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At least 289 records · Page 16Linked to original sources

PubMed1999

Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro.

In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.

Animals↗

PubMed1994

Collagen type IV gene expression in human optic nerve heads with primary open angle glaucoma.

Previous studies have demonstrated proliferation of basement membranes in the optic nerve head in primary open angle glaucoma (POAG). We used in situ hybridization (ISH) of a radiolabeled riboprobe specific for human collagen IV, a ubiquitous component of basement membranes, to identify cells actively synthesizing basement membranes in the optic nerve head in POAG. In addition, to detect and further characterize the collagen IV mRNA transcripts, we used reverse transcriptase-polymerase chain reaction (RT-PCR) in total RNA extracted from individual optic nerve heads with POAG and from age-matched normal controls. ISH results demonstrate that, in POAG, numerous astrocytes in the prelaminar region expressed collagen IV mRNA. Lamina cribrosa cells and astrocytes in the compressed lamina cribrosa hybridized the probe. Few astrocytes and lamina cribrosa cells hybridized the probe in the optic nerve head of normal age-matched controls. RT-PCR products for collagen IV and for glyceraldehyde-3-dehydrogenase (G3PDH), a reference gene, were detected by agarose electrophoresis as single bands of the expected sizes and positively identified by Southern hybridization using specific cDNA probes in normal and POAG samples. No additional products (bands) were observed in RT-PCR experiments, indicating that there was no genomic DNA contamination in the total RNA extract. The lack of additional bands suggests that, at least in the ten samples used in this study, there were no alternatively spliced RNA products in any of the amplified sequences. Semi-quantitative analyses using densitometry showed a two-fold increase in collagen type IV PCR present in POAG samples. No differences were detected in levels of G3PDH PCR products between POAG and normal samples. This investigation provides evidence of increased biosynthesis of collagen type IV at the mRNA level in optic nerve heads with POAG. Whether this phenomenon represents a response to elevated intraocular pressure or a reparative mechanism to the loss of axons remains to be determined.

Aged↗

PubMed1994

Analysis of epidermal growth factor receptor mRNA expression by polymerase chain reaction assay in 94 human breast adenocarcinoma tumors.

It is well known that breast cancer cells can synthesize and secrete various growth factors that are able to stimulate tumor growth through autocrine and/or paracrine mechanisms. EGF is one of these growth factors involved in normal breast epithelial development and tumor proliferation. EGF and TGF alpha (EGF-like peptide) are produced in variable amounts and both bind to the EGF receptor (EGF-R). Previous investigation in the laboratory measuring free and occupied EGF-R sites by differential ligand binding assays had demonstrated that non-occupied and total binding sites were present in 54 and 90% of 216 breast tumor biopsies respectively. EGF-R appeared to be totally masked by endogenous ligand in 40 and 21% of estrogen receptor positive and negative tumors respectively. The aim of the present study was to check by a molecular method the expression of the EGF-R gene. The PCR method was applied to 94 tumor samples of the previous series. Total RNA was treated with 0.5 units of Rnase-free Dnase/mg of RNA to remove any contaminating DNA. We simultaneously reverse transcribed and amplified another transcript (beta-actin) as an internal standard. Both signals were present in 88 of the 94 samples while the presence of EGF-R was detected in 74 of them when assessed by radioligand assay. The findings indicate that 93% of the tumors analysed in this series expressed EGF-R mRNA, in agreement with our previous data on occupied EGF-R sites, i.e. two-fold more than by using the standard binding assay. No significant correlation was observed between the expression of the EGF-R gene and the estrogen receptor content.

Adenocarcinoma↗

PubMed1994

Transfer RNA analysis during the reproductive cycle of a freshwater teleost, H. fossilis.

