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At least 91 records · Page 5Linked to original sources
PubMed1993

Analysis of Epstein-Barr virus gene transcription in lymphoma induced by the virus in the cottontop tamarin by construction of a cDNA library with RNA extracted from a tumour biopsy.

Inoculation of the cottontop tamarin with Epstein-Barr virus (EBV) gives rise to the development of mono- and/or oligoclonal large cell malignant lymphoma. A cDNA library was generated with the RNA extracted from an EBV-induced tamarin lymphoma biopsy in order to study the transcripts expressed in the tumour tissue. Fifteen EBV-specific cDNA clones were localized in the corresponding viral genomic fragments. Among them, two correspond to the EBNA-2 gene, and two others to the latent membrane protein gene. The majority of the cDNA clones were localized in the BamHI A fragment which has not been associated with latent expression. Furthermore, cDNAs were also found from the BamHI D and I fragments. Sequence analysis of the cDNAs localized in BamHI A showed that they correspond to a rightward transcript in the BALF-3 region, with the one clone that was sequenced containing four exons and three introns. The above results were confirmed by testing three different biopsies with the rapid amplification of cDNA ends-PCR method.

Animals↗
PubMed2002

Rapid detection of enterovirus infection by automated RNA extraction and real-time fluorescence PCR.

BACKGROUND: Molecular detection has been shown to be superior to tissue culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. OBJECTIVES: In this study, a qualitative molecular assay based on automated RNA extraction with the MagNA Pure LC and real-time PCR on the LightCycler (LC) instrument was evaluated and compared with an in-house molecular assay. STUDY DESIGN: A total of 109 CSF specimens were investigated for the comparative study. The detection limit of the new molecular assay was determined with 10-fold dilutions of two enterovirus strains and with the Third European Union Concerted Action Enterovirus Proficiency Panel. RESULTS: With the enterovirus strains, the detection limit of the LC assay was found to be 0.1 TCID(50) (50% tissue culture infective dose). When samples of the Third European Union Concerted Action Enterovirus Proficiency Panel were tested, both molecular assays gave identical results to the expected results, which were based upon the results of three reference laboratories using a total of four different molecular methods before distribution of the panel. When clinical specimens were tested, there was a correlation between the LC assay and the in-house assay in 105 of 109 cerebrospinal fluids. CONCLUSIONS: The new molecular assay allows rapid detection of enterovirus RNA in CSF. It was found to be labor saving and showed sufficient sensitivity.

Adult↗
PubMed2000

Heparin interference with reverse transcriptase polymerase chain reaction of RNA extracted from lungs after ischemia-reperfusion.

The reverse transcriptase polymerase chain reaction (RT-PCR) is a rapid and sensitive method for detecting gene expression. However, when we used this technique to study gene expression of cytokines in ischemic and ex-vivo-reperfused rat lungs as a model for transplantation, significant inhibition of RT-PCR reaction was observed. To optimize RT-PCR conditions, RNA was extracted from rat lungs after flushing, preservation, and reperfusion. RNA was further purified and PCR conditions were modified with various strategies. We found that heparinase I pretreatment completely overcame the inhibitory effects of RT-PCR using RNA extracted from lung tissues after ischemia-reperfusion. With this treatment, a dramatic increase in tumor necrosis factor-a (TNF-a) mRNA was revealed from lung tissues after ischemia-reperfusion. This result suggests that residual heparin in lung tissue interferes with RT-PCR. Because heparinization is routinely used during clinical and experimental organ transplantation, we recommend the treatment of RNA samples with heparinase prior to RT-PCR.

Animals↗
PubMed2004

Assessment of different commercial RNA-extraction and RT-PCR kits for detection of hepatitis A virus in mussel tissues.

In the present study, the efficiency of several nucleic acid extraction and RT-PCR commercial kits for the detection of hepatitis A virus (HAV) from seeded mussel tissue samples was evaluated in comparison with the "in-house" method used currently in our laboratory. The best results were achieved with Total Quick RNA Cells & Tissues version mini (Talent) for RNA extraction and the Superscript One-Step RT-PCR System (Life Technologies) for the RT-PCR reaction, obtaining a detection limit of 0.1-1pfu/mg of mussel tissue. A slightly lower sensitivity (in 1logunit) was achieved using the Rneasy plant mini kit (Qiagen) and the Total Quick RNA Cells & Tissues version maxi in combination with the Superscript RT-PCR system. The conventional method usually employed in our laboratory resulted in a sensitivity of 300pfu/mg of tissue. Taken together, these findings indicate that the combination of Total Quick RNA Cells & Tissues version mini and Superscript One-Step RT-PCR System cannot only improve significantly the sensitivity for the HAV detection from mussel, but are also labor and time saving and easy to standardize.

Animals↗
PubMed2003

[Manual microdissection of defined cells and RNA extraction for gene expression analysis of esophageal carcinoma progress].

