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PubMed1977

Factors affecting the extraction of intact ribonucleic Acid from plant tissues containing interfering phenolic compounds.

Using conventional methods it is impossible to extract RNA as uncomplexed intact molecules from the leaves of grapevines (Vitis vinifera L.) and from a number of woody perennial species that contain high levels of reactive phenolic compounds. A procedure involving the use of high concentrations of the chaotropic agent sodium perchlorate prevents the binding of phenolic compounds to RNA during extraction. Analyses of the phenolics present in plant tissues used in these experiments indicate that there is a poor correlation between the total phenolic content and the complexing of RNA. However, qualitative analyses suggest that proanthocyanidins are involved in the tanning of RNA during conventional extractions.

Journal Article↗

PubMed1974

Translation of vesicular stomatitis messenger RNA by extracts from mammalian and plant cells.

RNA was isolated from polyribosomes of vesicular stomatitis virus (VSV)-infected cells and tested for its ability to direct protein synthesis in extracts of animal and plant cells. In cell-free, non-preincubated extracts of rabbit reticulocytes, the 28S VSV RNA stimulated synthesis of a protein the size of the vesicular stomatitis virus L protein whereas the 13 to 15S RNA directed synthesis of the VSV M, N, NS, and possibly G proteins. In wheat germ extracts, 13 to 15S RNA also directed synthesis of the N, NS, M, and possibly G proteins. Analysis of extracts labeled with formyl [(35)S]methionine showed that the 28S RNA directed the initiation of synthesis of one protein, whereas the 13 to 15S RNA directed initiation of at least four proteins. It is concluded that the 28S RNA encodes only the L protein, whereas the 13 to 15S RNA is a mixture of species, presumably monocistronic, which code for the four other known vesicular stomatitis virus proteins.

Animals↗

PubMed1979

Poly(A)+ RNA from Tetrahymena: stimulation of protein synthesis in vitro.

Cell-free synthesis of high molecular weight polypeptides, programmed by RNA from Tetrahymena pyriformis strain W is reported, and methods for preparation of the RNA are described. The RNA was extracted by the SDS-phenol-chloroform-isoamyl alcohol technic. The bulk of extracted RNA was ribosomal and on sucrose gradients peaked at approximately 17S and 25S. After heat denaturation all the 25S RNA was converted to 17S, indicating the presence of hidden breaks, possibly the result of nuclease activity during extraction. Nevertheless, when poly(A) +/- RNA was collected using oligo-(dT)-cellulose column chromatography, it promoted a 15-fold increase in incorporation of [35S] methionine into TCA-precipitable material. Slab-gel electrophoresis and autoradiography of the product revealed 12 different major polypeptides, varying in weight from 28,000 to 65,000 Daltons. A method for preparation of translatable RNA from Tetrahymena will make possible the comparison of messenger RNAs associated with specific cell structures and with different developmental events.

Animals↗

PubMed1976

Surface immunoglobulins of lymphocytes in mouse plasmacytoma. IV. Evidence for the persistence of the effect of plasmacytoma-RNA on the surface immunoglobulins of normal lymphocytes in vivo and in vitro.

The previous findings were confirmed that RNA extracted from murine plasmacytoma alters the character of the lymphocyte surface immunoglobulins (SIg) to express the idiotypic specificity of the Ig of the plasmacytoma from which the RNA was derived (cell conversion). RNA extracted from spleens of plasmacytoma-RNA-injected BALB/c mice also had convering activity, and if injected into other mice, caused the appearance of RNA active in cell conversion in spleens of the second set of mice. This activity was lost only after two additional transfers. When splenic cells from animals 1 hr after injection with RNA extracted from MOPC 300, LPC-1 or MOPC 104E, were cultured for 7 days, the proportion of cells with the SIg specific for these tumours increased. The cell-converting activity of the RNA extracted from the cultured cells after 7 days incubation ('7-day' RNA) was higher than that of RNA extracted from cells after 1 hr incubation ('immediate' RNA). 'Seven-day' RNA could be used for sequential transfers without marked loss of activity in cell conversion for at least five transfers. The repetitive transferability of this phenomenon by the injection of plasmacytoma-RNA suggests the possibility of RNA replication in the recipient cells.

Animals↗

PubMed2005

Extraction of RNA from cells and tissue.