Transfer RNA was analyzed qualitatively as well as quantitatively from ovaries of the fresh water teleost Heteropneustes fossilis for twelve months. The tRNA samples were found to be pure and devoid of any high molecular weight RNA or DNA contaminations. The quantity of tRNA as well as its biological activity, assayed by in vitro aminoacylation using homologous aminoacyl tRNA synthetases, were found to be higher during resting and preparatory (pre-vitellogenic) phases, i.e. from November to March, as compared to vitellogenic and spawning phases of the fish, i.e. from April to October. The highest tRNA pool and its activity was found in the month of February, which coincides with the early preparatory phase. The results indicate that the accumulation of active tRNA starts in the resting phase. Such an accumulation of tRNA may be a part of the enrichment of mature eggs with complete translational machinery before ovulation in order to cope with the high rate of protein synthesis after fertilization.

Amino Acyl-tRNA Synthetases↗

PubMed1996

Bombyxin gene expression in tissues other than brain detected by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization.

Bombyxin is a 5 kDa insulin-related peptide produced in four pairs of medial neurosecretory cells in the brain of the silkmoth Bombyx mori. We demonstrate here the presence of bombyxin mRNA in tissues other than brain: ganglia, epidermis, testis, ovary, fat body, silk gland, Malpighian tubule, midgut, and hindgut of the Bombyx fifth instar larvae. Bombyxin mRNA was detected by Oligotex reverse transcription-polymerase chain reaction (RT-PCR), a rapid and simple procedure of reverse transcription-PCR, and in situ hybridization. The Oligotex RT-PCR method effectively eliminated the contaminating DNA in RNA samples and amplified bombyxin mRNA efficiently. In situ hybridization of the Bombyx ovary clearly demonstrated the localization of the bombyxin mRNA in the ovariole. The present study is the first demonstration of expression of brain neurosecretory peptide in tissues other than the central nervous system in insects at RNA level.

Animals↗

PubMed1996

Identifying the sex of human preimplantation embryos in X-linked disease: amplification efficiency of a Y-specific alphoid repeat from single blastomeres with two lysis protocols.

INTRODUCTION: Preimplantation diagnosis involves detecting genetic defects in one or two blastomeres biopsied from cleavage stage embryos following a vitro fertilization (IVF). For X-linked recessive disease, identification of the sex of embryos allows transfer of only unaffected females. To examine how critical the preparation of the single blastomere is for amplification of a Y chromosome specific repeat sequence using the polymerase chain reaction (PCR), the incidence of amplification failure has been examined following two lysis protocols. MATERIALS AND METHODS: Amplification of a Y alphoid repeat sequence from single blastomeres disaggregated from cleavage stage embryos was examined after either (1) lysis in distilled water and freeze-thawing twice or (2) a two-step lysis protocol involving an initial treatment in potassium hydroxide and dithiothreitol. Some of the embryos had been previously sexed by cleavage-stage biopsy and fluorescent in situ hybridization with X- and Y-specific probes. RESULTS: Amplification failure occurred in 6 of 50 (12%) and 4 of 60 (7%) single blastomeres from male embryos following lysis in distilled water or using the two-step protocol, respectively. Conversely, amplification from contaminating DNA occurred in 5 of 63 (8%) single blastomeres from female embryos and 6 of 94 (6%) of control medium-blanks. CONCLUSIONS: The incidence of amplification failure was improved but not eliminated using the two-step lysis protocol. At least two cells, therefore, would be necessary for accurate identification of males by amplification of Y-specific repeat sequences alone. Nevertheless, this protocol for preparing cleavage-stage blastomeres is likely to give more consistent amplification of any unique or repeat sequences.

Blastomeres↗

PubMed1995

Semi-quantitative analysis of cytokine gene expression in blood and cerebrospinal fluid cells by reverse transcriptase polymerase chain reaction.

