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PubMed1995

[The 69th annual meeting president lecture. Application of molecular biology for rapid detection of mycobacteria].

The application of molecular biology techniques for the rapid detection of mycobacteria in clinical specimens was evaluated. The DNA probe method was found to be accurate and rapid for identification of mycobacteria, but its low sensitivity needs improving. The nested polymerase chain reaction (PCR) targeting Pab gene was specific and sensitive enough for rapid detection of Mycobacterium tuberculosis in clinical specimens. The overall sensitivity and specificity of this method were excellent, 97% and 92%, respectively. Additionally, a novel method for the rapid detection of mycobacteria other than M. tuberculosis was developed using a combined method of PCR and DNA probe. The gene encoding 16S ribosomal RNA in mycobacteria was first amplified by PCR, and then PCR products were identified using an acridinium-ester labeled DNA probe. This method was as useful as that of the PCR for M. tuberculosis described above. The DNA probe method was also applied for drug sensitivity testing, which needs three to four weeks by conventional methods. The quantity of ribosomal RNA in mycobacteria cultured with or without the addition of drugs was compared. Three days after the start of culture, the strains sensitive for isoniazid or rifampicin showed remarkable decreases of ribosomal RNA, compared with the drug-free samples, while the resistant strains showed no difference. Another method for determining the drug resistance of mycobacteria consists of detection of the gene which is related to the resistance. The deletion of catalase-peroxidase gene suggested a gene related to isoniazid resistance was observed in 15% of isoniazid-resistant strains.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Bacterial↗
PubMed2001

Transcript quantification based on chemical labeling of RNA associated with fluorescent detection.

A general method for RNA measurement, based on chemical labeling of RNA with digoxigenin (without retrotranscription), has been established. Labeled RNA is hybridized with nylon membranes containing spot blots of PCR-amplified gene fragments and the fluorescence detection is mediated via specific anti-digoxigenin antibody coupled to alkaline phosphatase. The method was optimized in order to be quantitative, and high precision (less than 24% error) was obtained, allowing analysis of relatively small changes in gene expression. When the quantity of cellular RNA used in this method is maintained constant and the amount of RNA in the cell determined, the true intracellular transcript concentrations can be determined, rather than simple abundance of a messenger in RNA population. This RNA quantification technique was extended to macroarrays blotted automatically and the validity of the method was tested by comparison with expression data obtained by Northern blotting.

Blotting, Northern↗
PubMed1966

Genetic relatedness among mycoplasmas as determined by nucleic acid homology.

Reich, Paul R. (National Institutes of Health, Bethesda, Md.), Norman L. Somerson, James A. Rose, and Sherman M. Weissman. Genetic relatedness among mycoplasmas as determined by nucleic acid homology. J. Bacteriol. 91:153-160. 1966.-A sensitive membrane filter method to detect nucleic acid homology was used to determine genetic relatedness among mycoplasma isolates. Deoxyribonucleic acid (DNA) was isolated from mycoplasmas and used as a primer for synthesis of tritium-labeled, complementary ribonucleic acid (RNA) by the enzyme RNA polymerase. DNA from each mycoplasma isolate tested was reacted separately with complementary RNA synthesized with homologous or heterologous DNA as primer. The quantity of DNA-RNA hybrids formed was assayed by the nitrocellulose membrane filter method. The amount of radioactivity bound to the membrane filter was used to measure the degree of homology between the nucleic acids. The three mycoplasma isolates from human oral cavities (DC 63, V2785, Botteicher) and the prototype strain PG21 placed in the Mycoplasma hominis type 1 group by gel diffusion and complement-fixation testing were investigated with this technique. Analysis of the data confirmed their immunological grouping with the M. hominis type 1 and their distinction from other human mycoplasmas. In contrast to the data from immunological studies, none of the four isolates tested appeared to be identical to any other. Preliminary experiments with DNA from four other mycoplasma isolates from tissue cultures inoculated with human material revealed them to be closely related, and possibly identical. The advantages of this nucleic acid homology technique for the study of relatedness among mycoplasmas are described.

