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At least 775 records · Page 43Linked to original sources
PubMed2004

Evaluation of automated sample preparation and quantitative PCR LCx assay for determination of human immunodeficiency virus type 1 RNA.

Efforts have been made to achieve full automation of molecular assays for quantitative detection of human immunodeficiency virus type 1 (HIV-1). In the present study, the Abbott LCx HIV RNA Quantitative assay was evaluated in conjunction with automated HIV-1 RNA extraction on the MagNA Pure LC instrument and compared to the conventional LCx HIV RNA Quantitative assay, which uses a manual nucleic acid extraction protocol. Accuracy, linearity, and interassay and intra-assay variations were determined. The performance of the assay in a routine clinical laboratory was tested with a total of 105 clinical specimens. When the accuracy of the LCx HIV RNA Quantitative assay with the automated sample preparation protocol was tested, all results were found to be within +/- 0.5 log unit of the expected results. Determination of linearity resulted in a quasilinear curve over 3.5 log units. For determination of interassay variation, coefficients of variation were found to be between 21 and 66% for the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and between 10 and 69% for the LCx HIV RNA Quantitative assay with manual sample preparation. For determination of intra-assay variation, coefficients of variation were found to be between 7 and 25% for the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and between 7 and 19% for the LCx HIV RNA Quantitative assay with manual sample preparation. When clinical samples were tested by the LCx HIV RNA Quantitative assay with the automated sample preparation protocol and the results were compared with those of the LCx HIV RNA Quantitative assay with manual sample preparation, 95% of all positive results were found to be within +/- 0.5 log unit. In conclusion, the assay with automated sample preparation proved to be suitable for use in the routine diagnostic laboratory and required significantly less hands-on time.

Automation↗
PubMed1990

A new method for extracting DNA or RNA for polymerase chain reaction.

The use of glass powder suspension for the extraction of RNA or DNA was studied to simplify the procedures of polymerase chain reaction (PCR). Using this procedure, proviral DNA of human T-lymphotropic virus type-1 (HTLV-1) in the blood of an asymptomatic virus carrier and viral RNA of human immunodeficiency virus (HIV) in the blood of an AIDS patient were easily detected by PCR employing glass powder. The use of glass powder is a simple and highly efficient procedure for the extraction of DNA or RNA, and can be applied for routine PCR.

Acquired Immunodeficiency Syndrome↗
PubMed2005

QIAamp MinElute virus kit effectively extracts viral nucleic acids from cerebrospinal fluids and nasopharyngeal swabs.

BACKGROUND: Nucleic acid preparation from a variety of clinical specimens requires efficient target recovery and amplification inhibitor removal and is critical for successful molecular diagnosis. The QIAamp MinElute Virus kit (Qiagen Inc., Valencia, CA) was compared to the two existing methods currently used in our laboratory, IsoQuick (Orca Research Inc., Bothell, WA) for DNA extraction and RNAzol B (Leedo Laboratories Inc., Houston, TX) for RNA extraction, of viral nucleic acids. STUDY DESIGN: A total of 150 clinical specimens, including cerebrospinal fluid (CSF) and nasopharyngeal swabs (NPS), were used to determine the extraction efficiency of the MinElute compared to the other two methods. Nucleic acid recovery, hands-on time, turn-around-time and cost were compared across all kits. RESULTS: There was complete concordance between the MinElute and IsoQuick/RNAzol kits when herpes simplex virus (HSV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), influenza A virus or enteroviruses were detected using a colorimetric microtiter plate PCR system. The kits were equivalent in their ability to detect either DNA or RNA with superior ability to recover a high quality and quantity of RNA. With the potential to process larger specimen volumes, the MinElute kit can significantly shorten processing time from 2h to 50-55min. CONCLUSIONS: Although relatively high test kit costs were noted, the MinElute kit provides another rapid and user-friendly specimen processing tool in the diagnostic molecular microbiology laboratory.

Cerebrospinal Fluid↗
PubMed1990

Protooncogene expression in normal and psoriatic skin.