Total undegraded cellular RNA, largely free of contaminating DNA, can be rapidly and easily isolated from homogenized tissue or cultured cells using acid pH guanidinium thiocyanate/phenol/chloroform extraction. Commercially available acid phenol/guanidinium thiocyanate solutions allow one-step complete solubilization of tissue or cells under conditions that inhibit RNAse activity. We describe here a detailed procedure for total RNA extraction from homogenized tissues or cultured cells using such reagents, together with additional purification steps by passage through Phase-Lock gel tubes, and recommend optional chromatographic poly(A+) RNA enrichment procedure using Mini Oligo(dT) spin columns, together with the spectrophotometric determination of RNA yield and purity.

Cells, Cultured↗

PubMed2003

Early diagnosis of SARS coronavirus infection by real time RT-PCR.

BACKGROUND: A novel coronavirus was recently identified as the aetiological agent of Severe Acute Respiratory Syndrome (SARS). Molecular assays currently available for detection of SARS-coronavirus (SARS-CoV) have low sensitivity during the early stage of the illness. OBJECTIVE: To develop and evaluate a sensitive diagnostic test for SARS by optimizing the viral RNA extraction methods and by applying real-time quantitative RT-PCR technology. STUDY DESIGN: 50 nasopharyngeal aspirate (NPA) samples collected from days 1-3 of disease onset from SARS patients in whom SARS CoV infections was subsequently serologically confirmed and 30 negative control samples were studied. Samples were tested by: (1) our first generation conventional RT-PCR assay with a routine RNA extraction method (Lancet 361 (2003) 1319), (2) our first generation conventional RT-PCR assay with a modified RNA extraction method, (3) a real-time quantitative RT-PCR assay with a modified RNA extraction method. RESULTS: Of 50 NPA specimens collected during the first 3 days of illness, 11 (22%) were positive in our first generation RT-PCR assay. With a modification in the RNA extraction protocol, 22 (44%) samples were positive in the conventional RT-PCR assay. By combining the modified RNA extraction method and real-time quantitative PCR technology, 40 (80%) of these samples were positive in the real-time RT-PCR assay. No positive signal was observed in the negative controls. CONCLUSION: By optimizing RNA extraction methods and applying quantitative real time RT-PCR technologies, the sensitivity of tests for early diagnosis of SARS can be greatly enhanced.

Humans↗

PubMed1992

Improved detection of hepatitis C virus RNA by reverse transcription and polymerase chain reaction.

The polymerase chain reaction with prior reverse transcription of RNA into cDNA was applied to hepatitis C virus RNA detection in human serum samples of different origin. In order to eliminate false negative results, the following steps were optimized: RNA extraction, reverse transcription, and oligonucleotide primer selection. We compared different RNA extraction methods using guanidinium salt/detergent and proteinase K digestion/phenol extraction, and tested virus particle enrichment with polyethylene glycol precipitation and ultracentrifugation. RNA extraction with guanidinium salt/detergent was the most efficient method. Ultracentrifugation of single samples did not improve hepatitis C virus RNA detection. Polyethylene glycol precipitation performed poorly. Recombinant thermostable reverse transcriptase produced cDNA from fewer samples than did Moloney murine leukaemia virus reverse transcriptase. Nested oligonucleotide primers from the 5'-terminal non-coding region of the hepatitis C virus genome amplified cDNA from more samples than did primers from the coding regions. Thirty six anti-hepatitis C virus antibody positive samples were tested; nested primers (nucleotides 6 to 327 and 15 to 288) yielded 21 amplificates, whereas primers from the coding region produced 16 amplificates (nucleotides 4684-5276) and 5 amplificates (nucleotides 5166-5270), respectively. The most efficient combination of steps was RNA extraction with guanidinium salt solution, reverse transcription with Moloney murine leukaemia virus reverse transcriptase and nested polymerase chain reaction primed with primers from the 5'-terminal non-coding region of the hepatitis C virus genome. Other combinations produced more false negative results. Three different groups of anti-hepatitis C virus antibody positive individuals had markedly different viraemia patterns: Hepatitis C virus RNA was detected in the sera of only 10% of anti-hepatitis C virus antibody positive blood donors, but in 90% of anti-hepatitis C virus antibody positive patients with clinically manifest hepatitis C, and 90% of anti-hepatitis C virus antibody positive haemophiliacs who had received plasma products in the past which had not been virus-inactivated. No hepatitis C virus RNA could be detected in the sera of 450 anti-hepatitis C virus antibody negative blood donors with elevated serum alanine aminotransferase catalytic concentrations.

