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At least 451 records · Page 25Linked to original sources
PubMed2006

Assessment of the use of RNA quality metrics for the screening of articular cartilage specimens from clinically normal dogs and dogs with osteoarthritis.

OBJECTIVE: To assess 2 methods of RNA purification by use of different quality metrics and identify the most useful metric for quality assessment of RNA extracted from articular cartilage from dogs with osteoarthritis. SAMPLE POPULATION: 40 articular cartilage specimens from the femoral heads of 3 clinically normal dogs and 37 dogs with osteoarthritis. PROCEDURES: RNA was extracted from articular cartilage by 2 purification methods. Quality metrics of each sample were determined and recorded by use of a UV spectrophotometer (Spec I; to determine the 260 to 280 nm absorbance ratio [A(260):A(280) ratio]), a second UV spectrophotometer (Spec II; to determine A(260):A(280) and A(260):A(230) absorbance ratios), and a microfluidic capillary electrophoresis analyzer (to determine the ribosomal peak ratio [RR], degradation factor [DF], and RNA integrity number [RIN]). The RNA was extracted from affected (osteoarthritic) articular cartilage and assessed with the same quality metrics. Metric results were compared with visual analysis of the electropherogram to determine the most useful RNA quality metric. RESULTS: No differences in methods of RNA purification were determined by use of quality metrics. The RNA extracted from unaffected (normal) cartilage was of higher quality than that extracted from affected (osteoarthritic) cartilage, as determined by the RIN and Spec II A(260):A(230) ratio. The RIN and RR were the most sensitive metrics for determining RNA quality, whereas the DF was most specific. A significant proportion (32%) of RNA extracted from osteoarthritic articular cartilage specimens was determined as being of low quality. CONCLUSIONS AND CLINICAL RELEVANCE: No single metric provided a completely sensitive and specific assessment of the quality of RNA recovered from articular cartilage.

Animals↗
PubMed2004

Assessment of human papillomavirus mRNA detection over time in cervical specimens collected in liquid based cytology medium.

Little is known about the stability of human papillomavirus (HPV) RNA within cervical samples collected in liquid based cytology (LBC) preservation media. We addressed this by analysing patient LBC specimens for the presence of HPV RNA over a prospective time course. LBC samples in PreservCyt were obtained from seven women referred to colposcopy due to a cytological diagnosis of moderate or severe dyskaryosis. Aliquots were removed and subject to RNA extraction at, 6h (base-line), 4, 7 and 14 days, post-collection. HPV mRNA was detected using the PreTect HPV Proofer, which detects HPV 16, 18, 31, 33 and 45 E6/E7 transcripts and human small ribonucleoprotein U1A mRNA as a sample control. HPV DNA genotyping was also performed at base-line to assess the range of types in our group. In addition to assessment of viral RNA, overall integrity of the cellular RNA extract was analysed by the RNA 6000 pico assay. Control human RNA was amplified successfully in all seven samples at each time point. Five of the seven women were HPV positive for E6/E7 viral transcripts at base-line and positivity was maintained in all five up to 14 days. Although the pattern of cellular RNA profiles generated from the samples was variable, results indicated that this extract could be amenable to gene expression profiling and that degradation did not increase as a result of storage time. It is concluded that HPV RNA in routinely collected LBC specimens in PreservCyt can be detected for at least 14 days from sample collection.

Cervix Uteri↗
PubMed1996

Activin-like signal activates dorsal-specific maternal RNA between 8- and 16-cell stages of Xenopus.

