Document Sample
5.Article+for+mRNA+Expression Powered By Docstoc
					Analysis of mRNA expression
1. RT-PCR procedure (I–IV)
Total RNA was isolated according to the manufacturer’s procedure of Trizol® method (GibcoBRL, Life Technologies). The first strand cDNA was synthesised from 0.2–11 µg of total RNA using 200 units of Superscript™ II RnaseH– Reverse Transcriptase (GibcoBRL, Life Technologies) and random hexamer primers. cDNA was amplified with 1 unit of Dynazyme™ (Finnzymes) or 2.6 units of Expand™ High Fidelity PCR System enzyme mix (Boehringer Mannheim Gmbh) using specific primers for each mRNA under study (see I–IV) The amplification included cycles of denaturation, annealing and extension under the conditions required for the primers and templates. In semi-quantitative PCR (II) serial dilutions of cDNA were amplified, and products from linear reaction rate were quantified with an image processing and analysing program (ScionImage PC, Scion Corporation).

2. Quantitative analysis of mRNA (I)
For quantitation of the mRNA amount of both proα1(I) collagen and β -actin in samples, cRNA controls for proα1(I) collagen and β -actin were used (Tasanen et al. 1996). These cRNA molecules contained small deletions, and served as the internal controls for a reverse transcription and for amplification efficiency. Due to the size difference between cRNA and endogenous mRNA, PCR products were easily separated by gel electrophoresis. In the method 0.1–2 µg of total cellular RNA and 108–109 molecules of cRNA were reverse transcribed in combination into cDNA. Serial dilutions of the cDNA mixture were amplified by specific primers, of which the upstream primer was 32P end-labeled. Products were resolved by gel electrophoresis and autoradiographed. The amounts of radioactivity in the corresponding bands were determined by liquid scintillation, and plotted against the cRNA and RNA concentrations of samples. The copy number of target mRNA was obtained by extrapolating against the standard curve drawn with the Excel program (Microsoft) as described before (Tasanen et al. 1996).

3. Real-Time PCR (IV, Figures 5–14)
Specific TaqMan® primers and probes were designed with the Primer Express 1.0 software (Applied Biosystems). Sequences for the human MMP probes and primers are under the copyright of Applied Biosystems. The internal fluorogenic probes were labeled at the 5’ end with the reporter dye FAM, at the 3’ end with the quencher dye TAMRA and phosphateblocked at the 3’ end to prevent extension. The 18S rRNA probe was labeled with the VIC reporter dye at its 5’ end and the TAMRA quencher dye at its 3’ end. The amplified PCR products were quantified by measuring the accumulation of fluorescence during the amplification in each PCR cycle with the ABI Prism 7700 sequence detection system (Applied Biosystems). A cycle threshold (CT) describes a cycle when the reporter fluorescence dye of a given sample becomes significantly different from the baseline signal. The CT values obtained were plotted against log input RNA concentration in samples in serially diluted total RNA, and used to generate standard curves for all mRNAs analyzed. The amount of specific mRNA in samples were calculated from the standard curve, and normalized with the 18S rRNA.

4. Ribonuclease protection assay (RPA) (III)
The method of RPA III™ Ribonuclease Protection Assay (Ambion Inc.) was used for the study of the expression of MMP-14 mRNA in growth factor stimulated odontoblasts and pulp tissue. 3 to 5 µg of total RNA was hybridized with [α-32P]-UTP- labeled MT1-MMP antisense RNA probe (nucleotides 218–638), treated with RNase A/Rnase T1 mixture and electrophoresed by a 5% denaturing PAGE gel. The gel was exposed to the X-ray film and bands were analyzed with an image processing and analysing program (ScionImage PC, Scion Corporation).

5. Verifying amplification products (II, III)
Either Southern blotting procedure or sequencing was used for validating identities of amplification products as follows.

5.1. Southern blot (II)
The specific MMP-8 cDNA probe was prepared by amplifying cDNA, synthesized from total RNA of gingival fibroblasts, with MMP-8 specific primers (Hanemaaijer et al. 1997). The product (522 bp) was purified with Qiaex Gel Extraction -method (QIAGEN) and labeled with [α-32P]-dCTP. PCR products were fractioned on 1.5% agarose gel, transferred to nylon filter (Amersham) and hybridized to the MMP-8 probe.

