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At least 271 records · Page 15Linked to original sources
PubMed2003

Evaluation of the MagNA pure LC instrument for extraction of hepatitis C virus RNA for the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0.

The COBAS AMPLICOR system has played a major role in the transition of molecular diagnostics from research to routine clinical laboratory use by automating the nucleic acid amplification and detection processes. However, sample preparation remains a labor-intensive portion of the procedure. In this study, we evaluated the performance of the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0 (Roche Molecular Systems, Branchburg, N.J.) following manual hepatitis C virus (HCV) RNA extraction versus automated extraction with the MagNA Pure LC instrument (Roche Applied Science, Indianapolis, Ind.). Parallel replicate testing was performed with standard dilutions of 100, 75, 60, and 0 HCV IU/ml and 153 clinical specimens. An analytical sensitivity of 75 IU/ml was achieved with either the manual or the standard-volume (200 microl) automated extraction methodologies (25 of 26 [96.2%]; 95% confidence interval [95% CI], 80.4 to 99.9), whereas the clinical sensitivity and specificity were both 100% with either extraction method. A large-volume (1 ml) automated extraction method was also evaluated with standard dilutions of 40, 25, 10, and 0 IU/ml and the same 153 clinical specimens. The analytical sensitivity of the COBAS AMPLICOR assay with the large-volume extraction method was 25 HCV IU/ml (26 of 26 [100%]; 95% CI, 86.8 to 100), whereas the clinical sensitivity and specificity were both 100%. The MagNA Pure LC instrument is a versatile, labor-saving platform capable of integration with minimal modification of the existing assay procedure. The increased sensitivity of the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0 performed in conjunction with large-volume HCV RNA extraction may be important in HCV diagnostic testing as new therapeutic strategies evolve.

Hepacivirus↗
PubMed2001

[Extraction of RNA from Stored Bone Marrow Smears for Detecting Expression of the WT1 Gene in MDS Patients]

RNA was extracted from stored bone marrow smears, expression of WT1 gene in the patients with MDS was examined by means of nest RT-PCR. The results showed WT1 gene was highly expressed in some cases of MDS. Expression rates were higher in the patients with RAEB and RAEB-t than those patients with RA and RAS. WT1 expression is related to the advance of MDS. The detection of WT1 gene expression by RT-PCR might be useful for assessing disease progress in the patients with MDS.

Journal Article↗
PubMed1975

Translation of silk fibroin messenger RNA in an Ehrlich ascites cell-free extract.

RNA identified by its base composition and T1 RNase oligonucleotide pattern as the message for silk fibroin was purified from mature posterior silk glands of Bombyx mori larvae and used to direct polypeptide synthesis in an Ehrlich ascites cell-free extract. Fibroin mRNA stimulated [3-H]alanine incorporation about 3- to 4-fold in the presence of 80 mM K+ and 4 mM Mg-2+. The stimulation was reduced in the presence of 5 times 10-minus 6 to 10-minus 4 M aurintricarboxylic acid, an inhibitor of the initiation of protein synthesis. The cell-free products were heterogeneous in size, including peptides as large as 100,000 daltons. They co-precipitated with carrier fibroin sequences after digestion with trypsin. A large fraction of the polypeptides synthesized in response to fibroin mRNA was precipitated by antiserum directed against amino acid sequences in noncrystalline region polypeptides of fibroin. Furthermore, after digestion with chymotrypsin, a major fraction of the cell-free products specifically co-precipitated with crystalline region sequences of native fibroin. The size and amino acid composition of the fibroin crystalline region polypeptides isolated from the cell-free products were similar to those from native fibroin.

Amino Acids↗
PubMed2006

Extraction of high-quality, melanin-free RNA From Mycosphaerella fijiensis for cDNA preparation.

High-quality RNA preparations are critical for further applications such as reverse transcriptase-polymerase chain reaction (RT-PCR) transcript amplifications, and elaboration of cDNA and expressed sequence tag libraries. Melanins are phenolic compounds present in many fungi and apparently play key roles in fungi pathogenesis and survival. However, during RNA extraction these compounds constitute a significant challenge to extraction of substantial quantities of high-quality RNA, and consequently to preparation of cDNA libraries. No method currently exists for RNA extraction from Mycosphaerella fijiensis that produces high quantities of melanin-free RNA. This fungus is the most important pathogen of cultivated Musa sp. varieties. A comparison is made between results obtained from the Trizol and RNeasy protocols for RNA extraction, two commercially available methods commonly used to obtain RNA from various sources. An improved methodology is described that allows isolation of intact RNA and elimination of melanins from M. fijiensis mycelium. RNA quality is evaluated by electrophoresis in formaldehyde-agarose gels, RT into cDNAs, and subsequent PCR amplification using primers designed against actin and beta- tubulin from fungi.