An easy, reproducible and semi-quantitative, non-radioactive method for the analysis of mRNA expression for various cytokines, (i.e., Interleukin (IL)-1 beta, IL-4, IL-6, tumor necrosis factor (TNF)-alpha, lymphotoxin (LT), transforming growth factor (TGF)-beta, interferon (IFN)-gamma and endothelin-1 (ET-1)) in cells from cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) has been established. By means of polymerase chain reaction primers that cover a splice junction, amplification of contaminating DNA was omitted. Densitometric scanning of ethidium bromide-stained agarose gels proved to be very sensitive for semiquantitative analysis of PCR products. Serial tenfold dilutions of cDNA revealed a log-linear regression from 10(6) to 10(2) cells under optimal cycle conditions. The intra- and inter-assay variability of the method was below 10%. With this assay, the cytokine expression pattern of as few as 10(4) mononuclear cells from blood or CSF was determined. This method made it possible to detect differences in the cytokine gene expression pattern of mononuclear cells from patients with different neurological diseases. CSF cells from 43 patients with various neurological diseases were analyzed. TNF-alpha, LT, and IL-1 mRNA were prominent in the CSF cells of most patients with bacterial meningitis. TNF-alpha, LT, IFN-gamma and IL-6 mRNAs were detected in patients with active multiple sclerosis, whereas TNF-alpha, IL-6, and endothelin-1 mRNA expression was found frequently in patients with HIV encephalitis. Pro-inflammatory cytokines were rarely detected in CSF cells from patients with non-inflammatory diseases of the central nervous system. In blood mononuclear cells from patients with multiple sclerosis, TNF-alpha mRNA expression was associated with disease activity. The sensitivity, specificity, velocity and reliability of this assay considerably facilitates the analysis of cytokine production in mononuclear cells even in conditions where only a limited number of cells is available for analysis.

Base Sequence↗

PubMed2002

Identification of Agrobacterium spp. present within Brassica napus seed by TaqMan PCR--implications for GM screening procedures.

A fluorogenic probe (fliG-P), designed within a chromosomal DNA sequence, was used in a TaqMan PCR assay to identify Agrobacterium spp. The TaqMan assay detected 58 of 59 Agrobacterium strains tested, but did not detect 13 other Rhizobiaceae strains. Seedlings were grown from seven lots of surface-sterilised Brassica napus seed. Seedlings from these samples were placed in phosphate buffer and the resulting suspensions used to inoculate broth media selective for Agrobacterium biovars 1 and 2. Lysed broths (after 48 h incubation) were used as template in the fliG TaqMan PCR to detect Agrobacterium sp. in one of the seed samples. Individual Agrobacterium strains were isolated from this sample and tested by three Ti-plasmid conventional PCR assays. None of the strains possessed a plasmid. This is the first report of Agrobacterium sp. present within the seed of B. napus, a crop routinely screened for genetically modified DNA contamination using PCR assays with Agrobacterium sequences as targets.

Brassica napus↗

PubMed1997

Dual blastomere analysis improves reliability of preimplantation trembler mouse diagnosis.

Dual blastomere biopsy and independent blastomere analysis dramatically improved preimplantation diagnostic reliability as confirmed by testing the remaining biopsied eight-cell mouse embryo. The autosomal dominant trembler mouse point mutation was selected as a model for human preimplantation diagnosis because: (1) single cell assay failure is predicted to be the highest when testing autosomal dominant mutations; (2) point mutations represent the most common of all mutation categories and the most demanding mutation to assay reliably; and (3) the trembler mouse point mutation in peripheral myelin protein 22 (Pmp22) is a model of human Charcot-Marie-Tooth type 1A disease. Mathematical models predict our experimental results assuming amplification of 80% of each target allele as well as trembler sperm DNA contamination in 1 of 44 normal biopsied single blastomeres. Single blastomere analysis correctly predicted the genotype in only 84% of embryos that would have been implanted as normal. In contrast, when independent tests of both biopsied blastomeres agreed, test results were confirmed in 20 of 21 (95.2%) of the remaining six-cell biopsied embryos designated as normal. Thus, biopsied six-cell embryo confirmation demonstrated that dual biopsied blastomere analysis improved test reliability remarkably.