5.2. Sequencing (II, IV)
PCR products were sequenced with sense or antisense primer using DNA Sequencing Kit according the manufacturer’s instructions (Applied Biosystems). The data was analyzed with BLAST search tool provided by NCBI.
Source -

Cytokine mRNA expression
Expression of mRNA for eight cytokines was analyzed in an in vitro response-proliferation and Ig-secretion--of normal human B lymphocytes. This was made possible by the use of murine thymoma cells as helper cells in conjunction with human T cell supernatant, and the design of human DNA sequence-specific primers for RT-polymerase chain reaction. mRNAs for interleukin (IL)2 and IL-4, but also for IL-1 alpha and IL-1 beta remained undetectable during the whole culture period in highly purified B cells prepared by a three-step purification protocol. However, tumor necrosis factor alpha and IL-6 mRNAs peaked during days 1-3 after culture start and became undetectable after 5-6 d, shortly before bulk B cell proliferation started to decline. In contrast, transforming growth factor beta 1 mRNA, after a progressive increase during the first few days, and IL-10 mRNA, after a peak on days 1-3, remained detectable in immunoglobulin (Ig)-secreting cultures throughout the observation period of 22 d. Clonal analysis on 8-d cultures that had been seeded with single B cells by autocloning with the cell sorter, revealed that 85% of 77 B cell clones studied, expressed TGF-beta 1

mRNA, and only 19% IL-10 mRNA. These findings show a differentiation stage-related cytokine program during a B cell response, whereby (a) B cells can become activated without IL-1 alpha or IL-1 beta expression; (b) mRNA for positive (IL-10) and negative (TGF-beta 1) autoregulatory factors coexists in cell populations during the later phase of the response, although not necessarily in all B cell clones; and (c) normal Ig-secreting cells cease IL-6 expression in contrast to their malignant counterparts, myeloma cells Source-

Receptor mRNA Expression
Irritable bowel syndrome (IBS) is largely viewed as a stress-related disorder caused by aberrant brain-gut–immune communication and altered gastrointestinal (GI) homeostasis. Accumulating evidence demonstrates that stress modulates innate immune responses; however, very little is known on the immunological effects of stress on the GI tract. Toll-like receptors (TLRs) are critical pattern recognition molecules of the innate immune system. Activation of TLRs by bacterial and viral molecules leads to activation of NF-kB and an increase in inflammatory cytokine expression. It was our hypothesis that innate immune receptor expression may be changed in the gastrointestinal tract of animals with stressinduced IBS-like symptoms.

Methodology/Principal Findings
In this study, our objective was to evaluate the TLR expression profile in the colonic mucosa of two rat strains that display colonic visceral hypersensivity; the stress-sensitive WistarKyoto (WKY) rat and the maternally separated (MS) rat. Quantitative PCR of TLR2-10 mRNA in both the proximal and distal colonic mucosae was carried out in adulthood. Significant increases are seen in the mRNA levels of TLR3, 4 & 5 in both the distal and proximal colonic mucosa of MS rats compared with controls. No significant differences were noted for TLR 2, 7, 9 & 10 while TLR 6 could not be detected in any samples in both rat strains. The WKY strain have increased levels of mRNA expression of TLR3, 4, 5, 7, 8, 9 & 10 in both the distal and proximal colonic mucosa compared to the control Sprague-Dawley strain. No significant differences in expression were found for TLR2 while as before TLR6 could not be detected in all samples in both strains.

These data suggest that both early life stress (MS) and a genetic predisposition (WKY) to stress affect the expression of key sentinels of the innate immune system which may have direct relevance for the molecular pathophysiology of IBS.