Ascomycota↗
PubMed1983

The cytoskeletal framework of sea urchin eggs and embryos: developmental changes in the association of messenger RNA.

Extraction of sea urchin eggs and embryos with Triton X-100 generated a cytoskeletal framework (CSK) composed of a cortical filamentous network and an internal system of filaments associated with ribosomes. The CSK contained only 10-20% of the cellular protein, RNA, and lipid. A specific subset of proteins was enriched in the CSK. Several lines of evidence suggest that mRNA is a component of the CSK of both eggs and embryos. First, the CSK contained poly(A) sequences which hybridized with [3H]poly(U). Second, the CSK contained polyribosomes. Finally, RNA extracted from the CSK showed translational activity in an in vitro system. The nonhistone messages present in the CSK were qualitatively similar to those solubilized by detergent, as determined by separation on polyacrylamide gels of the products of in vitro translation. In the unfertilized egg, most mRNA was present as nonpolyribosomal messenger ribonucleoprotein complexes which, along with monoribosomes, were efficiently extracted by Triton X-100. The converse was found in blastulae, as most of the mRNA was present as polyribosomes associated with the CSK, although monoribosomes were still efficiently extracted by detergent. These results indicate a correlation between the activation of protein synthesis in eggs and the association of polyribosomes with the CSK.

Animals↗
PubMed2005

Gene expression profiling of HIV-1 infection using cDNA microarrays.

To illustrate the methods employed in gene expression profiling using cDNA microarrays, infection of CD4+ T cell lines with HIV-1LAI is used to identify expression changes relevant to in vitro HIV-1 infection. Cell lines are infected at a high multiplicity of infection to ensure a population of near-synchronously infected cells to be compared to uninfected cells. Infection status is verified using flow cytometry to determine the intracellular expression of the viral gag p24 protein before samples are harvested for total RNA extraction. Total RNA is extracted and amplified using commercially available kits, and RNA quality is verified using Bioanalyzer technology. To obtain fluorescently labeled cDNA probes, the amplified RNA is reverse-transcribed to yield cDNA, using random nonamers in the presence of dye-labeled dCTP. After first-strand cDNA synthesis, RNA is degraded and the probes are purified. For each infection condition (LAI and mock), two slides are hybridized with identical probes generated from the same RNAs, but with fluorescent labels reversed on one of the slides to control for dye-specific effects. Troubleshooting strategies and issues to consider prior to starting the experiment are discussed in detail in the notes section.

CD4-Positive T-Lymphocytes↗
PubMed1975

[Biochemical and cytospectrophotometric analysis of the extraction of RNA from spinal cord cell structures].

By means of two-wavelength spectrophotometry, according to Tsanev and Markov, a stability of RNA content has been demonstrated in rabbit spinal cord sections treated with cold perchloric acid: it was only after 18 and particularly 48 hr incubation of the section in a 16% perchloric acid solution that the total tissue RNA began to be extracted. Cytospectrophotometrical study of the motoneurons of spinal cord anterior horns and perineuronal glial cells in gallocyanin -- chrome alum stained sections has shown a rapid loss of RNA under effect of the cold perchloric acid: as early as after a 2 hr treatment, about 2/3 of the whole cellular RNA was extracted from the motoneurons, while about 1/2 from their glial satellite cells. Hydrolysis of the rest of RNA was found out in the neurons and in the neuroglia only after a 18 hr extraction with the perchloric acid. Similarities and differences in the features of neuronal and glial RNA are discussed.

Animals↗
PubMed1976

Surface immunoglobulins of lymphocytes in plasmacytoma. V. The effect of RNA-rich extract from mouse plasmacytoma MOPC 104E on the immune response.