DNA, Bacterial↗
PubMed2000

Iron enhances hepatitis C virus replication in cultured human hepatocytes.

BACKGROUND: Iron overload in the presence of increasing concentrations of iron is one of the indicators of poor response to interferon therapy in chronic hepatitis C. In order to analyze the effect of iron on hepatitis C virus (HCV) replication, we measured replication in an HCV-infected cell line. METHODS AND RESULTS: Cells from a non-neoplastic HCV-infected human hepatocyte line (PH5CH8) susceptible to HCV infection and supportive of HCV replication were used in this study. The replication of HCV RNA was measured by reverse transcription-nested polymerase chain reaction (RT-nested PCR). PH5CH8 cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. PH5CH8 cells were incubated with 0, 1, 10, 50, and 100 microM of FeSO4 at 37 degrees C with 5% CO2. Forty-eight hours after iron supplementation, the quantity of HCV RNA in the cells incubated in 50 and 100 microM of FeSO4 was approximately ten times that of the cells with no iron supplementation. Similar changes were observed beginning at 12 h from supplementation with FeSO4 and continued for at least 72 h after supplementation. MTT assay indicated that iron did not have cytotoxic effects on the PH5CH8 cells. CONCLUSION: Iron enhances HCV replication in a hepatocyte cell line. The results suggest that iron deposition in hepatocytes could facilitate HCV infection in the liver.

Cell Line, Transformed↗
PubMed1993

Changes in levels of mRNA encoding myosin heavy chain in porcine trachealis during ontogenesis.

We determined the steady-state level of mRNA for myosin heavy chain (MHC) from airway smooth muscle during maturation in domestic swine. Tissues were excised, and airway smooth muscle was dissected from three neonatal (NEO), three 2-wk-old swine (2ws), three 10-wk-old swine (10ws), and three adult swine. Total RNA was isolated, fractionated, and transferred to a nitrocellulose membrane (Northern blot). A single-stranded oligonucleotide of 63-nt was synthesized corresponding to the 3' coding region of the chicken gizzard MHC cDNA. This region appeared to be highly conserved (92% nucleotide sequence homology with the corresponding portion of rabbit uterine smooth muscle MHC cDNA). Northern blots, which were loaded with equivalent quantities of total RNA, were probed with gamma 32P-labeled synthetic oligonucleotide, and, under stringent washing conditions, the 5' end-labeled DNA was hybridized to a single band of the expected molecular weight. The mRNA for total myosin was quantified using autoradiograms of blots, and signal intensity was measured as integrated areas expressed as arbitrary densitometric units x mm (AU). The content of mRNA for MHC was substantially greater in NEO than in more mature animals; maximal area was 1.33 +/- 0.15 AU for NEO, 0.33 +/- 0.05 AU for 2ws, 0.30 +/- 0.04 AU for 10ws, and 0.34 +/- 0.08 AU for adult swine (P < 0.05, NEO versus 2ws, 10ws, and adult). Rehybridization of each blot with a 28S ribosomal RNA probe confirmed comparable total RNA loadings for all tissue samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1998

The gene for lysosomal protein CD63 is normal in patients with Hermansky-Pudlak syndrome.