Animals↗

PubMed2006

Increased levels of CK19 mRNA in oral squamous cell carcinoma tissue detected by relative quantification with real-time polymerase chain reaction.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumour in the oral and maxillofacial region and has a poor prognosis. Cytokeratin 19 (CK19) is a component of cytoskeleton protein. Previous studies have reported abnormal expression of CK19 protein in OSCC tissue. This study is to investigate the quantitative level of CK19 gene transcript in OSCC tissue as well as its clinical significance. Thirty-one OSCC patients (26 males and 5 females) took part in the present study, aged 34-78 years (mean 58.2 years). The level of CK19 mRNA was detected using fluorescent quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) in cancerous and paracancerous tissues. The relative quantification in cancerous tissue compared with paracancerous tissue was calculated using the 2(-DeltaDeltaCt) equation. The level of CK19 mRNA in cancerous tissue from OSCC patients was 2.21-fold higher than that in paracancerous tissue (P=0.020), and the amplicon was specific without genomic DNA contamination. The level of CK19 mRNA correlated significantly with the pathological differentiation grade of OSCC tissue (P=0.025), with poorer differentiation indicating a higher level of CK19 mRNA. These results suggest that fluorescent quantitative real-time RT-PCR is accurate and reliable for the detection of CK19 gene transcript levels in OSCC tissue. The level of CK19 mRNA was increased in OSCC tissue, and this was significantly correlated with the pathological differentiation grade.

Adult↗

PubMed2004

Single muscle fiber gene expression in human skeletal muscle: validation of internal control with exercise.

Reverse transcription and real-time PCR have become the method of choice for the detection of low-abundance mRNA transcripts obtained from small human muscle biopsy samples. GAPDH, beta-actin, beta-2M, and 18S rRNA are widely employed as endogenous control genes, with the assumption that their expression is unregulated and constant for given experimental conditions. The aim of this study was to determine if mRNA transcripts could be performed on isolated human single muscle fibers and to determine reliable housekeeping genes (HKGs) using quantitative gene expression protocols at rest and in response to an acute exercise bout. Muscle biopsies were obtained from the gastrocnemius of three adult males before, immediately after, and 4 h following 30 min of treadmill running at 70% of VO2max. A total of 40 single fibers (MHC I and IIa) were examined for GAPDH, beta-actin, beta-2M, and 18S rRNA using quantitative RT-PCR and SYBR Green detection. All analyzed single fiber segments showed ribosomal RNA (28S/18S). No degradation or additional bands below ribosomal were detected (rRNA ratio 1.5-1.8). Also, no high or low-molecular weight genomic DNA contamination was observed. For each housekeeping gene the duplicate average SD was +/-0.13 with a CV of 0.58%. Stable expression of GAPDH was observed at all time points for each fiber type (MHC I and IIa). Inconsistent expression of beta-actin, beta-2M, and 18S rRNA was observed during the post-exercise time points for each fiber type. These data indicate that successful extraction of high quality RNA from human single muscle fibers along with quantification of mRNA of selected genes can be performed. Furthermore, exercise does influence the expression of certain HKGs with GAPDH being the most stable.

Adaptation, Physiological↗

PubMed2005

A microsystem compatible strategy for viable Escherichia coli detection.

This study delineates a microsystem compatible strategy that enables the rapid determination of Escherichia coli viability for the application in food and water monitoring. This approach differentiates the living cells from the dead ones by detecting the presence of a "viability indicator", i.e. mRNAs of a common E. coli GroEL heat shock protein (hsp). Our method starts with a stimulated and controlled transcription of hsp mRNA under an elevated temperature (47 degrees C) for 20min. Following that, the short-life mRNA is rapidly extracted using streptavidin-modified magnetic particles containing biotin-labeled DNA probes complementary to a specific region of the mRNA. The quantification of mRNA by gel electrophoresis and Ag/Au-based electrochemical detection is done after the amplification of mRNAs by reverse transcription-polymerase chain reaction (RT-PCR). Heat shock temperatures and durations that have profound effect to the mRNA transcription were studied and it was found that the mRNA undergoes a rapid minute-by-minute self-degradation after the environment resumes room temperature. Issues such as the DNA contamination that interfere the magnetic particle-based mRNA extraction technique were tackled. A sensitive Ag/Au-based electrochemical analysis method was used to detect the RT-PCR products and a cell concentration as low as 10(2)cfu/ml can be achieved by the electrochemical method, but not by the conventional gel electrophoresis. The strategy demonstrated in this study can be readily implemented in a microsystem and is a step forward for the realization of an integrated bioanalytical microsystem (lab on a chip) for the viable cell detection.