BALB/c mice with the plasmacytoma MOPC 104E producing monoclonal IgM-lambda with antibody activity to alpha-1,3 dextran were found to have B lymphocytes with surface immunoglobulins with the immunochemical characteristics of 104E IgM capable of binding alpha-1,3 dextran. RNA extracted from this plasmacytoma induced the synthesis of such surface immunoglobulins on normal B lymphocytes in vitro and in vivo. Injection of 200 mug of MOPC 104E RNA into normal mice 72 hr prior to the administration of the antigen kept the immune response to dextran-S intact, but suppressed that to other antigens, such as DNP-Ficoll and LPS, T cell-independent antigens, and SRBC and BSA which are T cell-dependent. The effect of the RNA was abolished by RNase but not by pronase and DNase. RNA extracted from LPC-1 tumour (gamma2a-k without known antibody activity) significantly suppressed the immune response to dextran-S and to other antigens in normal mice. Thus, opposite effects of MOPC 104E RNA on the response to specific and non-specific antigens strengthen the hypothesis that the immune deficiency in plasmacytoma bearing mice is due to the conversion of normal surface immunoglobulin of a population of B lymphocytes to the idiotype of the respective myeloma globulin.

Animals↗
PubMed1991

The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. III. Extraction and purification of RNA and application to slot-blot hybridization analysis.

RNA was extracted from tissues processed by a new fixation and paraffin-embedding method (the AMeX method) and examined by Northern blot analysis and slot-blot analysis. The RNA extraction method for AMeX-processed tissue sections after the deparaffinization step was the same as that for fresh materials. The total amount of cellular RNA extracted from AMeX-processed mouse liver tissue was slightly less than that extracted from fresh tissue. In tissues of malignant lymphoma, the total amount of cellular RNA extracted from 25 sections each 20 microns thick was about 1.6-1.8 micrograms/mm2, regardless of the histological subtype and period of storage. The extracted RNA was moderately degraded, and usually could not be used for Northern blot hybridization analysis. The intensity of ethidium bromide staining and the hybridization signals of RNA extracted from AMeX-processed tissues were usually reduced in comparison with RNA from fresh material, but specific signals could be detected by slot-blot hybridization analysis. We have demonstrated previously that the AMeX method preserves high-molecular-weight DNA and various antigens. Since the present study showed that information on mRNA can be obtained from AMeX-processed tissue, the versatility and usefulness of this method were further proven.

Animals↗
PubMed1981

Meta-phosphoric acid-its use in the selective extraction of RNA from fixed tissue sections.

This communication presents a new quick method for selective extraction of RNA from formalin-fixed tissues, such as kidney, intestine, ovary and testis of white rat and liver of rodents, Tatera indica and Millardia meltada and of the frog, Rana tigrina as well as of human wart. Sections of pancreas and kidney fixed in acetic acid-alcohol were also tried. The method is to treat deparaffinised sections in 20% and 15% meta-phosphoric acid at 5 degrees C for 10-20 and 20-30 min, respectively, and then to stain nuclear DNA with 0.5% aqueous solution of methyl green, methylene green, Giemsa, toluidine blue O or 0.125% pyronin G for 2 min, rinsed with water, treated with n-butanol for 2-3 min, cleared in xylene and mounted. It has been found that liver sections of rodents require 24 hours of treatment in 15% cold meta-phosphoric acid for complete removal of RNA, whereas those of the frog require only 20 min at 20% acid. It has been concluded that following treatment of sections in cold meta-phosphoric acid, RNA is extracted selectively leaving DNA in a native state. Therefore, staining of DNA in tissue sections from which RNA has been extracted is due to binding of its negatively charged phosphate groups with the positively charged dye molecules.

Animals↗
PubMed2003

[A novel method of extracting total RNA from rice embryo samples].

A novel and simple CTAB-LiCl-based extraction method for high-quality and total RNA of rice embryo samples is developed. This method can efficiently eliminate the interference of polysaccharide and lipids rich in rice (Oryza sativa L.) embryo obtained under room temperature without using liquid nitrogen. The results of ultraviolet spectrophotometer and agarose gel electrophoresis analysis show that the obtained RNA has no obvious degradation and a good purity sufficient for further RT-PCR and RNA gel blotting. Therefore, it is also especially useful for the RNA extraction of plant material plenty of polysaccharide and lipids.

English Abstract↗
PubMed1993

Ultra-rapid, simple, sensitive, and economical silica method for extraction of dengue viral RNA from clinical specimens and mosquitoes by reverse transcriptase-polymerase chain reaction.

A rapid, simple and efficient single-tube procedure is described for the isolation of dengue virus RNA from small amount of serum (10 microliters) followed by a reverse transcriptase-polymerase chain reaction (RT-PCR). Recovery of RNA is based on the lysing and nuclease-inactivating properties of guanidinium thiocyanate in the presence of silica. The silica RT-PCR can be completed within 5 hours starting from RNA extraction to agarose gel electrophoresis. All of the 63 dengue-3 culture-positive sera were RT-PCR-positive (virus titres: < 10(2) to 11(10.69.). Of 33 culture-negative acute sera from serologically confirmed dengue fever patients collected during dengue-3 epidemic, 4 were RT-PCR-positive. RT-PCR was also positive in 29 of 30 dengue-1 culture-positive sera (virus titres range: < 10(2) to 10(8.69). Dengue-1 virus was also detected in field-caught Aedes aegypti mosquitoes by silica RT-PCR.