Source -

mRNA expression correlates with the central nervous system
Nestin is a recently discovered intermediate filament (IF) gene. Nestin expression has been extensively used as a marker for central nervous system (CNS) progenitor cells in different contexts, based on observations indicating a correlation between nestin expression and this cell type in vivo. To evaluate this correlation in more detail nestin mRNA expression in developing and adult mouse CNS was analysed by in situ hybridization. We find that nestin is expressed from embryonic day (E) 7.75 and that expression is detected in many proliferating CNS regions: at E10.5 nestin is expressed in cells of both the rostral and caudal neural tube, including the radial glial cells; at E15.5 and postnatal day (P) 0 expression is observed largely in the developing cerebellum and in the ventricular and subventricular areas of the developing telencephalon. Furthermore, the transition from a proliferating to a post-mitotic cell state is accompanied by a rapid decrease in nestin mRNA for motor neurons in the ventral spinal cord and for neurons in the marginal layer of developing telencephalon. In contrast to these data we observe two proliferating areas, the olfactory epithelium and the precursor cells of the hippocampal granule neurons, which do not express nestin at detectable levels. Thus, nestin mRNA expression correlates with many, but not all, regions of proliferating CNS progenitor cells. In addition to its temporal and spatial regulation nestin expression also appears to be regulated at the level of subcellular mRNA localization: in columnar neuroepithelial and radial glial cells nestin mRNA is predominantly localized to the pial endfeet. Source -

mRNA Expression Using an In Vitro Approach
So far, only a few industrial chemicals have been identified as developmental neurotoxicants. Because the current developmental neurotoxicity (DNT) guideline (Organisation for Economic Co-operation and Development TG 426) is based entirely on in vivo studies that are both time consuming and costly, there is a need to develop alternative in vitro methods for initial screening to prioritize chemicals for further DNT testing. In this study, gene expression at the mRNA level was evaluated to determine whether this could be a suitable endpoint to detect potential developmental neurotoxicants. Primary cultures of rat cerebellar granule cells (CGCs) were exposed to well known (developmental) neurotoxicants (methyl mercury chloride, lead chloride, valproic acid, and tri-methyl tin chloride) for different time periods. A significant downregulation of the mRNA level for the neuronal markers (NF-68, NF-200, N-methyl D-aspartate glutamate receptor, and gamma-amino butyric acid receptor) was observed after exposure to methyl mercury chloride, valproic acid, and tri-methyl tin

chloride. Moreover, a significant increase of the neural precursor marker nestin mRNA was also observed. The mRNA expression of the astrocytic markers (glial fibrillary acidic protein [GFAP] and S100β) was unchanged. In contrast, exposure to lead chloride significantly decreased the mRNA level of the astrocytic marker GFAP, whereas the neuronal markers were less affected. These results suggest that gene expression could be used as a sensitive tool for the initial identification of DNT effects induced by different mechanisms of toxicity in both cell types (neuronal and glial) and at various stages of cell development and maturation. Source -

mRNA expression analysis by Real-Time PCR

Relative mRNA expression of the indicated genes in the indicated conditions was quantified using a 7500 Fast Real-Time PCR System (Applied Biosystems). To adjust for possible differences in the amount of template added to the reaction, 18s RNA served as an endogenous control (expression levels of the endogenous control were subtracted from expression levels of target genes). Each sample was assessed in replicate for both the target gene and the endogenous control. Each experiment was repeated at least three times to ensure statistical significance. Real-Time PCR primers (18 – 20 nucleotides) for the indicated genes were designed with Primer 3 ( according to stringent product size (75 – 150 nucleotides) and annealing temperature (58 - 63°C) specifications. Predicted unspecific primer binding and secondary structure formation were excluded using the BLAST and IDT ( tools.

1.Extract total RNA from at least 2 × 106 cells using TRIZOL reagent (Ambion) and the appropriate RNeasy RNA extraction kit (Qiagen). 2.Reverse-transcribe 60 ng of total RNA with a High-capacity cDNA Reverse Transcription kit (Applied Biosystems) in a reaction volume of 200 microl. 3.Prepare a reaction mix (25 μl) according to the following recipe: 2X Sybr Green PCR Master Mix - 12,5 microl 1 μM primer mix - 2,5 microl cDNA (5 ng/microl) - 2 microl water - 8 microl 4.Perform Real-Time PCR on 10 ng of cDNA using a Power Sybr Green PCR Master MIX (Applied Biosystems). 5.Start the amplification procedure according to the following standard 7500 run protocol: 50°C (2min) - 95°C (10min) - 95°C (15sec), 60°C (1min), 40 cycles - 95°C (15sec), 60°C. 6.Perform a dissociation assay to evaluate any problem related to primer unspecific annealing or secondary structure formation. 7.Analyze data with built-in SDS Analysis Software.

