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At least 289 records · Page 16Linked to original sources
PubMed1988

Production of monoclonal antibody against electrophoretically purified RNA polymerase II subunits using in vitro immunization.

A procedure has been developed for the production of MAb against weakly immunogenic subunits of a multisubunit enzyme. This procedure takes into account the problems of insufficient antigen, single epitope immunodominance and the difficulty of mapping non-sequential determinants. Small quantities of mammalian RNA polymerase II subunits were purified by SDS-polyacrylamide gel electrophoresis and were used to immunize splenocytes in vitro. After fusion with plasmacytoma cells, the hybrid cells were cloned and screened by ELISA utilizing native RNA polymerase II. This procedure is biased towards the production of MAb directed against sequential epitopes accessible on the native enzyme. Monoclonal antibodies, produced by in vitro immunization, were shown to be useful in protein transblot analyses, to inhibit enzyme activity in vitro and to have binding affinities comparable with MAbs produced by in vivo immunization.

Animals↗
PubMed2002

Blood-sample processing for the study of age-dependent gene expression in peripheral blood mononuclear cells.

Although most new biogerontological studies seeking to identify longevity candidate genes and factors involved in successful human aging are population based, and likely to involve the collection of blood from extremely old individuals, to our knowledge no unified protocols have yet been published to describe a methodology permitting the simultaneous generation of different kinds of biological specimens derived from a single source of a very small volume of peripheral blood. Here we describe a method permitting the simultaneous generation of plasma, RNA, DNA, protein, fixed lymphocytes, and frozen blood aliquots from a single 10- to 30-ml blood sample obtained from donors of any age (10-102 years old), and we show that the quality and quantity of DNA, RNA, protein, and fixed lymphocytes obtained do not vary significantly with age. As is frequently observed, the older individuals have higher plasma proportions.

Adolescent↗
PubMed1999

Long-term administration of cyclosporin A to HCV-antibody-positive patients with dermatologic diseases.

BACKGROUND: Cyclosporine A (CYA) is an immunosuppressive agent which is being used in the treatment of an increasingly wide range of dermatologic diseases, but its use has been avoided in carriers of hepatitis C virus (HCV). METHODS: We administered small doses of CYA (maximum, 3 mg/kg/day) for a long time to treat dermatologic diseases in one HCV-antibody-positive patient with no HCV-RNA in the blood, one patient with a small amount of HCV-RNA in the blood, and two patients with large amounts of HCV-RNA in the blood. RESULTS: Skin lesions improved in all patients, but recurred upon complete or partial withdrawal of CYA. In the absence of HCV-RNA in the blood, or when only a small quantity of HCV-RNA was present in the blood, HCV-RNA load showed no apparent change. In one patient with a large blood HCV-RNA load, CYA dosage reduction was followed by increases in alanine aminotransferase (ALT) levels and decreases in blood HCV-RNA. Aggravation of hepatitis due to immunologic reactivation was suspected in this patient. CONCLUSIONS: The reduction of CYA dosage is a key element in the use of this agent for cutaneous diseases.

Aged↗
PubMed1978

Measurement of antigen-specific IgG autoantibody production in vitro.

This report describes the development of a direct, highly sensitive and reproducible microassay for measuring picogram amounts of IgG antibody produced in spleen cultures of NZB/NZW female mice and specific for a well defined nucleic acid antigen (native ssRNA). The spontaneously synthesized antibodies were extensively purified from the culture supernatants. The isolated IgG anti-RNA antibodies had a high affinity, limited heterogeneity, and were specific for RNA as compared with DNA. Spleen cell cultures produced quantities of anti-RNA antibodies sufficient to account for a large proportion of the circulating anti-RNA antibodies in the whole animal. However, our results provide no evidence for the recently published suggestion (Sawada) et al., 1977. J. Immunol. 119:355) that autoreactive lymphocytes are released from normal immunoregulatory control during in vitro culture conditions.

Animals↗
PubMed1979

Selective staining by 4', 6-diamidine-2-phenylindole of nanogram quantities of DNA in the presence of RNA on gels.