Aedes↗
PubMed2004

Immunotherapy of intracranial G422 glioblastoma with dendritic cells pulsed with tumor extract or RNA.

OBJECTIVE: To investigate the anti-tumor efficacy of dendritic cell (DC)-based vaccines pulsed with tumor extracts or RNA in a mouse model of intracranial G422 glioblastoma. METHODS: Bone marrow-derived DCs were pulsed ex vivo with tumor extracts or RNA. Ninety female mice harboring 4-day-old intracranial G422 glioblastomas and 126 normal mice were treated with three spaced one week apart subcutaneous injections either with PBS, unpulsed DCs, G422 tumor extracts, RNA, DCs pulsed with G422 tumor extracts (DC/extract) or with RNA (DC/RNA). Seven days after the third immunization of normal mice, the spleens of 36 of them were harvested for cytotoxic T lyphocyte (CTL) assays and the others were challenged in the brain with G422 tumor cells. All the treated mice were followed for survival. Some mice brains were removed and examined pathologically when they died. RESULTS: Immunization using DC/extract or DC/RNA significantly induced G422-specific CTL responses compared with control groups (P<0.01). Vaccination with DC/extract or DC/RNA, either prior to G422 tumor challenge or in tumor-harboring mice, significantly prolonged survival compared with other control groups (P<0.01). CONCLUSION: DCs pulsed with tumor extracts or RNA derived from autologous tumors has potential antitumor effects via activation of cell-mediated immunity. Our results suggest a useful therapeutic strategy against gliomas.

Animals↗
PubMed1994

Detection of bcr/abl mRNA in stained bone marrow smears.

We describe the polymerase chain reaction (PCR) method using stained bone marrow smears as sources of RNA. The amount of extractable RNA decreased during the process of making and staining bone marrow smears. The sensitivity of the reverse transcriptase-based polymerase chain reaction (RT-PCR) method for detecting target mRNA-positive cells in 5 x 10(5) suspended cells and stained bone marrow smears were 1:10(5) and 1:5000, when we used K562 cells. The bone marrow smears of 21 patients with chronic myelogenous leukemia (CML) were examined using this method. We extracted RNA from stained specimens stored at room temperature for 5-14 years. Twelve of 21 (57%) smears showed positive results for bcr/abl. The carrier RNA improved the recovery when added at the step of RNA extraction. These data indicate that mRNA is present in stained bone marrow smears for at least 14 years and that the sensitivity of RT-PCR is adequate for molecular analysis.

Base Sequence↗
PubMed2001

High-throughput detection of West Nile virus RNA.

The recent outbreaks of West Nile virus (WNV) in the northeastern United States and other regions of the world have made it essential to develop an efficient protocol for surveillance of WNV. In the present report, we describe a high-throughput procedure that combines automated RNA extraction, amplification, and detection of WNV RNA. The procedure analyzed 96 samples in approximately 4.5 h. A robotic system, the ABI Prism 6700 Automated Nucleic Acid workstation, extracted RNA and set up reactions for real-time reverse transcription (RT)-PCR in a 96-well format. The robot extracted RNA with a recovery as efficient as that of a commercial RNA extraction kit. A real-time RT-PCR assay was used to detect and quantitate WNV RNA. Using in vitro transcribed RNA, we estimated the detection limit of the real-time RT-PCR to be approximately 40 copies of RNA. A standard RT-PCR assay was optimized to a sensitivity similar to that of the real-time RT-PCR. The standard assay can be reliably used to test a small number of samples or to confirm previous test results. Using internal primers in a nested RT-PCR, we increased the sensitivity by approximately 10-fold compared to that of the standard RT-PCR. The results of the study demonstrated for the first time that the use of an automated system for the purpose of large-scale viral RNA surveillance dramatically increased the speed and efficiency of sample throughput for diagnosis.

Animals↗
PubMed1981

Phosphoric acid-its use in the extraction of RNA: staining of DNA in mammalian tissue sections.