The expression of the c-myc, c-fos, c-jun, c-erbB, and c-Ha-ras protooncogenes was compared by Northern blot analysis of total RNA extracted from keratome biopsies of normal skin and psoriatic plaques. Isolation of intact RNA from frozen tissue required careful attention to technique during the early stages of extraction. Densitometric analysis revealed 1.5- to 2.5-fold elevations of c-myc transcript levels in lesional psoriatic relative to normal epidermis. Similar increases in cyclophilin and lipocortin II transcripts were also observed and may reflect characteristic differences in RNA preparations from normal and psoriatic epidermis. C-myc, c-jun, c-erbB, c-fos, and c-Ha-ras transcript levels were not significantly increased in lesional psoriatic epidermis when protooncogene mRNA levels were normalized to those of the cyclophilin or lipocortin genes. In contrast, transforming growth factor-alpha (TGF-alpha) transcripts were significantly increased (10- to 20-fold) with or without prior normalization. C-myc, c-fos, and c-jun transcripts were significantly induced over in vivo levels 2-4 h after organ culture of normal or psoriatic keratome biopsies, demonstrating that these genes can be highly expressed in the context of tissue injury. Our results suggest that overexpression of these protooncogenes per se is not central to the pathogenesis of psoriatic epidermal hyperplasia.

Epidermis↗
PubMed2001

Expression of glyoxalase I and II in normal and breast cancer tissues.

The present work aimed to study the activities of glyoxalase system enzymes, glyoxalase I (G I) and glyoxalase II (G II), as well as the expression of their genes in human breast carcinoma. Samples of tumoral tissue and normal counterparts were drawn from several patients during surgery. They served either for preparing extracts to be used in enzyme activity evaluations or for RNA extraction and subsequent northern blot analysis. A far higher activity level of G I and G II occurs in the tumor compared with pair-matched normal tissue, as shown by both spectrophotometrical assay and electrophoretic pattern. Such increased activities of G I and G II likely result from an enhanced enzyme synthesis as a consequence of increased expression of the respective genes in the tumoral tissue, as evidenced by northern blot. The present findings confirm a key-role of glyoxalase system to detoxify cytotoxic methylglyoxal and modulate S-D-lactoylglutathione levels in tumor cells. Moreover, they suggest a possible employment of GI inhibitors as anti-cancer drugs.

Adult↗
PubMed1996

The mode of action of hirsutellin A on eukaryotic cells.

A 16-kDa protein toxin was purified from Hirsutella thompsonii var thompsonii and named hirsutellin A (HtA). At 0.5 and 5.0 microM concentrations, HtA caused detectable cytopathic effects on Spodoptera frugiperda cells (Sf-9) within 2-4 hr and completely inhibited Sf-9 cell growth at 4 days posttreatment. Electron microscope data showed that the HtA treated Sf-9 cells became hypotrophied and internal organelles and cell membranes were disrupted. At the same concentration, HtA effectively inhibited Brome mosaic virus protein synthesis of both rabbit reticulocyte and wheat germ in vitro translation system. The ribosomal RNA extracted from HtA treated Sf-9 cells produced a smaller RNA (approximately 528 bases) than untreated Sf-9 cells. In summary, HtA is the first mycotoxin of a invertebrate mycopathogen determined to possess ribosomal inhibiting activity and appears to possess some specificity to invertebrate cells.

Animals↗
PubMed1976

RNA, RIBOSOMAL/*BIOSYNon of ribosomal 5S RNA genes in Chironomus thummi by in situ hybridization of iodinated 5S RNA.

5 S RNA of Chironomus thummi larvae was purified from total phenol extracted RNA by gel filtration and labelled to about 10(7) dpm/mug with carrier-free iodine-125. After hybridization in situ of 125I-5 S RNA and autoradiography only region B3c-e (containing two "normal" and two very faint bands) of chromosome II of salivary gland cells was highly labelled. In chromosomes of an animal showing pairing discontinuities a clearly "heterozygous" labelling of the 5 S RNA region was found. Region B3c-e shows no clearcut morphological signs of puffing or autoradiographically detectable 3H-uridine incorportion in spite of a continuous synthesis of 5 S RNA in salivary gland cells.

Animals↗
PubMed1991

Detection of transmissible gastroenteritis virus using cDNA probes.