4', 6-Diamidine-2-phenylindole.2HCl (DAPI) forms fluorescent complexes with double-stranded (ds) DNA but not with ds RNA as shown by fluorescence titration. The widely used dye ethidium bromide (EB) forms fluorescent complexes with both types of nucleic acids. Also, in contrast to EB, DAPI forms much weaker fluorescent complexes with single-stranded DNA than with ds DNA. These observations were utilized to develop staining procedures for the selective visualization of ds DNA on gels. The use of DAPI in addition to EB for staining makes possible the localization of ds DNA and other species of nucleic acids on a single gel.

DNA↗
PubMed1988

Cell lines containing varicella-zoster virus open reading frame 62 and expressing the "IE" 175 protein complement ICP4 mutants of herpes simplex virus type 1.

Vero cells were cotransfected with pSV2neo and a recombinant plasmid containing the varicella-zoster virus (VZV) open reading frame 62 (ORF62). Three neomycin-resistant cell lines were isolated and shown to complement two different ICP4 mutants (tsB21 and d120) of herpes simplex virus (HSV) type 1 (HSV-1). VZV-specific RNA could not be detected in these cell lines, but following infection with tsB21, a 4.3-kilobase VZV transcript was detected. This RNA increased in quantity when cells were infected in the presence of cycloheximide. A VZV-specific protein of 175 kilodaltons was detected in extracts of all three cell lines following infection with wild-type HSV-1 but not in uninfected cells. That VZV RNA and protein were detected only in HSV-1 infected cells suggests that a component of the HSV virion activates the expression of VZV ORF62. The increase in RNA expression seen in the presence of cycloheximide indicates that the protein encoded by VZV ORF62, "IE"175, may be autoregulatory. These data provide further evidence that VZV "IE"175 is the functional analog of the HSV ICP4.

Animals↗
PubMed1976

Assessment of the quantity of biologically available phosphorus in yeast RNA and single-cell protein.

Two assays were conducted with chicks fed a phosphorus-deficient crystalline amino acid diet from day 8 to 17 posthatching to evaluate the phosphorus availability of purified torula yeast RNA and single-cell protein (Torula yeast). Tibia bone ash of the chicks was the criterion used in both assays, and regression analysis was used to evaluate bone ash as a function of phosphorus intake. A standard curve was established by feeding graded levels of KH2PO4 in each assay. Weight gain, tibia weight and tibia ash all responded to phosphorus supplementation whether provided by KH2PO4, RNA or single-cell protein. Weight of tibia ash provided the best linear fit when evaluated as a function of phosphorus intake. Availability in both assays estimated using the slope-ratio technique wherein milligrams of available phosphorus per gram of RNA or single-cell protein was calculated. In assay 1 the available phosphorus in RNA was found to be 93.2 mg/. In assay 2 the available phorphorus in single-cell protein was found to be 14.1 mg./g.

Animal Feed↗
PubMed1979

In vitro synthesis of RNA by Xenopus spermatogenic cells I. Evidence for polyadenylated and non-polyadenylated RNA synthesis in different cell populations.

Premeiotic and postmeiotic (haploid) gene expression during spermatogenesis in the anuran, Xenopus laevis, was studied by analyzing the accumulation of radioactively labelled cytoplasmic polyadenylated [poly (A +)] and non-polyadenylated [poly (A -)] RNAs. Dissociated spermatogenic cells were labelled and maintained in an in vitro system capable of supporting cell differentiation. Labelled cells were separated by density gradient centrifugation into subpopulations enriched for individual spermatogenic stages. RNA was extracted and purified from each cell fraction, and separated into poly (A +) and poly (A -) species. Comparison of poly (A +) to non-poly (A) radioactivity in cells labelled with tritiated uridine or adenosine demonstrated that (1) all cell fractions produced significant quantities of polyadenylated RNA relative to total RNA synthesis; and (2) that a cell fraction enriched for pachytene spermatocyte RNA contained up to 15% of total cytoplasmic and 35% of total polysomal RNA labelled as poly (A +) containing species. RNA was also characterized by sucrose density gradient centrifugation and polyacrylamide gel electrophoresis. All cell types showed typical poly (A -) peaks of 4S, 18S and 28S, corresponding to tRNA (4S) and rRNAs (18, 28S) respectively. Spermatids and spermatozoa had additional absorbance peaks at 13 and 21S which cosedimented with Xenopus oocyte mitochondrial rRNA. Patterns of incorporation of uridine and adenosine into poly (A +) RNA in all germ cell fractions tested were complex. In all cases, major areas of radioactivity were found in a broad band sedimenting between 6-17S. Spermatid fractions showed a prominent peak of incorporation at 6-8S, while pachytene cells also showed heavier poly (A +) peaks in the 17-25S region. A non-polyadenylated RNA species sedimenting at 6-8S with a relatively rapid rate of turnover was also observed in spermatids. From these results it is concluded that synthesis of transfer, ribosomal, and putative messenger RNA species continues in spermatogenic cells throughout all but the very last stages of spermatogenesis in Xenopus.