Hermansky-Pudlak syndrome (HPS) is one of the few genetic disorders associated with severe pulmonary fibrosis. Fifty percent of affected patients die as a result of respiratory insufficiency. Fibrosis is thought to be caused by the accumulation of ceroid, an insoluble fluorescent lipoprotein, both extracellularly and in the lysosomes of alveolar macrophages. In addition to pulmonary fibrosis, HPS is characterized by oculocutaneous albinism and a reduction in the number of platelet dense bodies. CD63 is a protein that was described originally in platelet lysosomes. It localizes to the membranes of melanosomes and platelet dense bodies. CD63 is decreased dramatically in the lysosomes and dense bodies of patients with HPS. We theorized that CD63, a membrane protein common to lysosomes, melanosomes, and platelet dense bodies, may play a role in HPS. We sought to characterize the gene coding for this protein in HPS lymphoid cell lines. The coding region for CD63 was sequenced in control and HPS cell lines. Messenger RNA from HPS and normal cell lines was examined by Northern analysis. Genomic DNA from the same cell lines was examined by Southern analysis and polymerase chain reaction (PCR). CD63 protein in lymphoid cell lines and peripheral blood monocytes was compared by Western analysis. We found no mutations in the coding region of CD63 in an HPS cell line. We also found no diminution in the quantity of CD63 RNA by Northern analysis and no gross defects in the structural gene by PCR and Southern analysis, suggesting that the CD63 structural gene, promoter, and untranslated regions were normal. Western analysis showed that the 43-kDa protein was present in control and HPS lymphoid cell lines and peripheral blood monocytes in equivalent amounts. Although CD63 is an attractive candidate for the primary defect of HPS, the disease is probably not caused by a mutation in the CD63 gene.

Adult↗
PubMed1995

Comparative study of conventional and novel strategies for the detection of hepatitis C virus RNA in serum: amplicor, branched-DNA, NASBA and in-house PCR.

The aim of this study was to compare the sensitivity and specificity of conventional procedures (in-house one-stage polymerase chain reaction (PCR) and in-house nested PCR) and of new technologies (rTth DNA polymerase (Amplicor), branched-DNA, NASBA (nucleic acid amplification system)) for the qualitative detection of hepatitis C virus (HCV) RNA in serum of HCV-infected individuals. Serum samples from 37 anti-HCV-positive individuals (15 with a normal alanine aminotransferase (ALT) level, 22 with an elevated ALT level) and 10 anti-HCV-negative individuals as negative controls were studied. A second panel, including 9 diluted serum samples (from 1/10 to 1/100,000) was constituted to establish the differences of sensitivity of the 5 procedures with small quantities of HCV RNA in the serum. The anti-HCV-positive individuals with elevated ALT gave positive results with all 5 procedures. In patients with a normal ALT level, the assays with the highest sensitivity were Amplicor, NASBA and nested RT-PCR, followed by one-stage RT-PCR, then branched-DNA. One false-positive result was observed with Amplicor, and two with in-house nested PCR. On diluted samples, Amplicor, NASBA and nested PCR appeared more sensitive than one-stage PCR and branched-DNA. It is concluded that new procedures have satisfactory sensitivity and specificity and could advantageously replace the conventional PCR procedures for the routine qualitative detection of serum HCV RNA.

Base Sequence↗
PubMed2004

A rapid, reliable method to isolate high quality endothelial RNA from small spatially-defined locations.

Phenotypic heterogeneity of the endothelium has important implications for cell sourcing for cardiovascular tissue engineered devices and is fundamental to many cardiovascular diseases. A critical first step to identifying genetic regulators associated with particular endothelial phenotypes is reliable isolation of pure RNA from the cell subpopulations of interest. We present here a rapid method for the isolation of endothelial RNA from small spatially-defined locations, illustrated for two sides of the porcine aortic valve. Endothelial cells were retrieved from fresh tissue by freezing them to a glass substrate, from which they were lysed in guanidine thiocyanate buffer for RNA isolation. Valve endothelial cells isolated by this technique stained positively for CD31 and von Willebrand factor, consistent with an endothelial phenotype, with no evidence of contamination by alpha-smooth muscle actin-positive valve interstitial cells or CD45-positive leukocytes. RNA integrity was excellent in 80% of the samples, with over 100 ng of total RNA typically obtained from each side of the valve. This rapid method yields high quality pure endothelial RNA in sufficient quantities for amplification and subsequent use in device-, cell-, and location-specific transcriptional profiling by microarray technologies, and thus facilitates studies of spatial gene regulation.