In many animals the dorsal-ventral axis forms by an initial localization of maternal molecules, which then regulate the spatial location of signals that directly influence the expression of axis-specific fates. Several recent studies have demonstrated that dorsal-animal blastomeres of the Xenopus morula (8-32 cells) are biased toward dorsal fates prior to mesoderm inductive signaling. In this study we ask whether the dorsal bias is the result of autonomous expression of maternal molecules specifically localized within dorsal cells or of early activating signals. It was found that although 16-cell dorsal-animal blastomeres (D1.1) can differentiate into dorsal tissues when cultured alone, the 8-cell mothers (D1) can not. Likewise, although RNA extracted from D1.1 can induce an extra dorsal axis when injected into vegetal blastomeres, RNA extracted from D1 can not. However, D1 does express dorsal tissues if co-cultured with dorsal-vegetal cells or with culture medium containing a mixture of activins (PIF-medium). Furthermore, short-term culture of D1 in PIF-medium enables the D1 RNA to induce an ectopic dorsal axis. Ventral-animal blastomeres also can express dorsal axial tissues when co-cultured with dorsal-vegetal blastomeres or in PIF-medium, but the RNA from the activin-treated ventral cells cannot induce ectopic dorsal axes. These studies demonstrate that there are maternal RNAs that, shortly after fertilization, are present only in the dorsal-animal region. They do not act cell autonomously, but require an activin-like signal. These RNAs may function by increasing the responsiveness of dorsal-animal blastomeres to the mesoderm inductive signals present in both the morula and the blastula.

Activins↗
PubMed1991

Cytokine messenger RNA stability is enhanced in tumor cells.

Hematopoietic growth factors are produced by a number of human tumors. We extracted RNA from selected human tumor cells known to produce at least one hematopoietic growth factor and found high levels of abnormally stable cytokine messenger (m)RNA. Half-life experiments performed after preventing RNA synthesis by exposing cells to actinomycin D before RNA extraction showed stabilization of cytokine messages in tumor cells in liquid culture as well as in human tumor xenografts grown in mice. Exposure to the phorbol ester phorbol 12-myristate 13-acetate (TPA) caused enhancement of granulocyte-macrophage colony-stimulating factor (GM-CSF) message level in lung cancer cells and in control fibroblasts but elevated levels persisted far longer in the tumor cells. In normal cells, an AU-rich sequence in the 3' untranslated region of cytokine mRNAs confers lability to the message. Although a beta-globin gene expression vector containing this region appears to produce unstable mRNA in lung cancer cells, cytokine mRNAs, which also contain this sequence, are very stable in the tumors we studied. This may indicate that another region of the cytokine mRNA molecule is of greater importance than the AU-rich region in determining mRNA stability in tumor cells.

Adenocarcinoma↗
PubMed1994

Supraspinal delta opioid receptor mRNA levels are not altered in [D-Ala2]deltorphin II tolerant mice.

Delta opioid receptor (DOR) mRNA levels were studied in mice rendered tolerant to [D-Ala2]deltorphin II by 4 days of repeated intracerebroventricular administration (10 micrograms, [tid]). ED50 determinations on day 5 revealed a 10-fold loss in [D-Ala2]deltorphin II potency with the tail-flick test. Utilization of a microdissection technique followed by quantitative solution hybridization of RNA extracts from mouse brain revealed mean levels of DOR mRNA ranging from 3.9 pg/micrograms RNA in the caudate-putamen to 0.4 pg/micrograms RNA in the cerebellum. DOR mRNA levels were not different when RNA extracts from tolerant and non-tolerant mice were compared. These data suggest that altered DOR mRNA levels are not one of the adaptive changes that occur with delta opioid ([D-Ala2]deltorphin II) tolerance.

Animals↗
PubMed1997

mRNA expression of ligands of the epidermal-growth-factor-receptor in the uterus.

Six different ligands of the epidermal-growth-factor receptor (EGFR) have been identified in the past. In some cervical squamous-cell carcinomas, an increased amount of proteins binding to the EGFR has been reported. In order to identify the mRNA of EGFR ligands (EGFRL), which might be overexpressed in cervical and endometrial cancers, we performed semi-quantitative reverse-transcription/polymerase chain reactions (RT-PCR) for all 6 EGFRL in RNA extracts of normal and malignant tissue samples of the human uterus. PCR products from RNA extracts of 83 patients were quantitated relative to the housekeeping gene and internal standard pyruvate dehydrogenase by analyzing the PCR kinetics of product synthesis. In extracts of normal cervix, the level of mRNA expression of the EGFRL was significantly higher than in endometrium. No significant difference was detected between normal cervix and cervical carcinomas. However, both in cervical and in endometrial cancers, mRNA expression was non-parametrically distributed and in some cervical cancers overexpression of transforming growth factor alpha (TGF-alpha), amphiregulin or EGF was observed. In endometrial cancers, mRNA levels of all EGFRL were higher than in normal endometrium. This increase was significant (p < 0.005) for TGF-alpha and amphiregulin. Thus, TGF-alpha mRNA is overexpressed in approximately 10% of cervical cancers and in the majority of endometrial cancers. Since TGF-alpha anti-sense therapy might represent a future strategy in such cancers, we also determined the absolute level of TGF-alpha mRNA expression by quantitative PCR using a cloned standard.