Animals↗
PubMedDate not supplied

Branched DNA for quantification of viral load.

This is a summary of a presentation made at the 13th International Convocation on Immunology. Nucleic acids in patient samples can be quantified directly using a solid phase nucleic acid hybridization assay based on branched DNA (bDNA) signal amplification technology. For example, HIV RNA is detected in a plasma sample by hybridization of multiple specific synthetic oligonucleotides to the target, 10 of which capture the target onto the surface of a microwell plate and 39 of which mediate hybridization of branched DNA molecules to the pol region of each HIV RNA molecule. Alkaline phosphatase-labeled probes bind to each arm of the branched DNA molecules. Detection is achieved by incubating the complex with a chemiluminescent substrate and measuring the light emission. The signal is directly proportional to the level of target nucleic acid, and the quantity of HIV RNA in a sample is determined by comparison with a 4-point standard curve. In order to ensure that different subtypes of HIV-1 were detected and quantified equally, in vitro RNA transcripts of the pol region of HIV subtypes A-F were purified and quantified by OD 260, phosphate analysis, and hyperchromicity. These characterized transcripts were then quantified using the bDNA assay. Comparisons were made using a ratio of signal per attomole for each transcript. Genetic subtypes A-F quantified within a factor of 1.5, indicating that the bDNA assay can be used to measure viral load in clinical samples regardless of genotype. Accuracy is important because several studies indicate that there may be a threshold level of virus which predicts progression of HIV disease. Detection of change in viral load is important in determining the efficacy of therapy. The bDNA assay for HCV RNA can be used to determine level of virus in HCV-infected individuals and assist in establishing prognosis prior to initiation of alpha-interferon therapy. Patients with lower levels of virus are more likely to have a sustained response to therapy. Patients who respond to treatment typically have a rapid decline in virus load within one to four weeks of the start of therapy. Many patients relapse when therapy is discontinued as evidenced by a rise in virus load to near pre-treatment levels. Sustained response is most often seen with patients who have lower pre-treatment levels of RNA.

DNA, Viral↗
PubMed2005

Accumulation of miR-155 and BIC RNA in human B cell lymphomas.

We show that the microRNA miR-155 can be processed from sequences present in BIC RNA, a spliced and polyadenylated but non-protein-coding RNA that accumulates in lymphoma cells. The precursor of miR-155 is likely a transient spliced or unspliced nuclear BIC transcript rather than accumulated BIC RNA, which is primarily cytoplasmic. By using a sensitive and quantitative assay, we find that clinical isolates of several types of B cell lymphomas, including diffuse large B cell lymphoma (DLBCL), have 10- to 30-fold higher copy numbers of miR-155 than do normal circulating B cells. Similarly, the quantities of BIC RNA are elevated in lymphoma cells, but ratios of the amounts of the two RNAs are not constant, suggesting that the level of miR-155 is controlled by transcription and processing. Significantly higher levels of miR-155 are present in DLBCLs with an activated B cell phenotype than with the germinal center phenotype. Because patients with activated B cell-type DLBCL have a poorer clinical prognosis, quantification of this microRNA may be diagnostically useful.

Cell Line, Tumor↗
PubMed2004

Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression.

BACKGROUND: RNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array) procedure for rapid and parallel gene expression analysis using fluorescently labeled probes. RESULTS: RNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples. CONCLUSION: RNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples.