This communication presents highly satisfactory methods for the demonstration of DNA in animal materials. The method involves selective extraction of RNA with concentrated, 90% or 75% phosphoric acid of 5 degrees C for 20, 40 and 120 min, respectively, followed by staining with aqueous solutions of basic dyes, such as setoglaucine, setocyanine, pinakryptol green 2) and alcoholic aniline blue without SO2. Perfect blue nuclei were seen when staining was performed with aqueous solutions of setoglaucine and setocyanine at pHs 3.5 and 4.0 to 4.5, respectively. Sections of tissues from which RNA has been extracted and then hydrolysed in 6 N HCl at 28 degrees C for 15 min followed by staining with these dyes also revealed perfect colouration of the nuclei. Acid-hydrolysed sections when stained with alcoholic aniline blue-SW2 prepared with N HCl and sodium thiosulphate revealed nuclei of magenta colour, and sections from whcih RNA has been extracted and then hydrolysed in hydrochloric acid and stained with this dye-reagent revealed nuclei of purplish colour. Sections of tissues fixed in Carnoy, 10% buffered neutral formalin as well as paraformaldehyde were found to be most suitable for staining with these dyes. The in situ absorption spectra of nuclei stained with aqueous solutions of setoglaucine, setocyanine and alcoholic aniline blue without SO2, after extraction of RNA as well as those of nuclei in tissue sections from which RNA has been extracted and then acid-hydrolysed and stained with alcoholic aniline blue-SO2 have been presented. Also presented herein are absorption data of nuclei in tissue sections which wee hydrolysed in hydrochloric acid and then stained with alcoholic aniline blue-SO2. Some implications of these findings have been discussed.

Aniline Compounds↗
PubMed1988

Immunochemical characterization and biosynthesis of pI-6.4 esterase, a carboxylesterase of rat liver microsomal extracts.

Rat liver pI-6.4 esterase was purified from microsomes (microsomal extracts) and used to generate antibodies in the rabbit. Two active enzyme forms, similarly sensitive to endo-H (endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), but differing slightly in polypeptide chain length, were present in the preparation. In microsomes, immunoblots revealed a single form, with Mr congruent to 62,000, identical with the large component of the purified enzyme, indicating that the second component is an artefact. Rabbit reticulocyte lysates and wheat germ extracts programmed with RNA extracted from total or bound polysomes synthesized a single immunoreactive 61 kDa polypeptide, which was not formed with RNA extracted from free polysomes. The immunoreactive product synthesized in the presence of dog pancreas microsomes was slightly larger (62 kDa); like the authentic enzyme, it bound to concanavalin A and was decreased in molecular size to 60 kDa by the action of endo-H. Thus the enzyme is synthesized with a short cleavable sequence and bears at least one high-mannose oligosaccharide chain. Metabolic labelling in hepatocytes cultured with [35S]methionine also generated a single immunoreactive polypeptide of 62 kDa, which was decreased to 60 kDa in size by treatment with endo-H or addition of tunicamycin to the culture medium. This confirms the molecular homogeneity and the glycosylation of the enzyme in the intact cell. Culture media contained no pI-6.4-esterase-related protein, whether tunicamycin was present or not. The processing steps in the synthesis of pI-6.4 esterase are thus, as for other esterases of the endoplasmic reticulum [Robbi & Beaufay (1986) Eur. J. Biochem. 158, 187-194; (1987) Biochem. J. 248, 545-550] indistinguishable from those occurring early in the synthesis of secretory proteins. Glycosylation is apparently not the sorting signal responsible for their retention in the endoplasmic reticulum.

Acetylglucosaminidase↗
PubMed1998

Extraction of RNA from single frozen sections.

The sensitivity of the reverse transcriptase-polymerase chain reaction (RT-PCR) makes it ideally suited for the detection of changes in gene expression. Unfortunately, traditional methods for RNA isolation require time-consuming procedures that are not appropriate for small samples, such as individual frozen sections. This report describes a new technique that permits the rapid extraction of RNA from individual frozen histological sections. RNA is extracted by incubating a frozen section in an RT-PCR compatible buffer solution containing RNase inhibitor and dithiothreitol. RNA isolated from frozen sections is stable at room temperature for up to 3 h under the conditions described. Alternatively, extracts can be frozen for later use. When maintained in a dry state at room temperature, RNA in sections remained stable for 2 weeks. Histological, immunohistochemical, or in situ analyses can be carried out with sections that are immediately adjacent to those used for extracting RNA. The simplicity and economy of this procedure may foster the development of prognostic screens that can be performed in parallel with traditional histopathological analysis.

Buffers↗