Alanine Transaminase↗

PubMed2000

[Detection of the genome of bovine viral diarrhea virus (BVDV) using the polymerase chain reaction after reverse transcription (RT-PCR): comparison of methods for the isolation of ribonucleic acid (RNA) from clinical samples].

The RT-PCR is an in vitro technique that is increasingly being used for diagnosis of viral animal pathogens. Due to its high sensitivity it is considered as an alternative to current standard methods for detecting BVDV especially in pooled samples, e.g. from bulk tank milk. A prerequisite for the performance of RT-PCR is an efficient and simple method for sample preparation. The aim of this work was to compare the efficiency of three commercially available kits for RNA extraction, and their suitability for sample preparation for the detection of the BVDV genome by RT-PCR in blood, milk and tissue samples. The kits were based on different methods for extraction of RNA and differed in costs, labour and time consumption. The most sensitive RT-PCRs (exception: heparinised blood) were obtained when sample preparation was performed by acidic guanidinium-isothiocyanate-phenol-chloroform extraction with the Trizol (Gibco) reagent. Using a kit based on the binding of RNA to silica membrane in a spin column, positive results in RT-PCR were obtained from all samples, but with lower sensitivity. The advantage of the column-based kits is that they are less time-consuming, easier to handle and suitable for automatisation of sample preparation. A kit using salt precipitation of the desoxribose nucleic acid (DNA) and proteins was unsuitable for the isolation of viral RNA from the samples.

Animals↗

PubMed2004

Impact of pre-analytical handling on bone marrow mRNA gene expression.

Large clinical trials on leukaemia, require the transport of bone marrow (BM) from participating clinics to central diagnostic laboratories. We have investigated the impact of RNA extraction protocols and time delays between sample aspiration and RNA extraction on RNA quality and gene expression profiles. Intact RNA can be extracted from BM samples stored at room temperature for up to 48 h. Gene expression analyses using Affymetrix U95Av2 GeneChips and a custom-designed cDNA array in parallel showed that even short-term storage of BM has dramatic effects on mRNA expression of individual transcripts. Many probe sets/genes showed either reproducible deregulation (18.8%, analysis of variance <0.05), or inconsistent expression that differed from patient to patient (38.4%). Moderate alterations were observed in 42.8% genes, with a maximum fold change <2.0 in all experiments and at all time points. These profound effects complicate the use of unstabilized, shipped BM samples for gene expression analyses. The comparison of a variety of RNA stabilization reagents (e.g. PAXgene) resulted in partial conservation of the mRNA expression patterns. Immediate density centrifugation or erythrocyte lysis and freezing at -80 degrees C represent simple procedures that reliably preserved mRNA gene expression patterns in BM.

Bone Marrow↗

PubMed2006

Automated extraction protocol for quantification of SARS-coronavirus RNA in serum: an evaluation study.

BACKGROUND: We have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS) based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay. METHODS: An automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics) was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit (QIAamp viral RNA mini kit, Qiagen) for quantitative performance, analytical sensitivity and precision. RESULTS: The newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method. CONCLUSION: As the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method.

Humans↗

PubMed1997

Interleukin (IL)-1 beta and IL-1 beta mRNA expression in normal and diseased skeletal muscle assessed by immunocytochemistry, immunoblotting and reverse transcriptase-nested polymerase chain reaction.

To confirm the production of IL-1 beta and to optimize detection and semiquantitation of IL-1 beta mRNA by polymerase chain reaction (PCR) techniques in skeletal muscle tissue, immunocytochemistry, immunoblotting and several procedures of RNA extraction and reverse transcription (RT)-PCR amplification were used on muscle samples from 12 patients with conditions associated with local production of IL-1 beta (AZT myopathy: 6 patients; sarcoid myopathy: 6 patients) and from 9 patients with normal muscle used as controls. Abundant IL-1 beta immunoreactivities, corresponding to both pro IL-1 beta and mature IL-1 beta as assessed by immunoblotting, were observed in all diseased muscles, either in inflammatory cells (sarcoid myopathy) or in atrophic muscle fibers (AZT myopathy). Acid guanidinium isothiocyanate phenol-chloroform extraction of RNA appeared less efficient for IL-1 beta mRNA detection by RT-PCR than proteinase K digestion followed by phenol-chloroform extraction. Even using the latter procedure, RT-single PCR for IL-1 beta mRNA was puzzlingly negative in all cases but one; in contrast, RT-nested PCR specified by DNA enzyme immunoassay yielded detection of IL-1 beta mRNA in all diseased muscles and in occasional controls, including the expected PCR product of 391 bp, but also another product of 935 bp, corresponding to IL-1 beta mRNA with unsplicing of the fourth intron. Semi-quantitative PCR showed that production of IL-1 beta mRNA was higher in sarcoid myopathy than in AZT myopathy, and in AZT myopathy than in controls. In conclusion, IL-1 beta expression can be reliably studied using immunocytochemistry, but assessment of IL-1 beta mRNA production in muscle tissue requires optimized extraction and RT-PCR procedures.