Animals↗
PubMed2004

Universal Reference RNA as a standard for microarray experiments.

BACKGROUND: Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR), developed with the goal of providing hybridization signal at each microarray probe location (spot). Measuring signal at each spot as the ratio of experimental RNA to reference RNA targets, rather than relying on absolute signal intensity, decreases variability by normalizing signal output in any two-color hybridization experiment. RESULTS: Human, mouse and rat URR (UHRR, UMRR and URRR, respectively) were prepared from pools of RNA derived from individual cell lines representing different tissues. A variety of microarrays were used to determine percentage of spots hybridizing with URR and producing signal above a user defined threshold (microarray coverage). Microarray coverage was consistently greater than 80% for all arrays tested. We confirmed that individual cell lines contribute their own unique set of genes to URR, arguing for a pool of RNA from several cell lines as a better configuration for URR as opposed to a single cell line source for URR. Microarray coverage comparing two separately prepared batches each of UHRR, UMRR and URRR were highly correlated (Pearson's correlation coefficients of 0.97). CONCLUSION: Results of this study demonstrate that large quantities of pooled RNA from individual cell lines are reproducibly prepared and possess diverse gene representation. This type of reference provides a standard for reducing variation in microarray experiments and allows more reliable comparison of gene expression data within and between experiments and laboratories.

Animals↗
PubMed2001

Comparison of techniques for HIV-1 RNA detection and quantitation in cervicovaginal secretions.

PRINCIPLES: HIV-1 in female genital secretions has been measured using swabs, Sno Strips (Akorn, Inc., Buffalo Grove, IL), and cervicovaginal lavage (CVL), but little is known regarding the comparability of these collection techniques. METHODS: We compared HIV-1 RNA detection and quantity in specimens obtained from HIV-1-seropositive women in Kenya using three sample collection techniques and three storage techniques and evaluated reproducibility in samples collected 5 days apart. Specimens were stored in no medium, freezing medium, or TRI Reagent (Molecular Research Center, Cincinnati, OH) for 2 to 15 months. RESULTS: HIV-1 RNA assays were conducted on 640 specimens from 20 antiretroviral naive women. Storage in TRI Reagent significantly enhanced detection of genital HIV-1 and yielded significantly higher mean log10 RNA levels than specimens collected in either no or freezing medium. The prevalence of HIV-1 RNA detection in TRI Reagent ranged from 50% to 80% depending on collection method and was highest in cervical swabs. Mean log10 HIV-1 RNA levels were 3.1 log10 copies/cervical swab, 2.6 log10 copies/cervical Sno Strip, 2.5 log10 copies/vaginal swab, 2.4 log10 copies/vaginal Sno Strip, 2.9 log10 copies/ml for cervicovaginal lavage (CVL) cell pellet, and 2.1 log10 copies/ml in CVL supernatant. Comparing specimens from days 1 and 6, there was significant concordance of HIV-1 RNA detection and correlation of HIV-1 RNA levels for cervical swabs, vaginal swabs, vaginal Sno Strips, and CVL cell pellets (kappa, 0.5-0.9; r, 0.5-0.9), but not for cervical Sno Strips or CVL supernatants. CONCLUSIONS: Cervical or vaginal swab, vaginal Sno Strip, and CVL collection led to reproducible measurement of genital HIV-1 RNA, despite storage for several months and international transport. Collection using swabs was simpler than Sno Strips or cervicovaginal lavage, and yielded the highest prevalence of HIV-1 RNA detection and reproducibility.

Cervix Uteri↗
PubMed2003

Short report: quantification of leishmaniavirus RNA in clinical samples and its possible role in pathogenesis.

Leishmaniavirus (LRV) is a double-stranded RNA virus that infects the protozoa Leishmania and has been identified in numerous strains of Leishmania braziliensis and L. braziliensis guyanensis. In general, the species of Leishmania dictates disease manifestation except in the case of L. braziliensis, which is capable of causing either cutaneous or mucocutaneous leishmaniasis. We wanted to determine 1) the quantity of LRV RNA present in a clinical sample and 2) if infection with LRV was associated with a specific disease manifestation. A real-time reverse transcriptase-polymerase chain reaction assay was used to assay clinical samples for the presence of LRV. Of 47 samples tested, 12 positive samples were obtained from patients with cutaneous lesions, lesions in the process of scarring, and cutaneous scars. This is the first study to examine the prevalence of LRV RNA within a small cohort from Brazil.