Five cDNA probes prepared from molecular clones representing genomic RNA sequences of the virulent Miller strain of transmissible gastroenteritis virus (TGEV) were used in a dot blot hybridization assay to detect TGEV in cell culture and fecal specimens. Two clones (pA2 and pB4) represent nucleotide base pairs at the 3' terminus of the Miller TGEV genome. The other three clones represent various portions of the 5' end of the E2 gene, which codes for the major surface glycoprotein of TGEV. Each of the 32P-labeled cDNA probes hybridized to the virulent Miller, attenuated Purdue and four field strains of TGEV. The probes detected 200 to 2000 pg of TGEV RNA extracted from density gradient purified virions and did not hybridize RNA from mock-infected cell cultures, porcine rotavirus or antigenically unrelated coronaviruses. The pB4 and Hpa-1600 probes detected TGEV RNA sequences in 79 and 88%, respectively of 34 field samples identified as TGEV positive by the immunofluorescence assay and electron microscopy (EM). The pD24 clone, which is able to differentiate TGEV from the antigenically related coronaviruses, also compared favorably with conventional methods of EM and immunofluoresence for the detection of TGEV in fecal specimens.

Animals↗
PubMed1988

Construction of a recombinant vaccinia virus expressing the Lassa virus glycoprotein gene and protection of guinea pigs from a lethal Lassa virus infection.

A cloned cDNA (1.65 kb) containing the complete glycoprotein gene of the Josiah strain of Lassa virus was inserted into the thymidine kinase (TK) gene of the New York Board of Health (WYETH) strain of vaccinia virus. The Lassa virus glycoprotein precursor, GPC, and the posttranslational cleavage products, G1 and G2, were shown by Western blot analysis to be properly expressed in cells infected with the recombinant virus. Northern blot hybridization of total cytoplasmic RNA extracted from recombinant virus infected cells demonstrated the presence of RNA transcripts of appropriate size considering the site of transcription initiation from the vaccinia P7.5 promoter, the size of the Lassa glycoprotein gene, and the presumed location of the transcription terminator in the vaccinia thymidine kinase gene. All guinea pigs vaccinated with the recombinant virus survived a lethal challenge infection with Lassa virus, whereas 80% of control animals died. The vaccinated guinea pigs did, however, develop transient, low-grade, fevers and detectable viremias following infection with Lassa virus, indicating that protection was not complete.

Animals↗
PubMed1995

Plasticity in the synthesis and storage of substance P and calcitonin gene-related peptide in primary afferent neurons during peripheral inflammation.

Several indices of peptidergic, primary afferent neural transmission in rat at the level of the lumbar spinal cord exhibited differential changes over time in response to adjuvant-induced inflammation of the hindpaw. The indices were measurements of the production of messenger RNA encoding the precursors for substance P and calcitonin gene-related peptide in dorsal root ganglia, the storage of substance P and calcitonin gene-related peptide in the dorsal spinal cord and the release of the peptides evoked by application of capsaicin to the dorsal spinal cord. A 47% decrease in the content of immunoreactive substance P in the dorsal half of the lumbar spinal cord, as determined by radioimmunoassay, was measured at 6 h following the injection of complete Freund's adjuvant into the hindpaw. Decreased content of immunoreactive SP persisted for four days, but was no longer present at eight days after the adjuvant injection. The content of immunoreactive calcitonin gene-related peptide in the dorsal spinal cord was decreased by 29% at one day following the injection of adjuvant into the rat hindpaw and 43% at two days; the content then increased to a level greater than that of control animals at eight days. The amount of messenger RNA encoding preprotachykinin and prepro-calcitonin gene-related peptide in L4-L6 dorsal root ganglia was determined from northern blot analysis of the total messenger RNA extracted from the dorsal root ganglia. Each species of messenger RNA had increased compared to the control animals at two days following the injection of adjuvant into the rat hindpaws and remained elevated after eight days. Thus, an increase in the messenger RNAs encoding substance P and calcitonin gene-related peptide in the dorsal root ganglia preceeded the recovery of the content of the peptides in the spinal cord. Morphometric studies of calcitonin gene-related peptide-immunoreactive perikarya in the L4 dorsal root ganglia indicated that the increase in messenger RNA occurred in neurons of the size that normally express calcitonin gene-related protein. Radioimmunoassay of the superfusate of the dorsal half of the lumbar spinal cord was used to measure the release of immunoreactive substance P and immunoreactive calcitonin gene-related protein in vitro. Although the basal release of immunoreactive substance P and immunoreactive calcitonin-gene related protein from the dorsal spinal cord was constant throughout the time points examined, changes occurred in the release of peptide evoked by 10 microM capsaicin. The capsaicin-evoked release of immunoreactive substance P was decreased at 6 h and eight days post-injection of adjuvant.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Gene expression profiling of adrenal cortical tumors by cDNA macroarray analysis. Results of a preliminary study.

