the precursor of ferredoxin-dependent sulfite reductase (Fd-SiR) has been isolated from a cDNA library of tobacco (Nicotiana tabacum cv. SR1). The identity of the cDNA was established by comparison of the purified protein and the predicted structure with the nucleotide sequence. The amino terminus of the purified enzyme was Thr62 of the precursor protein, and the mature region of NtSiR1 consisted of 632 amino acids. Tobacco Fd-SiR is 82, 77, and 48% identical with Fd-SiRs from Zea mays, Arabidopsis thaliana, and a cyanobacterium, respectively. Significant similarity was also found with Escherichia coli NADPH-SiR in the region involved in ligation of siroheme and the [4Fe-4S] cluster. On Northern blot analysis, a transcript of NtSiR1 was detected in leaves, stems, roots, and petals in similar amounts. We also isolated a genomic SiR clone named gNtSiR1. It consists of 8 exons and 7 introns. Genomic Southern blot analysis indicated that at least two SiR genes are present in the tobacco genome.

Amino Acid Sequence↗
PubMed2004

Evaluation of quality-control criteria for microarray gene expression analysis.

BACKGROUND: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. METHODS: In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A(260)/A(280) ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. RESULTS: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01). CONCLUSIONS: These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.

Analysis of Variance↗