OBJECTIVES: To isolate cells of interest from heterogeneous tissue blocks to obtain accurate representations of molecular alterations acquired by neoplastic cells so as to meet the demands of further study on gene expression patterns of the esophageal carcinoma (EC) evolution. METHODS: Blocks of EC were stored at -70 degrees C as close as possible to the time of surgical resection. The tissue block was embedded in OCT and frozen sections of 35 microns in thickness were cut in a cryostat under strict RNAse-free conditions. Individual frozen sections were mounted on plain glass slides and 30-gauge needle attached to a 1 ml syringe was used to microdissect defined cells in the sections. The procured cells were used for total RNA extraction. RESULTS: An optimized protocol of manual microdissection was developed successfully whereby regions with an area as small as 1/25 mm2 could be accurately dissected. The RNA recovered from procured cells was of high quality suitable for subsequent applications of molecular analysis as assessed of 18S and 28S rRNAs by electrophoresis on agarose gel. CONCLUSIONS: It is believed that manual microdissection is capable to procure defined cell populations from complex primary tissues, thus allowing investigation of tissue-, cell-, and function-specific gene expression patterns. The technique is simple, easy to perform, versatile, and of particular usefulness when laser capture microdissection (LCM) is practically unavailable.

Cell Separation↗
PubMed1985

Immunoactive products of placenta: VIII. Translation products of messenger RNA extracted from murine placenta or A6B9IC5 teratocarcinoma are immunosuppressive in vitro.

RNA was extracted from placentae of synpregnant C3H mice (day 14 of gestation) and from A6B9IC5 teratocarcinoma. After isolation of poly-(A)+ RNA on an oligo d(T) cellulose column, putative messenger RNA was injected into Xenopus laevi oocytes. Supernatants and homogenates from the oocytes were assayed for immunoregulatory activity in MLR, CML, and NK assays and found to be immunosuppressive. These data indicate that immunomodulators could be obtained from the placenta by molecular biology technology in sufficient quantities for eventual clinical applications.

Animals↗
PubMed2004

Development of a TaqMan RT-PCR assay without RNA extraction step for the detection and quantification of African Chikungunya viruses.

Chikungunya virus (CHIKV), a member of the alphavirus genus, is of considerable public health concern in Southeast Asian and African countries. However, despite serological evidence, the diagnosis of this arthropod-borne human disease is confirmed infrequently and needs to be improved. In fact, illness caused by CHIKV can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms, and laboratory confirmation of suspected cases is required to launch control measures during an epidemic. Moreover, no quantitative molecular tool is described to study CHIKV replication or detection in clinical samples and cell culture supernatants. In this study, a specific and sensitive CHIKV one-step TaqMan RT-PCR assay was developed as a tool for the diagnosis of African CHIKV as well as a rapid indicator of active infection by quantifying viral load. This study also showed that a simple heat viral RNA release during the reverse transcription step constituted an alternative to the conventional RNA extraction method.

Base Sequence↗
PubMed2000

Processing renal biopsies for diagnostic mRNA quantification: improvement of RNA extraction and storage conditions.

The goal of this study was to improve a procedure for the extraction and storage of RNA from minute quantities of human renal tissue in clinical practice, using kidney biopsies and cadaveric donor kidneys unsuitable for transplantation. Collagen alpha1(IV) mRNA was analyzed as a measure for RNA integrity. The results show that at least 3 h may pass between microdissecting the renal tissue and the onset of cDNA synthesis without degradation of the glomerular mRNA. To extract the glomerular mRNA, microdissected glomeruli were incubated in a permeabilization solution. Treating glomeruli with collagenase IV before permeabilization had a deteriorating effect on the mRNA yield. The addition of reverse transcription mixture to the permeabilization solution in the presence of the glomeruli resulted in the highest cDNA yields. Storage of glomerular tissue in the presence of Nonidet P-40-based buffer for 1 wk at -70 degrees C did not significantly affect the mRNA, but storage for 2 or 4 wk resulted in deterioration of the mRNA by approximately 40 and 95%, respectively. Furthermore, three methods for total RNA isolation from microdissected interstitial tissue were compared. An approximately 2.5 times higher yield of collagen alpha1(IV) mRNA was obtained with silica gel-based membrane spin technology than with a guanidine isothiocyanate/phenol chloroform or a lithium chloride/phenol chloroform method. Finally, this study shows for the first time reliable detection of collagen alpha1(IV) mRNA in biopsies that had been frozen for at least 10 yr at -70 degrees C. These experiments have helped to improve a procedure for the processing of glomerular and interstitial tissue acquired from human kidney biopsies for mRNA analysis. This method is suitable for implementation in routine clinical practice.

Biopsy↗
PubMed1977

In vitrocleavage of 45S pre-ribosomal RNA and of giant heterogeneous RNA extracted from human leukemic cells.

45S ribosomal precursor RNA and large heterogeneous RNA molecules (greater than 45S) extracted from human leukemic cells were incubated in vitro with purified RNase III, which specifically attacks double-helical RNA regions. About 50% of the ribosomal precursor was cleaved into two major fragments sedimenting at 28S and 32S respectively. A limited number of cleavages was also introduced in about 40% of heterogeneous RNA molecules sedimenting faster than 45S, causing a partial 'shift' to a polydisperse distribution in the 10S-45S range.