Atrophy↗

PubMed2007

Real-time RT-PCR (TaqMan) assays for the detection of Grapevine Leafroll associated viruses 1-5 and 9.

Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Grapevine Leafroll associated viruses 1-5 and 9 (GLRaV-1-5 and -9). The assays were evaluated against a wide range of geographically distributed isolates. Geographical locations included South Africa, Europe, Australia, Asia, Latin America and the United States. Sequences were piled up from the most conserved regions of these geographically diverse isolates and TaqMan primers and probes were designed, targeting the regions with 100% sequence identity. Improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan assay as an RNA specific internal control to validate the quality of the extracted RNA proved to generate better diagnostic assays. The real-time TaqMan RT-PCR assays were compared to the conventional RT-PCR assays for the detection of viruses using purified total RNA as well as crude extract. The data showed that when using total RNA extracted either by the Qiagen RNeasy method or by an ABI automated system more isolates were detected in comparison to crude extract. The optimum volume of crude extract prepared in GES for use in real-time TaqMan RT-PCR cocktail was determined to be 1 microl per reaction. In addition this report showed that TaqMan RT-PCR was more sensitive than conventional one-step RT-PCR for testing different isolates of these viruses either using RNA or crude tissue extract.

Closteroviridae↗

PubMed1998

Optimization of the tyrosinase mRNA probe to detect circulating melanocytes with reverse transcription and polymerase chain reaction.

It was recently suggested that reverse transcription polymerase chain reaction (RT-PCR)-based detection of tyrosinase messenger RNA (mRNA) in peripheral blood is useful in the early detection of circulating tumor cells, since tyrosinase is thought to be a melanocyte-specific marker. However, the sensitivity of detection of these cells in circulation is controversial, some authors reporting 0% effectiveness, others obtaining 100% efficacy. We developed a modification of a technique to process blood samples to detect tyrosinase mRNA, and tested the method with 50 samples from as many patients with histologically confirmed malignant melanoma in different stages. Whole blood was processed by discarding the plasma and extracting RNA from density gradient-isolated peripheral blood lymphocytes. The RNA samples were tested with a sensitive nested primer RT-PCR assay. Sensitivity was tested using RNA extracted from SK-mel-1 human melanoma cells diluted serially with peripheral blood obtained from healthy control subjects. A lymph node from a patient with confirmed disseminated melanoma served as the positive control. Our technique was able to detect tyrosinase mRNA in samples from the 37 patients with progressive metastatic melanoma. The test detected tyrosinase mRNA from both the melanoma cell line and the positive lymph node. Our method to extract RNA from whole blood improves the specificity and sensitivity of tyrosinase mRNA detection by RT-PCR. The test should be of use in determining the prognosis of patients with melanoma, and in deciding when to initiate early treatment in patients with malignant melanoma.

Humans↗

PubMed1978

Transfer of experimental allergic orchitis with immune RNA studies in vivo.

Ribonucleic acid extracts (RNA) obtained from the lymph nodes and spleens of guinea pigs, which were immunized with testicular antigen emulsified in Freund's complete adjuvant (FCA), were injected intraperitoneally into normal guinea pigs. The transferred guinea pigs developed a delayed hypersensitivity to sperm antigens and testicular lesions which resembled the lesions obtained in the donor RNA guinea pigs. When the transfer was performed with RNA extracted from guinea pigs immunized with FCA alone or with 'immune' RNA treated with Ribonuclease, neither cellular immunity nor testicular lesions were observed.

Animals↗

PubMed1990

Actin isoform mRNA alterations induced by doxorubicin in cultured heart cells.