Adolescent↗
PubMed1996

Quantification of residual disease in chronic myelogenous leukemia patients on interferon-alpha therapy by competitive polymerase chain reaction.

Interferon-alpha (IFN-alpha) induces cytogenetic responses of variable degree in patients with chronic myelogenous leukemia (CML). We sought to establish the relationship between BCR-ABL transcript numbers measured by competitive 2-step reverse transcription polymerase chain reaction (RT-PCR) and cytogenetic status in CML patients treated with IFN-alpha. A total of 250 peripheral blood and 55 bone marrow samples with 127 Philadelphia chromosome positive (Ph+) and 6 Ph-/BCR-ABL+ CML patients were investigated. Twenty-one patients were studied at diagnosis with IFN-alpha, 24 had a complete cytogenetic response, 21 a partial response, 12 a minor response, 26 no response, and 23 were unknown. Using nested RT-PCR, all 305 samples were positive for BCR-ABL transcripts. To standardize results for variability in RNA and cDNA quantity and quality, we quantified total ABL transcripts in each sample as internal control. The validity of ABL as internal control was shown by comparison with glucose-6-phosphate dehydrogenase transcript levels in 145 samples. The median BCR-ABL transcript numbers (and BCR-ABL/ABL ratios expressed as percentages) were 400/micrograms RNA (O.04%) in complete responders, 20,500/micrograms RNA (7.1%) in partial responders, 170,000/micrograms RNA (21.0%) in minor responders, and 430,000/micrograms RNA (58.7%) in nonresponders (P < .001). The cytogenetic results correlated with the BCR-ABL transcript numbers (r = .82; P < .001) and BCR-ABL/ABL ratios (r = .84; P < .001). Grouping the ratios BCR-ABL/ABL as less than 2%, 2% to 14% and greater than 14% to compare with cytogenetic complete response, partial response, and minor/nonresponse, the concordance between the two methods was 82% (chi2 P< .0001). We conclude that quantitative PCR with internal controls is as sensitive and reliable method for monitoring patients on IFN-alpha and reduces the need for repeated marrow investigations.

Adolescent↗
PubMedDate not supplied

Global gene expression changes during neoadjuvant chemotherapy for human breast cancer.

PURPOSE: The purpose of this study was to analyze global gene expression changes in serial tumor core biopsy specimens taken during neoadjuvant chemotherapy for primary breast cancer. PATIENTS AND METHODS: Core biopsy specimens from tumors were obtained before treatment and 24 and/or 48 hours after treatment from 21 women who were beginning chemotherapy for breast cancer. RNA was extracted, and radiolabeled complementary DNA was synthesized. The complementary DNA probes were hybridized to high-density microarray membranes that contained more than 25,000 human sequence clones. Hierarchical cluster analysis was used to compare the degree of similarity between expression profiles. RESULTS: Twenty-five (45%) of the 56 available core specimens yielded sufficient quantity and quality RNA for microarray analysis. Microarray profiles were performed only on samples from patients with pretreatment and posttreatment specimens, resulting in serial data sets for five patients (14 specimens). The serial samples from individual patients clustered more closely than the samples taken from different patients. Analyses of the variance of individual gene expression showed that there were significantly fewer genes with fivefold differences in expression in an individual tumor at different times (average, 359 genes) versus pretreatment samples of different tumors (average, 732 genes). Patients with a good pathological response to treatment had gene patterns that clustered distinctly from those of poor responders. Significant transcriptional response occurred in all patients during therapy. Surprisingly, all patients had different genes change after chemotherapy, with no single gene having a significant expression change in all five patients. DISCUSSION: This is the first report to show global gene expression changes during chemotherapy in a human solid tumor. Comprehensive gene expression profiles of more than 25,000 genes can be obtained from core biopsy specimens. A remarkable diversity in transcriptional response was observed for individual cases. Further data are needed to determine whether gene profiling can predict response to chemotherapy.

Analysis of Variance↗