Cells, Cultured↗
PubMed1983

Messenger RNA extracted from Schistosoma mansoni larval forms codes for parasite antigens when translated in vitro.

Intact messenger RNA was extracted from three stages of development of the parasite Schistosoma mansoni, cercariae, 3 h (mechanically prepared) schistosomula and adults. Some of the in vitro translation products are precipitable with immune rat and human serum showing that these sera recognize protein determinants. Whereas the patterns of the total translation products show major proteins expressed at all three developmental stages, immunoprecipitation delineates patterns unique to each stage. Serum from chronically infected patients is likely to preferentially recognize proteins expressed by the adult parasite yet precipitates more products of translation of cercarial RNA than of adult RNA, suggesting that many of the same epitopes are already coded by cercarial messenger RNA. It seems, however, that antigens appear on lower molecular weight species in the adult. The experiments described here open the way to cloning the genes of antigens from S. mansoni.

Animals↗
PubMed2004

Clinical usefulness of a new hepatitis C virus RNA extraction method using specific capture probe and magnetic particle in hemodialysis patients.

Hemodialysis patients are a high-risk group for hepatitis C virus (HCV) infection. Assessment of HCV infection using HCV-RNA assay among dialysis patients is important for the issue of safety and environmental protection. However, polymerase chain reaction (PCR)-based methods are unsuitable for analyzing samples from dialysis patients because the conventional centrifugal extraction method fails to eliminate heparin, a potent inhibitor of PCR. In this study, we evaluated the usefulness of a HCV-RNA extraction method using probes and magnetic particles for hemodialysis patients in comparison with the centrifugal method. The study population consisted of 17 HCV antibody-positive patients undergoing hemodialysis. These 17 patients consisted of 12 HCV carrier patients and five patients with past HCV infection. One hundred and two samples from these patients were measured using the centrifugal and magnetic methods. Moreover, we prepared five standards that included theoretically 5 KIU/mL of HCV. One was made from non-HD patient's serum and the other four were from hemodialysis patients' serum. These standards were measured using the two methods. False-negative results were not observed with the magnetic method, but were observed in five out of 102 samples with the centrifugal method. Studies using standard samples revealed that accurate HCV-RNA measurement is achieved using the magnetic method. In conclusion, the present study showed that this magnetic extraction method is a highly reproducible and reliable assay to obtain correct information about the presence of the infective virus itself in the hemodialysis setting. Precise identification of HCV-RNA using this specific method is considered to be useful in preventing HCV infection in hemodialysis units.

Aged↗
PubMedDate not supplied

Improved methods for extracting RNA from exfoliated human colonocytes in stool and RT-PCR analysis.

In order to diagnose colon cancer at an earlier, more localized stage, there is a need to develop diagnostic markers (genes) which can detect early patterns of gene expression in exfoliated colonocytes shed in the stool during routine screening for this disease. An RNA-based detection is more pertinent than either a DNA-based or a protein-based method as a screening procedure, but it has not been widely used as a cancer screen because of the difficulty of handling and stabilizing the RNA molecule. We describe a method that permits extraction of intact nondegraded total RNA from human colonocytes in stool and from normal and malignant colon tissues (which were employed for comparison with stool). Because it utilizes commercially available kits, this method is simpler than other published methods and does not require isolation of messenger (m)RNA, thereby reducing the chances of contaminating the preparations with degrading nucleases, and even a small amount of isolated total RNA can be adequately reverse transcribed, making high-quality copy (c) DNA. This is followed by PCR (either qualitative end point or semiquantitative real-time) using colon cancer-specific gene primers. By routinely and systematically being able to perform quantitative gene expression measurements on noninvasive samples, the goal of this pilot work is to lay the groundwork for conducting a large clinical study to identify groups of selected genes whose expression is consistently altered at an early stage in the neoplastic process. Such work will permit noninvasive monitoring of at-risk patients through the analysis of their stool samples. Correct diagnosis will allow for surgical and/or other interventions before the tumor is well established and, thus, should decrease mortality from this preventable disease.

Adenocarcinoma↗
PubMed1999

Comparison of seven RNA extraction methods on stool and shellfish samples prior to hepatitis A virus amplification.

When choosing an extraction method, two parameters have to be considered: recovery of the viral material and elimination or inactivation of inhibitory substances. Seven techniques for extracting hepatitis A virus (HAV) from stool and shellfish samples were compared, in order to identify the protocol most suited to both types of sample and with the best extraction yield. The protocols tested were either techniques for the recovery and purification of total RNA, such as RNAzol, PEG-CETAB, GTC-silica and Chelex, or techniques for isolating specifically HAV using a nucleotide probe or a monoclonal antibody. For stool samples, RNAzol, PEG-CETAB, and magnetic beads with antibody allowed detection of the virus in 11/12 and 12/12 of samples. For shellfish samples, three protocols allowed RNA to be extracted in 90% of cases, RNAzol, PEG-CETAB, and GTC-silica. Their rapidity and low cost make RNAzol and GTC-silica the most suitable for routine diagnostic testing. reserved.

Feces↗