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Results for “RNA extraction”

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At least 217 records · Page 12Linked to original sources
PubMed1999

A rapid novel method for the extraction of RNA from wild-type and genetically modified kanamycin resistant mycobacteria.

RNA extraction from mycobacteria can be a difficult and time consuming process due to their unique mycolic acid cell wall composition and the short half life of RNA. Rapid cell wall disruption is essential to isolate intact high quality RNA. Genetically modified Mycobacteria smegmatis resistant to kanamycin demonstrate an altered cell wall composition which impairs the quality of RNA obtained from these mycobacteria. In this report we describe a method of RNA extraction using the detergent Catrimox-14 resulting in high yields of pure, undegraded RNA in less than 1 h. Yields of 25-30 microg RNA per 1x10(9) cells were consistently obtained from both wild-type and genetically modified M. smegmatis. The integrity of this RNA is demonstrated by gel electrophoresis, Northern blot and cDNA analysis.

Blotting, Northern↗
PubMed1971

Eression of allelic immunoglobulin in homozygous rabbits injected with RNA extract.

Ribonucleic acid extracts obtained from lymph nodes of immunized rabbits homozygous for the b(4)or b(5)allele of light chain immunoglobulin allotypes were injected intravenously into nonimmunized rabbits homozygous for the alternate allele. Approximately 30 percent of the plaque-forming cells in the spleen yielded plaques with immunoglobulin M antibody possessing the allotype of the RNA donor. The allotype of the RNA donor was also found in the IgG immunoglobulin of lymphoid cell lysates as well as in the IgG isolated from the serum. These results suggest that the injected RNA has an informational role in the in vivo synthesis of immunoglobulins by host lymphoid cells.

Alleles↗
PubMed1973

Transcription of simian virus 40. II. Hybridization of RNA extracted from different lines of transformed cells to the separated strands of simian virus 40 DNA.

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and "revertants" isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and "revertant" cell lines. From 30 to 80% of the "early" strand and from 0 to 20% of the "late" strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.

Animals↗
PubMed2005

Comparison of 9 different PCR primers for the rapid detection of severe acute respiratory syndrome coronavirus using 2 RNA extraction methods.

The sensitivity and specificity of various severe acute respiratory syndrome coronavirus (SARS-CoV) PCR primer and probe sets were evaluated through the use of commercial kits and in-house amplification formats. Conventional and real-time PCR assays were performed using a heat-block thermocycler ABI 9600, the Roche LightCycler version 1.2, or the ABI 7000 Sequence Detection System. The sensitivity of all primers was between 0.0004 and 0.04 PFU with viral cell lysate and between 0.004 and 0.4 PFU in spiked stool specimen per PCR assay. The primer sets for real-time PCR assays were at one least 1 log more sensitive than the primer sets used in the conventional PCR. A panel of viruses including swine gastroenteritis virus, bovine coronavirus, avian bronchitis virus (Connecticut strain), avian bronchitis virus (Massachusetts strain), human coronaviruses 229E and OC43, parainfluenza virus (type III), human metapneumovirus, adenovirus, respiratory syncytial virus, and influenza A were tested by all assays. All real-time PCR assays used probe-based detection, and no cross-reactivity was observed. With conventional PCR, analysis was performed using agarose gel electrophoresis and multiple nonspecific bands were observed. Two commercial extraction methods, magnetic particle capture and silica-based procedure were evaluated and the results were comparable. The former was less laborious with shorter time for completion and can easily be adapted to an automated system such as the MagNa Pure-LC, which can extract nucleic acid from clinical samples and load it into the sample capillaries of the LightCycler. As exemplified by this study, the continued refinement and evaluation of PCR procedures will greatly benefit the diagnostic laboratory during an outbreak of SARS.

Animals↗
PubMed2001

Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies.

The archives of departments of pathology represent a unique source of morphologically defined biopsies derived from normal and pathologically altered tissues for which extensive clinical data are available. The exact quantification of nucleic acids in these biopsies offers a promising extension of current methodology to study the pathogenesis of many different diseases. The development of real-time PCR technology has greatly facilitated the realization of nucleic acid quantification. Now it is feasible to analyze large series of samples for the exact quantification of nucleic acids even if the number of target molecules is small and the amount of material available for analysis is limiting. This review focuses on our own experiences concerning the extraction of nucleic acids from fixed and embedded biopsies using both conventional approaches and laser-assisted microdissection and the subsequent application of real-time PCR methods for quantification of mRNA transcripts, gene copy number, and the methylation status. We provide a number of protocols to assist in the application of these techniques.

Biopsy↗
PubMed1992

[RNA extraction, in vitro translation and two-dimensional analysis of oncoproteins in cancers of the upper respiratory and digestive tracts].

After a review of the literature concerning oncogenes expression of in head and neck carcinomas, the authors studied RAN extraction, in vitro transduction and two dimensional analysis of oncoproteins in head and neck carcinomas. The results between the tumoral tissue and normal tissue were compared significantly more oncoproteins spots were found in the tumoral tissue analysis.

