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PubMed1997

Myocardial angiotensin receptor type 1 gene expression in a rat model of cardiac volume overload.

To investigate the regulation of the angiotensin receptor type 1 (AT1) in different organs in cardiac volume overload, we measured AT1 mRNA content in the atria, left and right ventricle, kidney and liver of rats with an aortocaval shunt, produced by infrarenal aortocaval puncture 4 weeks earlier. For angiotensin receptor mRNA quantitation a novel quantitative PCR procedure based on liquid phase hybridization was used that allowed the determination of absolute AT1 mRNA copy numbers and its comparison in different organs. Glyceraldehydephosphate dehydrogenase (GAPDH) mRNA was measured by RT-PCR to control externally equal mRNA content and quality of RNA extraction in shunt animals and controls. Heart weight was increased in the shunt animals, with the greatest increase in the atria. Blood pressure, plasma renin activity, plasma angiotensin I and II and aldosterone concentrations were not significantly altered. The AT1 mRNA content was significantly increased in the atria (shunt: 1167 +/- 350 copies AT1 mRNA/ng RNA vs controls: 803 +/- 240; p < 0.05). No change was found in the right or left ventricle, in the kidney and liver. The findings document that atrial hypertrophy in cardiac volume overload parallel with a significant increase in atrial AT1 mRNA content.

Animals↗

PubMed1995

Adequacy of mucosal biopsies for evaluation of intestinal cytokine-specific mRNA. Comparative study of RT-PCR in biopsies and isolated cells from normal and inflamed intestine.

Endoscopic biopsies are being increasingly utilized to investigate cytokine profiles in normal and diseased intestine. To evaluate the adequacy of mucosal biopsies as a source of cytokine-specific mRNA, we measured their content of interleukin-1 beta (IL-1 beta) and interleukin-2 (IL-2) mRNA by reverse transcription-polymerase chain reaction and compared it to that of autologous lamina propria mononuclear cells in control and inflammatory bowel disease-involved specimens. High-quality total RNA was recovered more consistently from cell isolates than from biopsies. Interleukin-1 beta mRNA was reliably detected in both cell and biopsy samples, whereas IL-2 mRNA was measurable in all lamina propria cells but not always in biopsies. Compared to controls, levels of IL-1 beta were significantly elevated in Crohn's disease and ulcerative colitis cells and biopsies, and a weak but significant correlation existed between values derived from the two sources. In contrast, only ulcerative colitis cell isolates but not biopsies contained significantly reduced concentrations of IL-2 mRNA compared to those of control and Crohn's disease samples, and no correlation was found between cell and biopsy contents. Widely different levels of cytokine-specific mRNA were present in closely adjacent biopsies, particularly for IL-2. These results suggest that mucosal biopsies are suitable to assess some but not all cytokine-specific mRNA due to variation in RNA recovery, intrinsic level of cytokine gene expression, and substantial variability of cytokine transcripts.

Adolescent↗

PubMed2003

Determination of optimal conditions for analysis of p53 status in leukemic cells using functional analysis of separated alleles in yeast.

Tumor suppressor p53 is transcription factor that participates in control of many cellular functions. Somatic mutations of the p53 gene are frequently detected in human cancers. Several methods can be used for identification of p53 mutations, including FASAY - functional analysis of separated alleles in yeast. FASAY distinguishes yeast colonies expressing functional p53 protein from colonies producing a dysfunctional p53 protein simply on the basis of color. The validity of the method depends on a low background level. There are several sources of background as PCR-induced point mutations, low quality of RNA and alternative splicing of intron 9 affecting the p53 carboxy-terminus. In the present work we show that FASAY can be successfully used for analysis of mRNA isolated from blood samples that were collected and stored for 24 hours at 0 degrees C without undesired increase of background. We also measured fidelity of several commonly used DNA polymerases and determined the most suitable kinds of Pfu DNA polymerases for FASAY. Reaction conditions described in this report allow routine analysis of p53 status in leukemic cells using FASAY.

Alleles↗

PubMed2026

Transcriptomic insights into thermal stress reveal physiological trade-off between thermal stress adaptation and reproductive investment in Spodoptera litura.