Animals↗
PubMed2003

A tissue fixative that protects macromolecules (DNA, RNA, and protein) and histomorphology in clinical samples.

Preservation of macromolecules (DNA, RNA, and proteins) in tissue is traditionally achieved by immediate freezing of the sample. Although isolation of PCR-able RNA has been reported from formalin-fixed, paraffin-embedded tissues, the process has not been shown to be reproducible because high molecular weight RNA is usually degraded. We investigated the potential value of a new universal molecular fixative (UMFIX, Sakura Finetek USA, Inc., Torrance, California) in preservation of macromolecules in paraffin-embedded tissue. Mouse and human tissues were fixed in UMFIX from 1 hour to 8 weeks. They were then processed by a rapid tissue processing (RTP) system, embedded in paraffin, and evaluated for routine histology as well as for the quality and quantity of DNA, RNA, and proteins. Formalin-fixed tissues were processed by RTP and evaluated in a similar manner. Fresh-frozen samples were used as controls. The morphology of UMFIX-exposed tissue was comparable to that fixed in formalin. High molecular weight RNA was preserved in tissue that was immediately fixed in UMFIX and stored from 1 hour to 8 weeks at room temperature. There were no significant differences between UMFIX-exposed and frozen tissues on PCR, RT-PCR, real-time PCR, and expression microarrays. Similarly, physical and antigenic preservation of proteins in UMFIX tissue was similar to fresh state. Both RNA and proteins were substantially degraded in formalin-fixed and similarly processed specimens. We concluded that it is now possible to preserve histomorphology and intact macromolecules in the same archival paraffin-embedded tissue through the use of a novel fixative and a rapid processing system.

Animals↗
PubMed1993

Detection and direct sequence identification of BCR-ABL mRNA in Ph+ chronic myeloid leukemia.

The reverse transcriptase-polymerase chain reaction (RT-PCR) for BCR-ABL mRNA is increasingly used to diagnose and monitor patients with Ph+ chronic myeloid leukemia (CML). We investigated an alternative approach to detect BCR-ABL mRNA in CML in order to overcome some of the potential drawbacks of RT-PCR. Nucleic acid sequence based amplification (NASBA) is a homogeneous, isothermal, in vitro process that provides the direct amplification of RNA. Peripheral blood from seven patients with Ph+ CML and Ph+ EM-2 cells were investigated by NASBA and RT-PCR. A nested set of four primers flanking the BCR-ABL junction was used in two serial NASBA reactions performed for 2 hours. The two methods were fully concordant for detection of transcripts with bcr3-abl2 and bcr2-abl2 junctions. Ethidium bromide fluorescence with NASBA indicated in repeated experiments that similar quantities of total RNA from patient material contained different amounts of BCR-ABL mRNA. The data suggest that direct amplification of RNA is suitable for identifying and monitoring patients with Ph+ CML and may provide a means to quantify BCR-ABL mRNA levels.

Base Sequence↗
PubMed2003

Donor corneoscleral buttons: a new source of trabecular meshwork for research.

The human trabecular meshwork (TM) is the major site of resistance for aqueous humor outflow. The purpose of this investigation was to determine the suitability of TMs harvested from donor corneoscleral buttons (buttons) for laboratory studies. Histologic examination using light and electron microscopy was performed. Additionally, the effect of incubation in serum free media on histologic appearance was studied. Total RNA extraction was performed on 45 buttons and four whole eyes. Some TMs were used as a source for establishing primary cell cultures of TM endothelial cells. Compared with donor whole eyes, light microscopy showed comparable TM cellularity in the juxtacanalicular and corneoscleral TM; there was some loss of cells in the inner portion of uveal TM that was adjacent to the anterior chamber. The TM cells appeared healthy and structurally normal on transmissive electron microscopy. Usable quantities of total RNA could be extracted from buttons that had been stored up to 5 weeks. The amount of total RNA extracted correlated well with the histologic appearance. Incubation in serum free media did not have an effect on the histologic appearance. All attempts at establishing primary cell cultures were successful. Unused, donor corneoscleral buttons are an excellent source of TM.