Adrenocortical carcinoma (ACC) are highly malignant tumors with poor prognosis. To verify if it is possible to assess their differential gene expression by a cDNA macroarray analysis using RNA extracted from paraffin sections, we analyzed two different cohorts of adrenal cortical adenoma (ACA) and ACC. Paraffin sections of seven ACC and seven ACA were analyzed. Transcriptional profiles were generated by commercially available c-DNA arrays testing 82 genes. Hybridization signals were quantified by densitometry and the intensity signal was compared for each gene between ACA and ACC cohorts. RNA was successfully extracted in only four out of 14 cases. Four genes displayed a significantly different expression (ACC/ACA ratio>1.5 or<0.6). Heat shock protein 60 (HSP-60) (ratio>2), Ciclin D1 and topoisomerase I (ratio>1.5) were overexpressed in the ACC cohort, while jun proto-oncogene was down-regulated. cDNA macroarray analysis from paraffin sections of adrenal tumors is feasible, despite with a low success rate. The different expression of HSP-60, Ciclin D1, jun proto-oncogene and topoisomerase I indicates that these genes may play a role in ACC pathogenesis and could represent potential diagnostic/prognostic/therapeutic target markers. Larger series of patients are necessary to confirm the biologic, diagnostic, prognostic and therapeutic implications of these findings.

Adrenal Cortex Neoplasms↗
PubMed2002

Sensitivity and reproducibility of HCV quantitation in chimpanzee sera using TaqMan real-time PCR assay.

The availability of molecular protocols for the detection and quantitation of very low numbers of hepatitis C virus (HCV) particles in biological samples is an issue of interest in both clinical and analytical fields of HCV research. A sensitive and reproducible assay is described for HCV RNA quantitation using the TaqMan PCR fluorogenic real-time detection system to establish the levels of HCV RNA in chimpanzee plasma. Our TaqMan PCR protocol and synthetic full length HCV RNA template show that the threshold of sensitivity for our TaqMan PCR is two copies per reaction. As few as 10 genome copies per reaction could be quantitated maintaining a linear range. The accuracy of the TaqMan PCR test was comparable to commercial bDNA and Amplicor tests. The RNA standards of the laboratory were tested in parallel with a World Health Organization (WHO) International Standard for HCV RNA obtaining ratios of 2.7+/-0.7 RNA copies per HCV international unit (IU). Our method using RNA extracted from chimpanzee samples had an estimated sensitivity of 200 RNA copies/ml of plasma (approximately eight copies/reaction or 74 WHO IU/ml). Serial plasma samples from HCV-infected chimpanzees were analyzed using this methodology to evaluate its applicability, and RNA profiles were observed consistent with the evolution of the pathology in each animal. The present study therefore illustrates the high reproducibility, sensitivity and reliability of our TaqMan methodology, providing a useful method for HCV research to consistently detect and quantify viral RNA throughout a range of concentrations.

Animals↗
PubMed1998

Modulation of the proteolytic balance plasminogen activator/plasminogen activator inhibitor by enhanced N-myc oncogene expression or application of genistein.

The aim of this study was to determine whether enhanced expression of N-myc in a neuroblastoma cell line affects the balance of plasminogen activator/plasminogen activator inhibitor (PA/PAI), a shift towards proteolysis having been observed in other malignant tissues. Two transfected neuroblastoma cell lines with (WAC2 cells) or without (SH-EP007 cells) enhanced expression of the N-myc oncogene were examined by zymography and RNA extraction to determine UPA and PAI enzyme activity and uPA RNA and PAI RNA expression, respectively. The effect of genistein, an inhibitor of tyrosine protein kinase, on uPA/PAI was also investigated. Both the uPA/PAI-1 ratio at mRNA level and the PA/PAI ratio at protein activity level were higher in the more malignant, WAC2 cell line. Genistein attenuated uPA activity and stimulated PAI activity in both cell lines, leading to a decrease in the PA/PAI ratio. This effect was more pronounced in the more malignant, WAC2 cell line.

Cell Division↗
PubMed1971

Transcription of the polyoma virus genome: synthesis and cleavage of giant late polyoma-specific RNA.