Spodoptera litura, a highly polyphagous lepidopteran pest, poses a major threat to agricultural productivity due to its remarkable adaptability to diverse environmental conditions. Although heat stress is known to trigger transcriptional reprogramming in insects, the molecular mechanisms underlying thermal stress responses in S. litura remain poorly understood. In the present study, fourth-instar larvae were exposed to acute heat stress (44 °C) and compared with control conditions (27 ± 1 °C) to investigate heat-induced transcriptional alterations affecting physiology and reproduction. High-quality RNA-Seq data achieved more than 80% mapping efficiency, with a total of 15,782 transcripts were identified. Transcriptome analysis of S. litura larvae showed 323 differentially expressed genes (DEGs), of which 262 genes were significantly upregulated and 61 were downregulated in heat-stressed larvae compared to the control group. The DEGs were associated with stress response, reproduction, signalling, proteostasis, detoxification, oxidative stress, metabolism, development, and chromatin regulation. Heat shock proteins genes, including HSP70, HSP90, and HSP27, together with co-chaperones such as TRET-1, STIP1, and Starvin, were strongly upregulated, indicating enhanced cellular protection against protein damage and oxidative stress under heat stress. Conversely, key reproductive and cell cycle-related genes, including BARR, CAPD2, FEO, CDK2 and MORULA, were significantly downregulated, suggesting reproductive impairment and developmental arrest. RT-qPCR validation corroborated the RNA-Seq findings, demonstrating a heat-induced physiological trade-off that prioritizes survival over reproduction. Consistent with these molecular responses, heat-stressed insects exhibited marked reproductive impairment, including significant reductions in gonadosomatic index, eupyrene sperm bundle count, mating frequency, mating success, female calling behaviour, copulation duration, fecundity, and egg fertility. Collectively, these findings provide comprehensive insights into the molecular basis of thermal adaptation in S. litura and demonstrate that acute heat stress compromises reproductive fitness while activating conserved stress-response pathways that promote short-term survival.

Animals↗

PubMed2003

Direct comparison of microarray gene expression profiles between non-amplification and a modified cDNA amplification procedure applicable for needle biopsy tissues.

Global gene expression profiling by cDNA microarray analysis has been used to discover the biomarkers for early diagnosis of various cancers, subclassing cancer type, and prediction of patient's treatment outcome. The information provided by gene expression profiling may contribute to the design of molecular mechanism-based strategies for cancer prevention and/or treatment. However, the standard procedure for cDNA microarray analysis requires 5mug of good quality total RNA as starting material for each target preparation reaction. Thus, there is a limit for needle biopsy samples, laser capture microdissected tissues, or flow-sorted cells to successfully utilize the microarray technology. In order to profile the gene expression of needle biopsy tissue, we have modified the standard protocol by carrying out cDNA amplification after cDNA synthesis. We compared percentage present calls, absent calls, reproducibility, and concordance in needle biopsy samples processed by standard microarray protocol (cDNA non-amplification method) and our modified protocol (cDNA amplification method). The results showed that cDNA amplification method provided high reproducibility, representation, and concordance with the standard cDNA non-amplification method. We have successfully analyzed the gene expression profiles of needle biopsy tissues using the modified method without significantly changing the expression profiles. These results suggest that the global gene expression profiles of small biopsy samples can be achieved by our modified method to facilitate the analysis of gene expression profiles for clinical application.

Biopsy, Needle↗

PubMed2003

Banking of fresh-frozen prostate tissue: methods, validation and use.

OBJECTIVE: To describe the establishment, methods, validation and use of a bank of fresh-frozen human prostate tissue. MATERIALS AND METHODS: On obtaining informed patient consent, protocols were followed for banking prostate tissue from any type of prostatectomy or cystoprostatectomy. A pseudobanking procedure was devised to determine the accuracy of assessing the histopathological status of the banked tissue. RNA was extracted, its quality assessed and used for quantitative real-time reverse transcription-polymerase chain reaction for the serine protease hepsin. RESULTS: To date prostate tissue from 112 patients has been banked, with pseudobanking in 58. The histopathological assessment showed pseudobanked tissue matched adjacent unbanked tissue in 98% of cases for benign vs malignant diagnoses, and in 92% of carcinomas for the Gleason score. Hepsin expression was significantly higher in malignant than in benign tissues (P < 0.0001). CONCLUSION: We established a validated method for banking human fresh-frozen prostate tissue and applied it successfully. Hepsin expression can be used to differentiate malignant and benign prostate tissue, and as an indicator of tissue heterogeneity.