Source -

Haloperidol changes mRNA

The quaking homolog, KH domain RNA binding (mouse) (QKI) is a candidate gene for schizophrenia. Disturbed QKI mRNA expression is observed in the prefrontal cortex of patients, and some of these changes correlate to treatment with antipsychotic drugs. To test if low doses of antipsychotic drugs can modify QKI mRNA expression, human astrocytoma (U343) and oligodendroglioma (HOG) cell lines were treated with five different antipsychotic drugs including Haloperidol, Aripiprazole, Clozapine, Olanzapine and Risperidone. Messenger RNA expression levels of splice variants QKI-5, QKI-6 and QKI-7 were measured by Real-Time PCR.

Haloperidol treatment (0.2 μM) doubled QKI-7 mRNA levels in U343 cells after 6 hours (pvalue < 0.02). The effect was dose dependent, and cells treated with ten times higher concentration (2 μM) responded with a five-fold and three-fold increase in QKI-7, 6 and 24 hours after treatment, respectively (p-values < 0.0001).

The results in U343 cells suggest that QKI-7 mRNA expression in human astrocytes is induced by Haloperidol, at concentrations similar to plasma levels relevant to clinical treatment of schizophrenia. The molecular mechanism of action of antipsychotic drugs after binding to receptors is not well known. We hypothesize that QKI regulation is involved in this mechanism. Source -

Analysis of global mRNA expression
To search for novel transcriptional pathways that are activated in skeletal muscle after endurance exercise, we used cDNA microarrays to measure global mRNA expression after an exhaustive bout of high-intensity cycling (~75 min). Healthy, young, sedentary males performed the cycling bout, and skeletal muscle biopsies were taken from the vastus lateralis before, and at 3 and 48 h after exercise. We examined mRNA expression in individual muscle samples from four subjects using cDNA microarrays, used repeated-measures significance analysis of microarray (SAM) to determine statistically significant expression changes, and confirmed selected results using real-time RT-PCR. In total, the expression of 118 genes significantly increased 3 h postcycling and 8 decreased. At 48 h, the expression of

29 genes significantly increased and 5 decreased. Many of these are potentially important novel genes involved in exercise recovery and adaptation, including several involved in 1) metabolism and mitochondrial biogenesis (FOXO1, PPARδ, PPARγ, nuclear receptor binding protein 2, IL-6 receptor, ribosomal protein L2, aminolevulinate δ-synthase 2); 2) the oxidant stress response (metalothioneins 1B, 1F, 1G, 1H, 1L, 2A, 3, interferon regulatory factor 1); and 3) electrolyte transport across membranes [Na+-K+-ATPase (β3), SERCA3, chloride channel 4]. Others include genes involved in cell stress, proteolysis, apoptosis, growth, differentiation, and transcriptional activation, as well as all three nuclear receptor subfamily 4A family members (Nur77, Nurr1, and Nor1). This study is the first to characterize global mRNA expression during recovery from endurance exercise, and the results provide potential insight into 1) the transcriptional contributions to homeostatic recovery in human skeletal muscle after endurance exercise, and 2) the transcriptional contributions from a single bout of endurance exercise to the adaptive processes that occur after a period of endurance exercise training. Source -

FoxP3 mRNA Expression in Regulatory T Cells
We read with great interest the article by Guyot-Revol and colleagues (1) assessing the role of T regulatory cells (Tregs) in tuberculosis (TB) pathogenesis. Guyot-Revol and coworkers found that in patients with TB, percentages of CD4+CD25high T cells and levels of FoxP3 mRNA expression in peripheral blood mononuclear cells were both significantly higher in comparison with controls. To better comprehend Tregs' mechanism of action, the authors investigated whether the increased levels of FoxP3 mRNA that they had observed were effectively caused by increased gene expression, or were simply the result of increased CD4+CD25high frequency. FoxP3 mRNA in CD4+CD25+ T cells was compared between patients with TB and control subjects. As no quantitative difference was observed, the authors suggest that the greater levels of FoxP3 mRNA are simply an effect of increases in Treg frequency rather than a result of an up-regulated gene expression. The transcription factor FoxP3 is the most specific molecular marker for Tregs available to date, and its correct evaluation is crucial (2–4). In the analysis of Treg dynamics, an assessment of what happens in the frequency and level of FoxP3 mRNA expression at the single cell level is mandatory. The recent literature demonstrates a strong correlation between levels of CD25 expression and the frequency of FoxP3-positive cells in healthy donors (4), and suggests that to correctly evaluate FoxP3 expression in Tregs, mRNA should be quantified in CD4+CD25high, rather than in CD4+CD25+ T cells (5). We are currently performing a prospective study to evaluate Tregs in patients with TB. Analysis of CD4+CD25high percentage on CD4 T cells at TB diagnosis showed similar levels