Adult↗
PubMed1996

Rapid reverse transcription-PCR detection of hepatitis C virus RNA in serum by using the TaqMan fluorogenic detection system.

We describe the application of a new fluorogenic probe-based PCR assay (TaqMan; Perkin Elmer Corp./Applied Biosystems, Foster City, Calif.) for the detection of hepatitis C virus RNA in serum and plasma. This assay allows for the direct direction of specific PCR products within minutes of completion of the PCR by monitoring the increase in fluorescence of a dye-labeled oligonucleotide probe. We evaluated this assay by comparing the results obtained by nested PCR with those obtained by TaqMan PCR. Test samples included two separate dilutions series of plasma samples from experimentally infected chimpanzees and a panel of 48 serum specimens from patients with community-acquired hepatitis C virus. The quantity of HCV RNA in each chimpanzee plasma sample was determined by using branched DNA (bDNA) signal amplification assay (Quantiplex HCV RNA assay; Chiron Corp., Emeryville, Calif.). Both PCR assays demonstrated similar levels of detection and could reliably detect 13 bDNA genome equivalents per sample. We found an overall concordance of 88% between results of two PCR assays with the community-acquired panel, which resolved to 100% when discrepant samples were retested by nested PCR. TaqMan compared favorably with nested PCR with key advantages of speed, increased throughput, and decreased opportunity for false-positive results because of elimination of second-round amplification.

Animals↗
PubMed2004

Selection of appropriate control genes to assess expression of tumor antigens using real-time RT-PCR.

Real-time reverse transcription PCR (RT-PCR) is a sensitive and accurate method to monitor gene expression and is often used to profile the expression of putative tumor antigens in the context of immunotherapy. However, this technique consists of several steps, including cell processing, RNA extraction, RNA storage, assessment of RNA concentration, and cDNA synthesis prior to PCR. To compensate for potential variability introduced in this procedure, the expression of housekeeping genes is commonly assessed in parallel with the expression of the gene of interest. In this study, the expression of a variety of housekeeping genes in a panel of 26 different human tumor and embryonal cell lines was assessed using real-time RT-PCR. For some control genes, the variability in expression was significant between different cell lines, despite the equalization of quantities of input RNA. The greatest variability was found for GAPDH. The lowest variability was found for beta-glucuronidase (GUS) and 18S rRNA. While real-time RT-PCR is a powerful tool for gene expression analysis, these results suggest that the choice of control genes to normalize the expression of the gene of interest is critical to the interpretation of experimental results and should be tailored to the nature of the study.

Animals↗
PubMed1976

[Embryotropic action of phosalone].

The study on the embryotrophic action of phosalon insecticide preparation is conducted against the background of its comprehensive and thorough hygiene-toxicologic assay. Pregnant white rats undergo treatment per os with 1/10 and 1/100 LD50 throughout the entire gravidity period. Recordings are made of the quantity of corpora lutea, pre- and postimplantation lethality; the pedigree development is traced up to the end of the second postnatal month with readings being made of weight and length in the first postnatal day, presence or not of external malformations, weight increase and survival up to the end of the lactation period, day of eye cleft opening and hairing, and sex ratio. The presence of phosalon and phosalon-oxone in whole embryo homogenates is estimated. Enzyme activity of GOT, GPT, catalase, LDH, G-6-PD, RNA and DNA quantity are studied in the liver of newborns on the second postnatal day. Within 21 days of birth, the peripheral blood picture and visceral weight coefficient are also studied. Investigation of the peripheral blood picture, activity of catalase, GOT and GPT in the liver and serum, alkaline and acid phosphatase, and peroxidase in the serum is performed at the end of the second month. The results point to the presence of an embryotoxic effect at the level of the investigated hepatic indicators, detected on the 2nd day of the postnatal period, only at 1/10 LD50 dose.