Cryopreservation↗

PubMed2002

Differential allograft gene expression in acute cellular rejection and recurrence of hepatitis C after liver transplantation.

Treatment of acute cellular rejection (ACR) is associated with increased viral load, more severe histologic recurrence, and diminished patient and graft survival after liver transplantation for hepatitis C virus (HCV). Recurrence of HCV may be difficult to distinguish histologically from ACR. Because the immunologic mechanisms of ACR and HCV recurrence are likely to differ, we hypothesized that ACR is associated with the expression of a specific subset of immune activation genes that may serve as a diagnostic indicator of ACR and provide mechanistic insight into the pathophysiology of ACR and recurrence of HCV. The goal of the study was to study intragraft gene expression patterns in ACR and during recurrence of HCV in HCV-infected recipients. High-density microarrays were used to determine relative intragraft gene expression in two groups of HCV-infected liver transplant recipients: four with steroid responsive ACR by Banff criteria and four age- and gender-matched HCV-infected recipients with similar necroinflammatory activity but without histological criteria for rejection (no cholangitis or endotheliitis). Immunosuppression was similar in both groups. Other etiologies of graft dysfunction were excluded by ultrasound, cholangiography, and cultures. High-quality total RNA was extracted from snap frozen liver biopsies, reverse transcribed, labeled with biotin, and fragmented according to established protocol. Twenty-five genes were relatively overexpressed, and 15 were relatively underexpressed by > or = twofold in the ACR when compared with the HCV group. ACR was most notably associated with the relative overexpression of genes associated with major histocompatibility complex I and II, insulin-like growth factor-1 expression, apoptosis induction, and T-cell activation. In HCV-infected liver transplant recipients, ACR is associated with an intragraft gene expression profile that is distinct from that seen during recurrence of HCV. These experiments provide evidence that alloimmunity, as indicated by expression of T-cell activation and apoptosis-inducing genes, is less important in recurrence of HCV than in ACR. Further studies are required to determine whether gene expression profiles, either intragraft or in serum, can be used for the diagnosis and differentiation of ACR from recurrence of HCV.

Adult↗

PubMed2006

Transcriptional profiling of enriched populations of stem cells versus transient amplifying cells. A comparison of limbal and corneal epithelial basal cells.

The basal layer of limbal and central corneal epithelium is enriched in stem cells and transient amplifying cells, respectively. This physical separation of stem and transient amplifying cells makes the limbal/corneal epithelium an exceptionally suitable system for isolating basal cells enriched in these two proliferative populations. Prior attempts to isolate epithelial stem cells used methods such as proteolytic tissue dissociation and cell sorting that could potentially alter their gene expression profile. Using laser capture microdissection, we were able to isolate resting limbal and corneal basal cells from frozen sections with minimal tissue processing, thereby improving the yield and quality of RNA. Analyses of RNA isolated from 300 limbal and corneal basal cells from eight mice revealed a set of approximately 100 genes that are differentially expressed in limbal cells versus corneal epithelial basal cells. Semiquantitative reverse transcription-PCR confirmed the up-regulation of three limbal and three corneal genes. LacZ identification of epiregulin from epiregulin-null mice and immunohistochemical staining of wild type mice confirmed that epiregulin, one of the limbal epithelium-enriched genes, was associated with the limbal epithelial basal cells. Within the limbal and corneal basal cells, we detected previously unknown genes that were differentially expressed in these two regions that contribute further to our understanding of the unique heterogeneity of these two closely related basal cell populations. Our findings indicate that we can obtain accurate gene expression profiles of the stem cell-enriched limbal basal cell population in their "natural" quiescent state.