Cloning, Molecular↗
PubMed1998

Eel (Anguilla japonica) testis 11beta-hydroxylase gene is expressed in interrenal tissue and its product lacks aldosterone synthesizing activity.

A recombinant expression vector containing Japanese eel (Anguilla japonica) testis cytochrome P450(11 beta) (11beta-hydroxylase) cDNA was introduced into COS-1 cells. Enzymatic activity of the expressed P450(11 beta) for corticosteroid synthesis was analysed by incubating transfected cells with 14C-labelled 11-deoxycorticosterone or 3H-labelled deoxycortisol as substrates. Thin layer chromatography of incubation medium revealed that a high percentage of 11-deoxycorticosterone was converted into corticosterone and 11-dehydrocorticosterone but no aldosterone was detected. Similarly, deoxycortisol was converted into cortisol and cortisone. These results show that eel P450(11beta) does not possess significant aldosterone synthesizing activity. Northern blot analysis detected a 1.8 kb transcript of P450(11beta) using RNA extracted from interrenals of untreated Japanese eel but no hybridization signal was apparent using RNA extracted from brain, spleen, heart, muscle or testis. Immunohistochemistry using an antiserum against P450(11beta) also revealed strong immunostaining in interrenal cells.

Aldosterone↗
PubMed1994

Search for persistent enterovirus infection of muscle in inflammatory myopathies.

To investigate the hypothesis that the inflammatory muscle diseases (IMD) polymyositis (PM) and dermatomyositis (DM) may be due to a chronic, persistent enterovirus (EV) infection we sought to determine the prevalence of these viruses in muscle tissue using both nested polymerase chain reaction (PCR) and dot-blot hybridization assays. Thirty-six frozen muscle biopsies from 32 adult cases of IMD and 42 biopsies from 36 control subjects with other neuromuscular disorders were studied. Primers for PCR were chosen to conserved regions of the 5'-untranslated region of the EV genome. Constitutive Ableson tyrosine kinase (ABL) mRNA was detected by PCR to confirm the integrity of muscle RNA extracts. The sensitivity of the EV PCR was determined to be 40-400 copies (12.5-125 ag) of synthetic EV RNA transcript against a background of 1 microgram of cellular RNA. The specificity was assessed using a range of enteroviral and unrelated viral isolates extracted from cell cultures. Of the 78 samples tested, ABL mRNA was successfully detected in all but four samples. The time the biopsies spent in ultracold storage (1-73 months) did not appear to influence the integrity of extracted RNA. When assayed for EV RNA by nested PCR, none of 29 IMD cases (i.e., 28 PM and 1 DM) nor sequential biopsies from 3 PM patients were found to be positive. All 42 control biopsies were also negative for EV RNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Detection of Gene Expression in Genetically Engineered Microorganisms and Natural Phytoplankton Populations in the Marine Environment by mRNA Analysis.

A simple method that combines guanidinium isothiocyanate RNA extraction and probing with antisense and sense RNA probes is described for analysis of microbial gene expression in planktonic populations. Probing of RNA sample extracts with sense-strand RNA probes was used as a control for nonspecific hybridization or contamination of mRNA with target DNA. This method enabled detection of expression of a plasmid-encoded neomycin phosphotransferase gene (nptII) in as few as 10Vibrio cells per ml in 100 ml of seawater. We have used this method to detect expression of the ribulose-1,5-bisphosphate carboxylase large-subunit gene (rbcL) in Synechococcus cultures and natural phytoplankton populations in the Dry Tortugas, Florida. During a 36-h diel study, rbcL expression of the indigenous phytoplankton was greatest in the day, least at night (1100, 0300, and 0100 h), and variable at dawn or dusk (0700 and 1900 h). These results are the first report of gene expression in natural populations by mRNA isolation and probing. This methodology should be useful for the study of gene expression in microorganisms released into the environment for agricultural or bioremediation purposes and indigenous populations containing highly conserved target gene sequences.