The size of virus-specific RNA synthesized in cultured mouse kidney cells infected with polyoma virus was estimated by electrophoresis and sedimentation analysis of RNA extracts from whole cells. Newly synthesized "late" polyoma-specific RNA appears as "giant" molecules of heterogeneous size, up to several times larger than a strand of polyoma DNA (1.5 x 10(6) daltons). Treatment with dimethylsulfoxide or urea showed that the large size of these molecules is not due to aggregation. Giant polyoma-specific RNA is strikingly similar in size distribution to "nuclear messenger-like" RNA ("heterogeneous nuclear" RNA) of the host cell. Subsequent to its synthesis, some of the giant polyoma-specific RNA appears to be cleaved to at least three smaller species.

Animals↗
PubMed1975

Rous sarcoma virus activates embryonic globin genes in chicken fibroblasts.

Complementary DNA (cDNA) specific for chick globin mRNA sequences fails to hybridize to total RNA extracted from chicken fibroblasts. After infection by Rous sarcoma virus, RNA complementary to globin cDNA is detectable in 100-500 copies per cell. Infection of fibroblasts with the transformation defective (td) deletion mutant of Rous sarcoma virus leads to normal virus production, but not to host cell transformation or accumulation of RNA sequences complementary to globin cDNA. Our evidence shows that the globin genes activated by Rous sarcoma virus are those specified by embryonic chick red cells; adult-specific globin sequences were not detected.

Age Factors↗
PubMed1975

Oncornavirus-like particles in human skin cancers.

A high-molecular-weight RNA encapsulated with an RNA-instructed DNA polymerase in particles possessing the density characteristic of the RNA tumor viruses has been detected in 13 out of 14 human malignant melanomas. The [3H]DNA synthesized by these particles in an endogenous reaction hybridizes to RNA extracted from the human melanoma particulate structures, but not to RNA from normal skin. Similar particles containing RNA and enzyme have been found in basal cell and squamous cell carcinomas of the skin. The RNA of the melanoma particles is easily distinguishable by hybridization from the RNAs found in the particles of the basal and squamous cell carcinomas.

Basal Cell Carcinoma↗
PubMed1994

Cellular retinoid binding proteins and nuclear retinoic acid receptors in endometrial epithelial cells.

Retinoic acid, one of the most potent of the naturally occurring retinoids (retinol and derivatives), is required in vivo for the maintenance of epithelial cell growth. This study describes the pattern of expression of nuclear retinoic acid receptors (RARs and RXRs), and cellular binding proteins for retinol and retinoic acid (CRBP I, CRABP I and II), in endometrial epithelial cells. The effects of retinoic acid on the expression of these receptors in endometrial epithelial cells have also been studied and compared with its effects in endometrial stromal cells. Messenger RNA for RAR-alpha, RAR-beta, RAR-gamma, RXR-alpha, CRBP I and CRABP II was detected by Northern blotting of total RNA extracted from cultured epithelial cells. In comparison with stromal cell RNA that was used as an internal standard, CRBP I appeared to be more abundant in epithelial cells, whereas CRABP II appeared to be more abundant in the stromal cells. This implies that the intracellular concentration of retinoic acid may be maintained at higher levels in epithelial cells compared to stromal cells. In addition, the response of the two cell types to retinoic acid differs: RAR-beta is induced in stromal cells treated with all-trans retinoic acid but not in epithelial cells. From these data we suggest that retinoid physiology differs between endometrial epithelial and stromal cells. Furthermore, by analogy with other studies, we propose that retinoic acid may be maintained at a higher intracellular concentration in endometrial epithelial cells to facilitate differentiation to a glandular phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1983

Analysis of histone H5 and globin mRNAs in chick-embryo development.

Transcripts from histone H5 and alpha and beta chicken globin genes were analysed in total RNA extracted from 2-6-day-old chick embryos. Denatured RNA, separated on gels, was transferred to either aminophenylthioether (APT) paper or to nitrocellulose and was detected with 32P-labelled recombinant DNA probes. Good resolution was achieved with methyl mercury hydroxide gels and aminophenylthioether transfer, allowing estimation of H5 and alpha and beta globin mRNA sizes. With glyoxal gels and nitrocellulose transfer, alpha and beta globin mRNAs were not resolved, but an increased sensitivity of detection of at least 10-fold was achieved. Levels of H5 and alpha globin mRNAs increased in parallel over 2-6 days. The appearance of definitive red cells in 6-day embryos was indicated by detection of adult beta globin transcripts at this stage.

Animals↗