of Tregs among patients with TB and controls (median percentage: 2.1% [IQR, 1.8–6.6] vs. 1.9% [IQR, 1.5–2.2]). Unlike Guyot-Revol and coworkers, we evaluated FoxP3 mRNA expression by real-time polymerase chain reaction in CD4+CD25high T cells, purified using an EPICS ALTRA Cell Sorter (Beckman-Coulter; purity > 93.6%). A considerable increase of FoxP3 mRNA expression in patients with TB with respect to control subjects was clearly observed (median: 39.1 [IQR, 5.1–109] vs. 1.6 [IQR, 0.8–2.8]; p < 0.0062). These data implicate FoxP3 gene overexpression as a probable mechanism involving Tregs in the pathogenesis of TB, and suggest a possible role of FoxP3 gene up-regulation in the outcome of chronic infections. Our results underscore the importance of using the correct methodology in evaluating Treg cell dynamics, and prove that an accurate evaluation of FoxP3 mRNA expression in Treg cells may lead to the identification of previously unknown mechanisms of Treg cell involvement in disease pathogenesis. Source -

mRNA Expression in Kidneys of Rats
The development of renal interstitial fibrosis (RIF) is related to the expression and excretion of cytokines and growth factors. Thus, we investigated the time course of mRNA expression of cytokines known as causative factors in a model of RIF in rats before and on day 10 after unilateral ureteral obstruction (UUO), when first signs of fibrosis were visible, as well as during progressive RIF. UUO causes a fivefold increase in mRNA expression of monocyte chemoattractant protein 1 15 days after surgery as compared with contralateral kidneys. The level remains elevated about three-fold up to day 25. The mRNA of the fibrogenic cytokine transforming growth factor beta 1 (TGF- 1) is increased two- to threefold during the time course, whereas the mRNAs of platelet-derived growth factor B chain (PDGF-B) and its receptor beta (PDGF-R ) increase after UUO, reaching their maxima on days 10-15. PDGFB mRNA increase up to day 15, marking the onset of fibrosis, and decreases thereafter, whereas the expression of the PDGF-R mRNA remains elevated more than threefold over the entire study period. Incubation of cultured renal fibroblasts with TGF- 1 and/or PDGF-B suggests that their specific action on cell growth and proliferation is maintained even when they are used in combination. The sustained elevation of TGF- 1 and PDGF-B/PDGF-R mRNA levels confirms the assumption of a particular involvement of these cytokines in the pathogenesis of RIF. The mRNA expression of the gap junctional protein connexin 43 in ureteral ligated kidneys is increased sixfold already 5 days after UUO. In this way, the increased connexin 43 mRNA levels indicate a possible function in the remodeling of the kidney tissue after tubular damage and fibrosis. Source -

mRNA expression in fatty liver and nonalcoholic steatohepatitis
Nonalcoholic fatty liver disease represents the hepatic manifestation of the metabolic syndrome. Nonalcoholic steatohepatitis (NASH) is the progressive form of liver injury. The pathophysiology that leads to NASH is not well understood. Objective We hypothesize that an altered cortisol metabolism in the liver may be a pathogenetic factor. Design and patients 75 patients (28 men, 47 women) underwent liver biopsy for elevation in liver enzymes. Histological diagnosis identified normal liver in eight, fatty liver in 20, NASH grade 1 in 22, grade 2 in nine, grade 3 in three patients, and other forms of hepatitis or cirrhosis in 13 patients. We quantified hepatic 11β-hydroxysteroid dehydrogenase type1 (11β-HSD1) and hexose-6-phosphate-dehydrogenase (H6PDH) mRNA expression by real-time PCR. In addition, analysis of 24 h urinary excretion of cortisol metabolites using GCMS was performed and compared with healthy controls. Results 11β-HSD1 mRNA expression correlated significantly (R2= 0·809; P < 0·001) with H6PDH mRNA expression, negatively with waist-to-hip ratio in women (R2= 0·394; P= 0·005), but not with urinary (THF + 5α-THF)/THE ratio, total cortisol metabolite excretion, age, BMI, degree of fatty liver or NASH stages. Total cortisol metabolite excretion was increased in patients with fatty liver or NASH compared with healthy controls. Conclusions Our data suggest that expression of hepatic 11β-HSD1 and H6PDH are closely interlinked. 11β-HSD1 gene expression does not seem to be involved in the pathogenesis of fatty liver or NASH. However, those patients showed an increased 5α- and 5β-reduction of cortisol leading to an increased cortisol turnover rate and an activation of the HPA axis. Source