Cultured rat myocardial cells (CMC) were incubated with adriamycin (ADR), an antineoplastic which causes dilated cardiomyopathy (COCM). CMC were exposed to 10(-7) M to 10(-5) M ADR for 24 hours, harvested, and CMC RNA was extracted. Extracted RNA underwent electrophoresis, transfer to nitrocellulose filters, and hybridization with radiolabeled cDNA probes which were specific for the actin isoforms (alpha, beta, and gamma) and isotypes (alpha sk = alpha skeletal; alpha c = alpha cardiac). RNA from CMC exposed to 10(-7) M ADR yielded a strong signal for mRNA coding for alpha c actin when probed with the isotype-specific cDNA. Hybridization signal was reduced in CMC extracts at 10(-6) M ADR. In CMC extracts at 10(-5) M ADR, the alpha c actin mRNA again hybridized. The cDNAs which were specific for beta and gamma actin isoforms yielded hybridization signals in extracts from CMC exposed to 10(-7) M and 10(-6) M ADR. These signals were reduced in extracts of CMC at 10(-5) M ADR. ADR's dose-related effect on the expression of alpha c actin in CMC is specific and suggests that ADR may have effects on regulation of CMC alpha c actin polypeptides and mRNA.

Actins↗

PubMed2006

Comparison of methods for detection of norovirus in oysters.

In the absence of culture methods for noroviruses, detection in foods relies on molecular techniques such as Reverse Transcription-Polymerase Chain Reaction (RT-PCR) on extracted viral RNA followed by PCR product confirmation by hybridisation and/or sequencing. However, in order to obtain a successful detection it is of great importance to remove the tissue inhibitors during the viral RNA extraction. To select the most efficient extraction procedure of oysters we have compared four protocols. A pool of digestive gland material from oyster samples was divided into 1.5 g portions and spiked with 10-fold dilutions of human faecal samples containing norovirus genogroup II. The samples were tested on three different occasions using four different sample treatment protocols. The protocols were assessed with regard to their ability to recover viral RNA and detect norovirus in spiked oysters and for their in-house reproducibility. One method using viral elution by a Mixer Mill Cell Disrupter resulted in a 10-fold better recovery than the other three protocols when an RT-seminested PCR (G2SKR/COG2F and G2SKR/G2SKF) detection approach was applied. Although less distinctive this was also the case when NoV was detected by a single round RT-PCR approach using the primers JV13i and JV12y. The second most efficient method was a method using chloroform extraction and polyethylene precipitation.

Animals↗

PubMed1980

Chromatin-associated RNA: differential extraction and characterization.

Mammalian cells in different states of cytodifferentiation exhibited different RNA-synthesizing and processing patterns that could be used as markers for phenotypic variability. Inherent in these patterns was an RNA class which was differentially extracted from the cellular homogenate by elevating the temperature and pH of the buffer used in the phenol procedure. This class of RNA was initially designated fraction B (chromatin-associated RNA). In the characterization of fraction B, human myeloma cells labeled for 3 and 24 h were fractionated into subcytoplasmic and subnuclear components and the [3H]-RNA was differentially extracted. After 3 and 24 h labeling 84% and 73%, respectively, of the labeled RNA in the chromatin was extracted in fraction B. Only 10-20% of the polysomal RNA was extracted in fraction B with little enrichment in poly(A) RNA. These and other observations suggested that fraction B was a subpopulation of heterogeneous nuclear RNA which was tightly bound to the chromatin complex.

Chromatin↗

PubMed2004

Determination of bovine rotavirus G genotypes in Kashmir, India.

Rotavirus ribonucleic acid (RNA) was extracted from ten faecal samples of diarrhoeic calves positive for group A rotavirus by enzyme-linked immunosorbent assay (ELISA). A portion of the extracted RNA was run in polyacrylamide gel to determine the presence of rotaviral RNA and the rest subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) to generate the near full length VP7 gene. Only six samples yielded the desired product. The amplified products were subjected to G-typing by PCR using a cocktail of G6, G8 and G10 typing primers. All of the six samples were characterised as G10 and none of the samples revealed mixed infection by twin G types. Four samples, despite possessing sufficient rotavirus particles as revealed by ELISA and polyacrylamide gel electrophoresis, did not yield any amplified product on RT-PCR. This could be due to non-specific inhibitors of the PCR reaction, present in the faecal samples, being carried through the extraction procedures.

Animals↗