Biosensing Techniques↗

PubMed2006

Detection of Mammaglobin A-mRNA-positive circulating tumor cells in peripheral blood of patients with operable breast cancer with nested RT-PCR.

OBJECTIVES: The development and validation of a nested RT-PCR methodology for the detection of Mammaglobin A-mRNA-positive circulating tumor cells in peripheral blood of patients with operable breast cancer and evaluation of its prognostic significance. DESIGN AND METHODS: Different combinations of specific primers were in silico designed and selected, so that false positive results due to genomic DNA contamination were avoided. The specificity of the primers used was evaluated in 30 healthy individuals, 20 patients with colorectal cancer and 20 patients with non-small cell lung cancer. The method was applied in 101 patients with operable breast cancer before the administration of adjuvant chemotherapy and 39 patients with metastatic breast cancer. RESULTS: Mammaglobin A-mRNA-positive cells were detected in 14/101 (13.9%) of early breast cancer patients but not in the control population studied (0%); 9 of them (64.3%) relapsed during the follow-up period. Mammaglobin A was detected in 7/39 (17.9%) of patients with verified metastasis. Multivariate analysis revealed the detection of Mammaglobin A-mRNA-positive cells, as an independent risk factor for reduced DFI. CONCLUSIONS: Mammaglobin A is a highly specific molecular marker for the detection of circulating tumor cells in operable breast cancer, with important prognostic applications.

Biomarkers, Tumor↗

PubMed2006

Rapid high-yield mRNA extraction for reverse-transcription PCR.

Reverse-transcription PCR (RT-PCR) is the gold standard for mRNA quantification. Efficient, rapid, and high-throughput mRNA extraction is a prerequisite to ensure PCR sensitivity and precision, particularly for quantification of low-abundance mRNAs, and for large numbers of samples. Many mRNA extraction methods entail meticulous handling of individual samples, and are not well suited for large sample numbers. To achieve simple separation of mRNA binding matrix and the medium from which mRNA is to be isolated, oligo (dT)(20)-coated silica beads were used. Simple centrifugation and decanting steps can be used throughout the extraction procedure to separate supernatant fluids from the silica beads. DNase treatment reduced clumping of sedimented beads, thus facilitating bead resuspension and avoiding repeated agitation. DNase treatment also significantly reduced contaminating DNA, increased mRNA purity, and enhanced mRNA PCR readout by approximately 5-fold. The number of target transcripts per sample aliquot was higher in DNase-treated mRNA than in non-treated mRNA or in total nucleic acids. Thus, use of DNase-treated mRNA increased sensitivity of detection and quantification of low-copy transcripts. In conclusion, we describe here a simple, rapid, and cost-effective method that facilitates convenient extraction of high-quality mRNA by minimizing cumbersome mechanical disruption and pipetting steps.

Animals↗

PubMed2006

Facilities for complainants of sexual assault throughout the United Kingdom.

BACKGROUND: A sexual assault referral centre (SARC) is a model of service established to address the forensic and therapeutic needs arising following sexual assault. As yet, only a minority of urban areas in England are served by one, the rest of the United Kingdom (UK) being served by police victim examination suites. OBJECTIVE: To examine variations in service offered to complainants of sexual assault within the United Kingdom in 2005. METHODS: A purpose designed questionnaire sent to all areas of the United Kingdom. Data were received and analysed from 12 of 13 SARCs and 54 of 58 non SARC services. RESULTS: Very wide disparities in service, most marked between SARC and non SARC services. SARCs see a proportion of complainants from non-police sources. The non SARC services do not usually offer a forensic examination without police involvement, and a significant minority have so few doctors that they cannot provide a 24h rota for examinations. Inadequate numbers of forensic physicians are available for child examinations, and a robust service for 'acute child sexual assault' is virtually absent. Photodocumentation with appropriate safe storage is available in all SARCs, while 45% of non SARCs have no facility for photodocumentation. DNA contamination issues were perceived to be significant in many of non SARC services. Most non SARC services for adults do not provide baseline screening for sexually transmitted infection (STI) or offer prophylaxis against STIs. Follow up is by referral to local clinics which complainants may have to arrange themselves. Funded counselling is rare in the non SARCs with the exception of Yorkshire. CONCLUSION: In the non SARC services, lack of co-operative working with local health services, lack of equipment, and lack of 'in house' medical follow up arrangements is the norm. Many areas rely on the good will of a small number of doctors to provide a service without a rota.