Animals↗

PubMed2006

Pilot study of mucosal genetic differences in early smokers and nonsmokers.

BACKGROUND: Global gene expression analysis is proving to be an important means of assessing human tumors and may identify key components of carcinogenesis or clinical prognosis. This technique has been successfully applied to head and neck squamous cell carcinoma (HNSCC) and thyroid carcinomas; however, little has been done to evaluate premalignant states. METHODS: Human buccal mucosal cells were sampled from smokers and nonsmokers using a noninvasive brush technique. The method was validated by assessing the quantity and quality of RNA obtained. The purified RNA was then assayed using cDNA microarrays containing 27,323 cDNA clones to examine the buccal mucosa in these patients for differences in gene expression patterns. Using unsupervised and supervised hierarchical clustering methods, we developed a gene profile signature for an initial training set of smokers and nonsmokers and then used this to predict smoking status in a subsequent test set of subjects. Selected genes were then cross-referenced with previously published gene sets found in HNSCC identified by our group. RESULTS: Nineteen subjects were used in this pilot analysis, 9 smokers and 10 nonsmokers. Smoking among the study group ranged from 1 to 60 pack years. RNA purified from buccal mucosal brushing demonstrated a high degree of similarity in gene expression profiles among independent samples. Through the application of supervised clustering techniques, we were able to identify 113 genes whose expression differed significantly between samples from smokers and nonsmokers (t test, P < .001). This expression signature was able to accurately predict who within the second set of subjects were smokers, with the exception of one person who had a minimal tobacco history and clustered with the nonsmokers. Cross-referencing data with that found in HNSCC, we were able to identify a tumor suppressor gene involved in the c-myc pathway (Mxi1) that was similarly under-expressed in smokers and cancer patients with progressive disease. CONCLUSIONS: Although the sample size was small in this preliminary dataset, our analysis revealed several groups of genes that were either over- or under-expressed in the smokers and which could be used to predict smoking exposure. Many of these represent genes of possible interest as early molecular markers for head and neck carcinogenesis.

Adult↗

PubMed2004

Use of RT-PCR and DNA microarrays to characterize RNA recovered by non-invasive tape harvesting of normal and inflamed skin.

We describe a non-invasive approach for recovering RNA from the surface of skin via a simple tape stripping procedure that permits a direct quantitative and qualitative assessment of pathologic and physiologic biomarkers. Using semi-quantitative RT-PCR we show that tape-harvested RNA is comparable in quality and utility to RNA recovered by biopsy. It is likely that tape-harvested RNA is derived from epidermal cells residing close to the surface and includes adnexal structures and present data showing that tape and biopsy likely recover different cell populations. We report the successful amplification of tape-harvested RNA for hybridization to DNA microarrays. These experiments showed no significant gene expression level differences between replicate sites on a subject and minimal differences between a male and female subject. We also compared the array generated RNA profiles between normal and 24 h 1% SLS-occluded skin and observed that SLS treatment resulted in statistically significant changes in the expression levels of more than 1,700 genes. These data establish the utility of tape harvesting as a non-invasive method for capturing RNA from human skin and support the hypothesis that tape harvesting is an efficient method for sampling the epidermis and identifying select differentially regulated epidermal biomarkers.

Actins↗

PubMed2002

Detection and differentiation of North American and European genotypes of porcine reproductive and respiratory syndrome virus in formalin-fixed, paraffin-embedded tissues by multiplex reverse transcription-nested polymerase chain reaction.

North American and European genotypes of porcine reproductive and respiratory syndrome virus (PRRSV) were distinguished in formalin-fixed, paraffin-embedded tissues from PRRSV-infected pigs by multiplex reverse transcription-nested polymerase chain reaction (RT-nPCR). A method based on xylene deparaffinization followed by proteinase K digestion yielded RNA of quality for reliable and consistent RT-nPCR analyses. The PRRSV nucleic acid was detected in lung, mediastinal lymph node, tonsil, and liver samples from pigs inoculated with North American strain virus, European strain virus, or both North American and European strains. All 30 archival formalin-fixed, paraffin-embedded lung tissues from pigs naturally infected with PRRSV were positive for PRRSV by RT-nPCR amplication and gave a pattern that corresponded to the North American genotype. Multiplex RT-nPCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for the detection and differentiation between North American and European genotypes of PRRSV.

Animals↗

PubMed2026

Transcriptomic analysis of eggs, rediae and cercariae reveal stage-specific adaptations in the rumen fluke Calicophoron daubneyi.

Rumen flukes, particularly the trematode Calicophoron daubneyi, are emerging parasites of livestock in Europe, yet transcriptomic insights into their environmental and intermediate host stages remain limited. Here, we present a comprehensive transcriptomic analysis of eggs at three distinct developmental stages (freshly excreted, early developmental and eye-spot stages), as well as rediae and cercariae, of C. daubneyi. High-quality RNA-sequencing (RNA-seq) datasets revealed both shared and stage-specific transcriptional profiles with each developmental stage exhibiting its own distinct expression pattern. Subsequent GO-Term enrichment analyses revealed that fully embryonated eggs in eye-spot-stage especially upregulated genes related to cilia assembly, movement and motility, reflecting preparation for miracidial hatching and host-seeking behavior. Rediae showed enhanced transcription of genes involved in diverse metabolic and biosynthetic processes, supporting rapid asexual proliferation within the snail intermediate host. Cercariae exhibited predominant upregulation of genes associated with signal transduction and energy metabolism, indicating the adaptation to its changing environmental conditions. These findings provide the first transcriptomic insights into the biology of C. daubneyi outside the definitive host, reveal molecular mechanisms underlying development, transmission and adaptation to a changing environment and identify stage-specific genes as potential targets for interventions aimed at disrupting the parasites life cycle and controlling rumen flukes in the future.

Animals↗

PubMed2004

Enhanced sensitivity RNA gel loading buffer that enables efficient RNA separation on native gels.

RNA gel analysis is essential for quality assessment of RNA preparations for subsequent analysis such as microarrays and real-time PCRs. The routinely used standard electrophoresis of RNA through formaldehyde-containing agarose gels is not only labor-intensive and time-consuming, but also involves sizeable quantities of hazardous materials. Above all, it is not sensitive, requiring more than 1 microgram of RNA for the assay. Current gene expression profiling with microarrays and real-time PCR often involves limiting amounts of RNA. It is therefore important to have a more sensitive way to analyze RNA. Here we report an improved ethidium bromide-based RNA gel analysis system with our Superload buffer that increases sensitivity to 12.5 ng of total RNA and allows RNA analysis on a regular native Tris-acetate EDTA (TAE) agarose gel.

Animals↗

PubMed1997

Generation of multiple mRNA fingerprints using fluorescence-based differential display and an automated DNA sequencer.

Differential display is a method for the survey, analysis and comparison of gene expression in eukaryotic cells and tissues. Differential display involves isolation of high-quality nondegraded RNA, selective reverse transcription of polyadenylated mRNA using specific anchored oligopolydeoxythymidine [oligo(dT)] primers, and the subsequent PCR amplification of the cDNA with the same oligo(dT), an arbitrary upstream primer and radioisotopes for labeling the PCR products. The radioisotopically labeled products are then separated on a sequencing gel. In this report, we describe a rapid, specific, nonradioactive fluorescent differential display methodology in which fluorescently differentially labeled anchored oligo(dT) downstream primers are used in the reaction, with subsequent analysis of fluorescently labeled PCR products on an automated sequencer. Complete gene expression profiles, containing multiple mRNA fingerprints are possible by the simultaneous comparison of the multicolored banding patterns of the fluorescently differentially labeled products from several primer combinations. This modification of the differential display technique simplifies the assay and increases the throughput of high sample volumes required for comparative gene expression studies in various clinical applications.

Antibodies↗

PubMed2006

beta-Glucuronidase is an optimal normalization control gene for molecular monitoring of chronic myelogenous leukemia.

Quantitative monitoring of breakpoint cluster region (BCR)-Abelson kinase (ABL) transcripts has become indispensable in the clinical care of patients with chronic myelogenous leukemia. Because quantity and quality of RNA in clinical samples are highly variable, a suitable internal normalization control is required for accurate BCR-ABL quantification. However, few studies have examined suitability of the control genes using criteria relevant to residual disease testing. In this study, we evaluated a number of control genes with the application of several novel criteria, including control gene performance on serial patient sample testing and in a residual disease model. We also examined expression of the control genes in BCR-ABL-positive K562 cells in response to Gleevec treatment. We found that beta-glucuronidase is the best control gene among those studied. Importantly, ABL, a widely used control gene, generates misleading BCR-ABL changes that potentially affect the clinical management of chronic myelogenous leukemia patients.

Benzamides↗

PubMed1991

Developmental capacity acquisition requires exposure of uncondensed chromosomes before maturation of bovine oocytes.

Immature bovine oocytes are forming an heterogeneous population since only one third of all morphologically normal cumulus-oocyte-complexes will result in a developing embryo after in vitro culture. To increase developmental competence, it is essential to improve the quality of the 2/3 not responding to the in vitro maturation context. To achieve this goal we can address the cytoplasmic domain or the nucleus capacity to control future events leading to normal fertilization and development. To influence for example the quantity and quality of RNA present at meiotic resumption (MR), we need access to uncondensed chromatin before that period. Maintenance of the germinal vesicle for a minimum of 24 h can be achieved with protein synthesis or phosphodiesterase (PI) inhibitors or with cAMP accumulation at a level higher that the one required in the cumulus-granulosa portion to stimulate MR. It is suspected that in vivo, the granulosa layer is responsible for the arresting signal since follicular fluid (BFF) alone is not sufficient for a complete effect in vitro. Low concentrations of granulosa cells in vitro (10(6)/ml) stimulate MR but monolayers with their conditioned medium provide inhibitory conditions (33% GV) to oocytes adhering to the monolayers. To achieve a complete meiotic arrest for 24 h (greater than 75% GV), a high concentration of granulosa cells must be used (10(7)/ml). The embedding of cumulus-enclosed oocytes in agar prior to culture with the cells prevents the significant effect of the cells on MR indicating either a need for contact stimulation or a very labile product.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

Measurement of H2'-C2' and H3'-C3' dipolar couplings in RNA molecules.

The quality of nucleic acid solution structures can be significantly improved using residual dipolar coupling data. However, many of the one-bond couplings that could be used for this purpose are difficult to measure. Conventional 2D experiments are often unable to reveal one-bond H2'-C2' and H3'-C3' couplings in large RNA molecules due to spectral overlap. Here we show how to use 3D HCcH-COSY and Relay HCcH-COSY to measure one-bond H2'-C2' and H3'-C3' couplings which improved the precision of the structures obtained recently for a 42 nucleotide RNA.

Carbon Isotopes↗

PubMed2005

RNA degradation by the exosome is promoted by a nuclear polyadenylation complex.

The exosome complex of 3'-5' exonucleases participates in RNA maturation and quality control and can rapidly degrade RNA-protein complexes in vivo. However, the purified exosome showed weak in vitro activity, indicating that rapid RNA degradation requires activating cofactors. This work identifies a nuclear polyadenylation complex containing a known exosome cofactor, the RNA helicase Mtr4p; a poly(A) polymerase, Trf4p; and a zinc knuckle protein, Air2p. In vitro, the Trf4p/Air2p/Mtr4p polyadenylation complex (TRAMP) showed distributive RNA polyadenylation activity. The presence of the exosome suppressed poly(A) tail addition, while TRAMP stimulated exosome degradation through structured RNA substrates. In vivo analyses showed that TRAMP is required for polyadenylation and degradation of rRNA and snoRNA precursors that are characterized exosome substrates. Poly(A) tails stimulate RNA degradation in bacteria, suggesting that this is their ancestral function. We speculate that this function was maintained in eukaryotic nuclei, while cytoplasmic mRNA poly(A) tails acquired different roles in translation.

Adaptor Proteins, Signal Transducing↗