Journal Article↗
PubMed2005

Global gene expression profiling of circulating tumor cells.

Metastases from primary tumors are responsible for most cancer deaths. It has been shown that circulating tumor cells (CTCs) can be detected in the peripheral blood of patients with a variety of metastatic cancers and that the presence of these cells is associated with poor clinical outcomes. Characterization of CTCs in metastatic cancer patients could provide additional information to augment management of the disease. Here, we describe a novel approach for the identification of molecular markers to detect and characterize CTCs in peripheral blood. Using an integrated platform to immunomagnetically isolate and immunofluorescently detect CTCs, we obtained blood containing > or = 100 CTCs from one metastatic colorectal, one metastatic prostate, and one metastatic breast cancer patient. Using the RNA extracted from the CTC-enriched portion of the sample and comparing it with the RNA extracted from the corresponding CTC-depleted portion, for the first time, global gene expression profiles from CTCs were generated and a list of cancer-specific, CTC-specific genes was obtained. Subsequently, samples immunomagnetically enriched for CTCs from 74 metastatic cancer patients and 50 normal donors were used to confirm by quantitative real-time reverse transcription-PCR CTC-specific expression of selected genes and to show that gene expression profiles for CTCs may be used to distinguish normal donors from advanced cancer patients as well as to differentiate among the three different metastatic cancers. Genes such as AGR2, S100A14, S100A16, FABP1, and others were found useful for detection of CTCs in peripheral blood of advanced cancer patients.

Breast Neoplasms↗
PubMed1983

Biochemical complementation with RNA in the Xenopus oocyte: a small RNA is required for the generation of 3' histone mRNA termini.

When the cloned h22 histone gene repeat unit of the sea urchin Psammechinus miliaris is injected into the frog oocyte nucleus, transcription initiates faithfully in the promoter region of the H3 gene, but transcription termination or RNA processing to yield genuine H3 mRNA 3' ends is very inefficient. The generation of 3' ends can be enhanced by the injection of a small sea urchin poly (A)- RNA into the oocyte cytoplasm one day prior to injection of the histone gene repeat unit. This RNA is approximately 60 nucleotides in length and appears to be present in relatively low abundance in embryos of both Psammechinus and Paracentrotus. RNA extracted from a nuclear DEAE fraction, previously shown to complement the lesion in H3 gene expression, also causes the appearance of true 3' ends of H3 mRNA. Furthermore, RNA extracted from our 12S "termination" factor includes an RNA species comigrating with the 60 nucleotide RNA from poly (A)- RNA. We suggest that the 12S nuclear "termination" component is in fact a small nuclear RNP.

Animals↗
PubMed1975

The contribution of RNA and non-histone proteins to the circular dichroism spectrum of chromatin.

This paper is an investigation of the contribution of low salt extractable RNA and non-histone proteins to the circular dichroism of chromatin. Circular dichroism (CD) of chromatin above 250 nm is due mainly to DNA and is different from that of DNA free in solution. In addition, to a smaller extent, we find that low salt extractable RNA and/or non-histone protein side chain chromophores contribute significantly to the spectra in this region and account for the major differences observed among the CD spectra of chromatins isolated from the five tissues studied; pig cerebellum, myeloma, calf thymus, chick embryo brain, and chick erythrocytes.

Animals↗
PubMed1994

Detection of enterovirus RNA in clinical samples by nested polymerase chain reaction for rapid diagnosis of enterovirus infection.

Employing a nested polymerase chain reaction with primers from the 5' non-translated region of the enterovirus genome, we detected enteroviral RNA from patients with a variety of enterovirus-associated clinical syndromes. This technique was found to be sensitive (detecting enteroviral RNA extracted from 0.1 50% tissue culture infectious dose) and specific; no specific PCR product was detected from RNA extracts of a variety of non-enterovirus isolates. Although the technique is of comparable sensitivity to single round polymerase chain reaction followed by Southern blot hybridization, it was more rapid, since it enabled a diagnosis to be made within 1 day. Thus, using nested polymerase chain reaction we were able to detect enteroviral RNA in 31 of 46 clinical specimens from 17 of 23 patients with suspected enterovirus infections. The samples included cerebrospinal fluid, throat swabs, stool, vesicle fluid, peripheral blood lymphocytes, whole blood and pericardial effusion. In contrast virus was isolated in only 17 of 42 clinical specimens from 12 of 22 these patients. In preliminary studies, restriction endonuclease analysis of polymerase chain reaction products enabled us to distinguish between non-polio enteroviruses and poliovirus types 1, 2, and 3. This additional technique may be useful in distinguishing between such infections in polio-endemic countries where rapid public health measures may be required.