mRNA expression levels of PXR splice variants in livers
We have documented the expression of PXR (NR1I2) alternatively spliced transcripts in a panel of 36 human tissues. PXR.1 was expressed in many more tissues than previously determined, including human bone marrow and select regions of the human brain. In each of these tissues, we observed alternative splicing of various exons of PXR that generated multiple distinct PXR isoforms. The most abundant PXR alternative mRNA transcripts lacked 111 nucleotides, deleting 37 amino acids from the PXR LBD (PXR.2), or lacked 123 nucleotides, deleting 41 amino acids from the PXR LBD (PXR.3). CYP3A4, a gene transcriptionally regulated by PXR, showed incomplete overlap with PXR in its tissue distribution. In this study, quantitation of PXR mRNAs in human liver demonstrated that PXR.2 and PXR.3 represented 6.7% and 0.32% of total PXR mRNA transcripts. Nicotine, the psychoactive and addictive chemical in cigarettes, and a known inducer of brain CYP2B6, has been shown to be efficacious activator of PXR and inducer of CYP3A4 transcription in

vitro. Since nicotine activation of PXR will enhance metabolism of nicotine to the nonpsychoactive cotinine, these results suggest a molecular mechanism for the development of tolerance to nicotine. Moreover, the identification of PXR in many human tissues, such as brain, and activation by tissue specific ligands (such as neurosteroids) suggests additional biological roles for this receptor in these tissues. Source

mRNA Expression of p53 and p21
DNA damage induced by benzene reactive metabolites is thought of as an important mechanism underlying benzene hematotoxicity and genotoxicity, and genetic variation in cell-cycle control genes may contribute to susceptibility to chronic benzene poisoning (CBP). Using a case-control study that included 307 benzene-poisoned patients and 299 workers occupationally exposed to benzene in south China, we aimed to investigate the association between genetic polymorphisms of p53 and p21 and the odds of CBP. To investigate whether benzene exposure may influence mRNA expression of p53 and p21 in benzene-exposed workers, we also chose 39 CBP workers, 38 occupationally benzene-exposure workers, and 37 nonexposure workers in the same region of China. PCR-restriction fragment length polymorphism technique was applied to detect polymorphisms of p53 (rs17878362, rs1042522, and rs1625895) and p21 (rs1801270 and rs1059234), and real-time PCR was applied to detect the quantity of gene mRNA expression. We found that p21 C98A variant genotypes (CA+AA) or C70T variant genotypes (CT+TT) were associated with decreased odds of CBP [odds ratio (OR), 0.51; 95% confidence interval (95% CI), 0.32-0.83, and OR, 0.53; 95% CI, 0.29-0.95, respectively. Further analysis showed the decreased odds of CBP in the subjects with p21 CC/AT diplotype (OR, 0.51; 95% CI, 0.30-0.85). In addition, p53 mRNA expression of CBP workers or benzene-exposure workers was significantly lower than that of nonexposure workers. Although these results require confirmation and extension, our results show that polymorphisms in p21 may be protective against the risk of CBP in the Chinese occupational population. Source -

mRNA expression in lung adenocarcinomas
The relationship between gene expression measured at the mRNA level and the corresponding protein level is not well characterized in human cancer. In this study, we compared mRNA and protein expression for a cohort of genes in the same lung adenocarcinomas. The abundance of 165 protein spots representing 98 individual genes was analyzed in 76 lung adenocarcinomas and nine non-neoplastic lung tissues using twodimensional polyacrylamide gel electrophoresis. Specific polypeptides were identified using matrix-assisted laser desorption/ionization mass spectrometry. For the same 85 samples, mRNA levels were determined using oligonucleotide microarrays, allowing a comparative