Autoradiography↗
PubMed1987

[In vitro translation of RNA extracted from corpuscules of Stannius of the eel (Anguilla anguilla); identification of the precursor of parathyrin in the corpuscules of Stannius (PCS)].

Corpuscles of Stannius (CS) of Teleosts secrete a PTH like hormone (Parathyrin of CS: PCS) which is involved in calcium metabolism by acting mainly at the gill level. Translated products encoded by mRNA extracted from Eel Corpuscles of Stannius and among these, translated products immunoprecipitated by anti b-PTH immunserum were compared to the products synthetized during incubation of the glands with labelled methionine. The analysis was performed by polyacrylamide gel electrophoresis and fluorography. Biosynthesis of the PCS involves a mRNA encoding for a 45 kD precursor which is immunoprecipitated by anti b-PTH immunserum and totally displaced from antibody, either by 1-34 h-PTH or by purified PCS.

Anguilla↗
PubMed2005

Gene expression profiling using RNA extracted from whole blood: technologies and clinical applications.

Gene expression profiling promises to provide an insight into normal biologic and pathologic processes with the hope of predicting disease outcome or indicating individualized courses of therapy. The entire process for gene expression profiling from clinical samples consists of sample collection, transport and storage of clinical sample, isolation of nucleic acids, enzymatic modification of nucleic acids, detection and data analysis. All steps exert an influence on the quality, accuracy and reliability of the final result. A standardization of the entire process from sample collection to nucleic acid analysis is therefore required in order to achieve reliable gene expression results. After providing a general overview of technologies for gene expression profiling and respective caveats, this review will focus on clinical applications of blood sample profiling.

Acute Disease↗
PubMedDate not supplied

RT-PCR amplification of RNA extracted from formalin-fixed, paraffin-embedded oral cancer sections: analysis of p53 pathway.

We present a new approach towards the detection of the mRNAs in formalin-fixed, paraffin-embedded samples using a reverse transcriptase (RT)-polymerase chain reaction (PCR). The total RNAs were extracted from 10-micron-thick sections and were reverse-transcribed, then the RT-products were subjected to PCR amplification of GAPDH mRNA for screening the mRNA degradation. Next, nested PCR was performed for examining the expression of p53-related genes, p21WAF1, MDM2, p33ING1 and p14ARF. GAPDH mRNA expression was detectable in 12 out of 21 oral squamous cell carcinoma (SCC) samples. p21WAF1 mRNA expression was detectable in 5 out of 12 SCC samples, MDM2 mRNA expression was detectable in 5 our of 12 SCC samples and p33ING1 mRNA expression was detectable in 6 out of 12 SCC samples. However, the expression of p14ARF mRNA was not detectable in any of the samples. Seven out of 12 oral SCC samples showed abnormal nuclear accumulation of p53 protein by immunohistochemical staining, whereas 5 out of 12 oral SCCs showed negative staining for p53 protein. Of of p33ING1 mRNA. One of these was a verrucous carcinoma in which the p53 gene products might be inactivated by the oncoprotein E6 of human papilloma virus. Thus, the p53 tumor suppressor pathway was disrupted in most oral SCCs at the cellular levels, due to either an abnormality in p53 itself or loss of expression of p53 regulatory factors. This method would assist in making diagnosis, determining therapeutic strategy and predicting the prognosis of various cancers including oral SCCs.

Animals↗
PubMed1993

Simple method for extracting RNA from cultured cells and tissue with guanidine salts.

We have developed a simple protocol for isolating RNA from both cell culture and tissue from human and animal sources, using guanidine thiocyanate and guanidine hydrochloride, but no organic solvents. The protocol reproducibly yielded 15 to 25 micrograms of high-quality RNA per 10(6) cells of human and animal origin and 1 to 1.1 mg of RNA per gram of human placental tissue. The RNA so obtained was ribonuclease-free and not contaminated by DNA. It was suitable for reverse transcription-polymerase chain reaction, Northern blot analysis, and in vitro expression of proteins. Thus, the molecular assessment of both research and clinical samples can be readily and reliably initiated by the application of this protocol.

Animals↗
PubMed1983

Efficient extraction of RNA from mammalian tissue.

RNA extraction from mammalian tissue has been compared using the different deproteinizing agents: a) guanidine-HCl, b) guanidinium-thiocyanate, c) buffer-saturated phenol, or d) buffer-saturated phenol followed by a proteinase K digestion of the aqueous phase. Both solid tissues (first, second, and third trimester fetal bovine pancreas), and human white blood cell populations were studied. Degradation, as seen in citric acid-urea agarose gels, and the ability to serve as templates for cell-free protein synthesis were used as criteria to assess the efficiency of the different methods. We conclude that employing buffer-saturated phenol with proteinase K digestion is a superior method for consistent extraction of relatively undegraded RNA in quantitative amounts from mammalian tissue.

Animals↗