Ambulatory Care Facilities↗

PubMed2006

Screening anion-exchange chromatographic matrices for isolation of onco-retroviral vectors.

The adsorption kinetics of retroviral vectors to several chromatographic media, DEAE FF, Streamlinetrade mark Q XL and CHTtrade mark Ceramic Hydroxyapatite, in batch mode was investigated. The effects of buffer type, pH and operational temperature were studied. A mathematical model describing viral adsorption kinetics that considers viral degradation in solution was developed. The best results, either in terms of speed and extent of adsorbed infectious particles, were obtained with DEAE FF and Streamlinetrade mark Q XL. Fixed-bed chromatography was further investigated using DEAE FF, Q XL and Q FF, for validation of the batch adsorption process. Fixed-bed DEAE FF and Q XL proved to be good candidates for purification of MoMLV derived vectors due to resulting high yields, 53+/-13% and 51+/-7%, respectively, while removing more than 99% of protein and 90% of the DNA contaminants.

Adsorption↗

PubMed2003

Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays.

Genome-wide expression studies of human blood samples in the context of epidemiologic surveillance are confronted by numerous challenges-one of the foremost being the capability to produce reliable detection of transcript levels. This led us to consider the Paxgene Blood RNA System, which consists of a stabilizing additive in an evacuated blood collection tube (PAX tube) and a sample processing kit (PAX kit). The PAX tube contains a solution that inhibits RNA degradation and gene induction as blood is drawn into the tube. The stability of RNA in PAX tubes under conditions for practical clinical applications has been determined by RT-PCR, but has not been assessed at the transcriptome level on Affymetrix microarrays. Here, we report a quality assured and controlled protocol that is capable of producing reliable gene expression profiles using the GeneChip system with RNA isolated from PAX tubes. Using this protocol, we compared quality metrics and gene-expression profiles of RNA, extracted from blood in PAX tubes that sat at room temperature for 2 h, with that of blood in PAX tubes incubated at room temperature for 9 h followed by storage at -20 degrees C for 6 days. Of numerous metrics, differences between the two handling methods were detected for the level of DNA contamination, RNA yield, and double stranded cDNA yield. Analysis of variance of gene-expression revealed small but significant differences between the handling methods. These results contribute to the determination of protocols for clinical studies and progress us towards the goal of using the transcriptome in diagnosis and surveillance.

Blood Specimen Collection↗

PubMed2004

Real-time RT-PCR: considerations for efficient and sensitive assay design.

Real-time RT-PCR has been recognised as an accurate and sensitive method of quantifying mRNA transcripts. Absence of post amplification procedures allows rapid analysis with a greater sample throughput, yet with less risk of amplicon carry-over as reaction tubes are not opened. In order to maximise sensitivity, careful reaction design and optimisation is essential. Several aspects of assay design for real-time RT-PCR are discussed in this paper. We demonstrate the effect of amplicon secondary structure on reaction efficiency and its importance for primer design. Taq-man probes with a deoxyguanosine base at the 5' end fluoresce weakly when labelled with FAM, although weak fluorescence is not a problem when probes are labelled with Texas Red. DNA contamination of RNA samples purified using silica membrane columns is a significant problem but DNase digestion can be used to reduce this, particularly in-solution. MMLV and AMV enzyme systems using a variety of RT priming methods are compared and the problem of primer-dimer formation associated with RT enzymes is described.

Animals↗