analysis of mRNA and protein expression among the 165 protein spots. Twenty-eight of the 165 protein spots (17%) or 21 of 98 genes (21.4%) had a statistically significant correlation between protein and mRNA expression (r > 0.2445; p < 0.05); however, among all 165 proteins the correlation coefficient values (r) ranged from -0.467 to 0.442. Correlation coefficient values were not related to protein abundance. Further, no significant correlation between mRNA and protein expression was found (r = -0.025) if the average levels of mRNA or protein among all samples were applied across the 165 protein spots (98 genes). The mRNA/protein correlation coefficient also varied among proteins with multiple isoforms, indicating potentially separate isoform-specific mechanisms for the regulation of protein abundance. Among the 21 genes with a significant correlation between mRNA and protein, five genes differed significantly between stage I and stage III lung adenocarcinomas. Using a quantitative analysis of mRNA and protein expression within the same lung adenocarcinomas, we showed that only a subset of the proteins exhibited a significant correlation with mRNA abundance. Source -

mRNA Expression during Experimental Murine Cryptococcal Meningoencephalitis
The immune events that take place in the central nervous system (CNS) during cryptococcal infection are incompletely understood. We used competitive reverse transcription-PCR to delineate the time course of the local expression of mRNAs encoding a variety of cytokines and inducible nitric oxide synthase (iNOS) during progressive murine cryptococcal meningoencephalitis and assessed the CNS inflammatory response using immunohistochemistry. Interleukin 18 (IL-18), transforming growth factor ß1, and IL-12p40 mRNAs were constitutively expressed in the brains of infected and uninfected mice; IL-2 mRNA was not detected at any time. Increased levels of transcripts corresponding to IL-1 , tumor necrosis factor alpha (TNF- ), and iNOS were detected as early as day 1 postinfection, with TNF- rising by 30-fold and iNOS increasing by 5-fold by day 7. Each remained at these levels thereafter. IL-4, IL-6, and gamma interferon transcripts were detected on day 5, and IL-1ß and IL-10 transcripts were detected beginning on day 7. Once detected, each remained at a relatively constant level through 28 days of infection. This cytokine profile does not suggest a polarized Th1 or Th2 response. Immunohistochemistry did not reveal inflammatory infiltrates before day 7, despite the presence of cryptococci. Intraparenchymal abscesses with inflammatory cells in their peripheries were found beginning on day 10. The infiltrates were comprised primarily of cells expressing CD4, CD8, or CD11b; low numbers of cells expressing CD45R/B220 were also present. The persistence of Cryptococcus observed in the CNS may result from an ineffective immune response, perhaps owing to an insufficient anticryptococcal effector function of endogenous glial cells resulting from competing pro- and anti-inflammatory cytokines. These data detail the immune response in the brain and could be important for the future design of specific immunomodulatory therapies for this important opportunistic infection. Source -

Gastrin-releasing peptide mRNA expression
Gastrin-releasing peptide (GRP) is a bombesin (BN)-like peptide widely distributed in the gastrointestinal tract and central nervous system. In the brain, GRP mRNA is located in the hypothalamic paraventricular nucleus (PVN), a region that receives neural input from the arcuate nucleus and plays a critical role in food intake and energy balance. Because GRP neurons are localized in the vicinity of projection sites in the PVN for peptides that participate in energy homeostasis, we investigated whether GRP mRNA expression in the PVN may be sensitive to challenges imposed by either 38 h food deprivation or stimulation of the melanocortin system by the melanocortin 3/4 receptor agonist, melanotan II (MTII). We found that food deprivation significantly decreased GRP mRNA expression, whereas lateral ventricular MTII administration increased GRP mRNA expression in ad lib fed rats 4 h after administration. Furthermore, administration of MTII at a dose that reduces 24 h food intake and body weight prevented the decrease in GRP mRNA expression observed in animals that were pair-fed to the amount of food consumed by those injected with MTII. These results demonstrate that food deprivation and stimulation of the melanocortin system produce opposing changes in GRP gene expression in the PVN suggesting that GRP-containing neurons in the PVN may be part of the hypothalamic signaling pathway controlling food intake and energy balance. Source -