Adult↗
PubMed2001

Theileria annulata: identification, by differential mRNA display, of modulated host and parasite gene expression in cell lines that are competent or attenuated for differentiation to the merozoite.

To identify both host and parasite genes that show altered expression during differentiation of Theileria annulata from the macroschizont to the merozoite stage of the life cycle, the RNA profiles of two T. annulata-infected clonal cell lines (D7 and D7B12) with the same genetic background have been compared by RNA display. In the cloned cell line D7, T. annulata differentiates from the macroschizont to the merozoite at 41 degrees C, whereas in the cell line D7B12, which was derived by recloning D7, the parasite does not differentiate. Therefore, genes that show altered expression levels in either clone could be modulated by the differentiation event and are possible candidates for regulators of this process. Differential display was carried out initially on RNA extracted from D7 and D7B12 macroschizont-infected cells cultured at 37 degrees C and secondly on RNA extracted from the two cell lines incubated at 41 degrees C to induce differentiation to the merozoite. The first procedure identified 29 cDNA fragments that displayed altered levels between D7 and D7B12, 9 of which were confirmed to be differentially expressed by Northern blot analysis. Of these 9 gene fragments, 8 were found to be of host origin, while 1 was parasite derived. The second RNA display analysis identified 14 transcripts that showed altered levels during a differentiation time course, of which 6 were confirmed to be differentially expressed between D7B12 cells and differentiating D7 cells by Northern blot analysis. Of these 6 gene fragments, 1 was of host and 5 were of parasite origin. The parasite genes either showed levels of RNA consistent with constitutive gene expression or, in one case, a genuine upregulation of mRNA associated with the differentiation process.

Animals↗
PubMed1993

Atrial natriuretic hormone: a paracrine or endocrine role within the human placenta?

There is evidence to suggest that the human placenta may synthesize atrial natriuretic hormone (ANH). To test this hypothesis we examined specimens of normal term placenta obtained at elective caesarean section for evidence of either storage or synthesis of this peptide. No immunocytochemical or ultrastructural evidence of ANH storage was found. RNA extracted from cultured cytotrophoblast cells, the chorionic or decidual plates, or from placental villi contained no ANH-encoding messenger RNA by Northern blot analysis. Polymerase chain reaction amplification of these RNA extracts using primers directed to the midportion of the ANH gene failed to demonstrate any evidence of ANH-encoding RNA. We conclude that ANH is neither stored nor synthesized within the normal term placenta, and thus a purely endocrine role for this peptide within the fetoplacental compartment is proposed.

Atrial Natriuretic Factor↗
PubMed1981

Induction of immunoenhancing factors for murine splenocyte cultures by Salmonella typhimurium ribosome and ribonucleic acid extracts.

Ribosomal and ribonucleic acid (RNA)-rich preparations derived from Salmonella typhimurium were examined for their ability to enhance the primary in vitro antibody response of normal mouse spleen cell cultures to sheep erythrocytes. Both of these fractions were consistently more active in elevating the antibody response of normal mouse splenocytes from lipopolysaccharide (LPS) responder mice than was LPS. Furthermore, injection of mice with either the ribosomal or RNA-rich fraction induced antibody response helper factor activity in 2-h post-treatment serum similar to that induced by LPS. Endotoxin low-responding C3H/HeJ mice were stimulated to release helper factors by ribosomes and the RNA extracts but not by LPS. Treatment of the ribosomes and RNA fractions with ribonuclease destroyed their ability to stimulate the production of the helper factor in serum of treated mice. Therefore, it appears likely that ribosomes and RNA-rich fractions stimulated an intermediate helper factor due to the presence of RNA and not LPS.

Animals↗