Animals↗
PubMed2000

Effective isolation of high-quality total RNA from human adult articular cartilage.

The isolation of large quantities of good-quality RNA from human articular cartilage has been a long-standing problem for researchers working with human articular cartilage. In this paper we report a protocol which we have developed based on the Qiagen RNeasy procedure to produce high yields of purified, DNA-free RNA from normal and osteosteoarthritic human articular cartilage. The average yield of RNA was 8.39 microg/g (n = 59) for normal and 6.69 microg/g (n = 58) for osteoarthritic cartilage (average ratio OD 260/280 = 1.8-1.9). Quantitative PCR, cDNA array technology, and Northern blot analysis were used to verify the quality of the RNA.

Adult↗
PubMed1993

In vitro destabilization of plant viruses and cDNA synthesis.

DNA copies of a wide range of RNA viruses can be made by the direct addition of appropriately treated, purified virus particles to a reverse transcription reaction. Therefore, many problems associated with RNA isolation can be circumvented. Virus particles can be sufficiently destabilized by adjustments of salt content, buffer, pH or by the use of physical force supplied by a freeze/thaw cycle so that RNA in sufficient quantity and physical condition is available for the synthesis of in some cases, full length cDNAs. cDNAs have been made of viruses in the bromo-, poty-, carla-, ilar-, potex-, tobra and tobamovirus groups. Reported here are experiments with cowpea chlorotic mottle virus and bean common mosaic virus.

Blotting, Southern↗
PubMed2006

Nuclear and cytoplasmic mRNA quantification by SYBR green based real-time RT-PCR.

Measurement of the steady-state abundance of nuclear and cytoplasmic RNA requires efficient subcellular fractionation and RNA recovery coupled with accurate quantification of individual RNA species. Detergent lysis of tissue culture cells provides a simple fractionation procedure that can be optimized to individual cell lines. The large dynamic range, extreme sensitivity, high sequence-specificity, and fast turn-around time has allowed real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to become a standard tool for mRNA quantification. Among the different chemistries used for PCR product detection during amplification, DNA binding dyes such as SYBR Green I are simple, versatile, and yet highly reliable and least expensive. With attention to primer design and cycling conditions, virtually any mRNA species can be accurately quantified from even minute quantities of starting RNA. This method provides an accurate and efficient procedure for estimating the relative ratios of nuclear and cytoplasmic RNA concentrations.

Benzothiazoles↗
PubMed1984

Adenovirus mutants with DNA sequence perturbations in the intragenic promoter of VAI RNA gene allow the enhanced transcription of VAII RNA gene in HeLa cells.

Ad2 VAI gene strongly competes for transcription with VAII gene in vitro. It has been suggested that this competition may be a basis for the large excess of VAI gene transcription in virus infected cells at late times. We have studied the effect of the DNA sequence perturbations of the intragenic promoter of the VAI gene on transcription of VAII gene at the level of viral chromosome. Several Ad5 mutants with mutations in the promoter of VAI gene were constructed and transcription of their VAI and VAII genes were analyzed in the infected cells. It was found that transcription of VAII gene increased dramatically when either Box A or Box B promoter sequences of VAI gene were mutated or when the entire VAI gene was replaced by a DNA segment with an unrelated DNA sequence. Thus, at late times, active transcription of VAI gene appears to partially repress transcription of VAII gene. Those mutants which synthesized large quantities of VAII RNA only grew more slowly yielding a titer which was 1/10 of that of their parent but 5 to 6 fold higher than that of an Ad5 mutant lacking both VAI and VAII genes.

Adenoviruses, Human↗