TAK1 mRNA Expression in the Tumor Tissue

Resistance to radio and chemotherapy is one of the major drawbacks in the progression of head and neck squamous cell cancer (HNSCC) patients, evidencing the importance of finding optimum molecular prognosis markers to develop personalized treatment schedules. TGF-β effector TAK1 activity has been related to a greater aggressiveness in several types of cancer (Kondo et al. 1998; Edlund et al. 2003; Kaur et al. 2005) and, although there has been described no significant implication of TAK1 in HNSCC development, we have further examined the role of its mRNA expression as a marker of prognosis in HNSCC. Fifty-nine advanced HNSCC patients were recruited for the study. The tumor expression of TAK1 mRNA was analyzed with RT-PCR using Taqman technology and its relationship with the clinical outcome of the patients studied. TAK1 mRNA expression was lower in patients that relapsed than in those that did not, but the difference was only significant between the patients that showed response to treatment (p < 0.001). ROC curve analyses pointed a 0.5 expression ratio TAK1/B2M value as an optimum cut-off point for relapse and response. Our data suggest the TAK1 mRNA analysis by Taqman RT-PCR can predict the risk of relapse in HNSCC patients. Source -

mRNA expression of genes
The expression of the oestrogen receptor (ER) is one of the more important clinical parameters of breast cancer. However, the relationship between the ER and its ligand, oestradiol, and the enzymes that synthesise it are not well understood. The expression of mRNA transcripts of members of the oestradiol metabolic and signalling pathways including the ER was studied in detail. Method mRNA transcripts for aromatase ( CYP19 ), 17-βhydroxysteroid dehydrogenase I, 17-β-hydroxysteroid dehydrogenase II, ERα , ERβ , steroid sulfatase ( STS ), oestradiol sulfotransferase ( EST ), cyclin D 1 ( CYCLD1 ) and ERBB2 were fluorometrically quantified by competitive RT-PCR using an internal standard in 155 breast carcinomas. In addition, the transcripts of CYP19 were analysed for alternative splicing/usage of exon 1 and an alternative poly A tail. Results A great variability of expression was observed, ranging from 0 to 2376 amol/mg RNA. The highest levels were observed for STS and EST , and the lowest levels (close to zero) were observed for the 17-βhydroxysteroid dehydrogenase isoenzymes. The levels of mRNA expression were analysed with respect to clinical and histopathological parameters as well as for disease-free survival. High correlation of the mRNA expression of STS , EST and 17-β-hydroxysteroid dehydrogenase in the tumours suggested a common regulation, possibly by their common metabolite (oestradiol). Hierarchical clustering analysis in the 155 patients resulted in two main clusters, representing the ERα-negative and ERα-positive breast cancer cases. The mRNA expression of the oestradiol metabolising enzymes did not follow the expression of the ERα in all cases, leading to the formation of several subclasses of tumours. Patients with

no expression of CYP19 and patients with high levels of expression of STS had significantly shorter disease-free survival time ( P > 0.0005 and P < 0.03, respectively). Expression of ERβ mRNA was a better prognostic factor than that of ERα in this material. Conclusion Our results indicate the importance of CYP19 and the enzymes regulating the oestrone sulfate metabolism as factors of disease-free survival in breast cancer, in addition to the well-known factors ER and ERBB2 . Source -

Microarray mRNA Expression Data Enhances Computational Identification of Active MicroRNAs
Elucidation of regulatory roles played by microRNAs (miRs) in various biological networks is one of the greatest challenges of present molecular and computational biology. The integrated analysis of gene expression data and 3′-UTR sequences holds great promise for being an effective means to systematically delineate active miRs in different biological processes. Applying such an integrated analysis, we uncovered a striking relationship between 3′-UTR AU content and gene response in numerous microarray datasets. We show that this relationship is secondary to a general bias that links gene response and probe AU content and reflects the fact that in the majority of current arrays probes are selected from target transcript 3′-UTRs. Therefore, removal of this bias, which is in order in any analysis of microarray datasets, is of crucial importance when integrating expression data and 3′-UTR sequences to identify regulatory elements embedded in this region. We developed visualization and normalization schemes for the detection and removal of such AU biases and demonstrate that their application to microarray data significantly enhances the computational identification of active miRs. Our results substantiate that, after removal of AU biases, mRNA expression profiles contain ample information which allows in silico detection of miRs that are active in physiological conditions. Source -

Shared By: