Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
TL;DR: A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described, providing a pure preparation of undegraded RNA in high yield and can be completed within 4 h.
About: This article is published in Analytical Biochemistry.The article was published on 1987-04-01. It has received 65881 citations till now. The article focuses on the topics: Acid guanidinium thiocyanate-phenol-chloroform extraction & Guanidinium thiocyanate.
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TL;DR: Serum leptin concentrations are correlated with the percentage of body fat, suggesting that most obese persons are insensitive to endogenous leptin production.
Abstract: Background Leptin, the product of the ob gene, is a hormone secreted by adipocytes. Animals with mutations in the ob gene are obese and lose weight when given leptin, but little is known about the physiologic actions of leptin in humans. Methods Using a newly developed radioimmunoassay, we measured serum concentrations of leptin in 136 normal-weight subjects and 139 obese subjects (body-mass index, >27.3 for men and >27.8 for women; the body-mass index was defined as the weight in kilograms divided by the square of the height in meters). The measurements were repeated in seven obese subjects after weight loss and during maintenance of the lower weight. The ob messenger RNA (mRNA) content of adipocytes was determined in 27 normal-weight and 27 obese subjects. Results The mean (±SD) serum leptin concentrations were 31.3±24.1 ng per milliliter in the obese subjects and 7.5±9.3 ng per milliliter in the normal-weight subjects (P<0.001). There was a strong positive correlation between serum leptin concentration...
6,350 citations
TL;DR: A simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine, based on the lysing and nuclease-inactivating properties of guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent.
Abstract: We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.
5,445 citations
Cites background from "Single-step method of RNA isolation..."
...cells combined with its potential to inactivate nucleases (6-8, 10, 15, 25, 28)....
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TL;DR: The cloning and expression of a complementary DNA that encodes a G protein-coupled receptor that is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana are suggested.
Abstract: Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.
4,806 citations
TL;DR: The messenger RNA expression of both ER subtypes in rat tissues by RT-PCR is investigated and the ligand binding specificity of the ER sub types is compared, revealing a single binding component for 16β-estradiol with high affinity.
Abstract: The rat estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand binding domain and in the N-terminal transactivation domain. In this study we investigated the messenger RNA expression of both ER subtypes in rat tissues by RT-PCR and compared the ligand binding specificity of the ER subtypes. Saturation ligand binding analysis of in vitro synthesized human ER alpha and rat ER beta protein revealed a single binding component for 16 alpha-iodo-17 beta-estradiol with high affinity [dissociation constant (Kd) = 0.1 nM for ER alpha protein and 0.4 nM for ER beta protein]. Most estrogenic substances or estrogenic antagonists compete with 16 alpha-[125I]iodo-17 beta-estradiol for binding to both ER subtypes in a very similar preference and degree; that is, diethylstilbestrol > hexestrol > dienestrol > 4-OH-tamoxifen > 17 beta-estradiol > coumestrol, ICI-164384 > estrone, 17 alpha-estradiol > nafoxidine, moxestrol > clomifene > estriol, 4-OH-estradiol > tamoxifen, 2-OH-estradiol, 5-androstene-3 beta, 17 beta-diol, genistein for the ER alpha protein and dienestrol > 4-OH-tamoxifen > diethylstilbestrol > hexestrol > coumestrol, ICI-164384 > 17 beta-estradiol > estrone, genistein > estriol > nafoxidine, 5-androstene-3 beta, 17 beta-diol > 17 alpha-estradiol, clomifene, 2-OH-estradiol > 4-OH-estradiol, tamoxifen, moxestrol for the ER beta protein. The rat tissue distribution and/or the relative level of ER alpha and ER beta expression seems to be quite different, i.e. moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ER alpha and prostate, ovary, lung, bladder, brain, uterus, and testis for ER beta. The described differences between the ER subtypes in relative ligand binding affinity and tissue distribution could contribute to the selective action of ER agonists and antagonists in different tissues.
4,367 citations
Cites methods from "Single-step method of RNA isolation..."
...Tissue samples were immediately processed for total RNA isolation according to the acid guanidinium thiocyanate-phenol-chloroform single-step extraction protocol (16)....
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TL;DR: It is demonstrated that the phenotype of APC resistance is associated with hetero-zygosity or homozygosity for a single point mutation in the factor V gene which predicts the synthesis of a factor V molecule that is not properly inactivated by APC.
Abstract: Activated protein C (APC) is a serine protease with potent anticoagulant properties, which is formed in blood on the endothelium from an inactive precursor During normal haemostasis, APC limits clot formation by proteolytic inactivation of factors Va and VIIIa (ref 2) To do this efficiently the enzyme needs a nonenzymatic cofactor, protein S (ref 3) Recently it was found that the anticoagulant response to APC (APC resistance) was very weak in the plasma of 21% of unselected consecutive patients with thrombosis and about 50% of selected patients with a personal or family history of thrombosis; moreover, 5% of healthy individuals show APC resistance, which is associated with a sevenfold increase in the risk for deep vein thrombosis Here we demonstrate that the phenotype of APC resistance is associated with heterozygosity or homozygosity for a single point mutation in the factor V gene (at nucleotide position 1,691, G-->A substitution) which predicts the synthesis of a factor V molecule (FV Q506, or FV Leiden) that is not properly inactivated by APC The allelic frequency of the mutation in the Dutch population is approximately 2% and is at least tenfold higher than that of all other known genetic risk factors for thrombosis (protein C (ref 8), protein S (ref 9), antithrombin10 deficiency) together
3,895 citations
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TL;DR: In this article, the rat pancreas RNA was used as a source for the purification of alpha-amylase messenger ribonucleic acid (RBA) using 2-mercaptoethanol.
Abstract: Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
19,805 citations
TL;DR: The present study arose from the observation that a more intense colour was sometimes produced if, instead of being heated at 1000 for 10 min., the reaction mixture was allowed to stand overnight at room temperature.
Abstract: Of the colour reactions available for the determination and identification of deoxyribonucleic acid (DNA), the reaction with diphenylamine in a mixture of acetic and sulphuric acids at 1000 (Dische, 1930) has been perhaps the most widely used. The present study arose from the observation that a more intense colour was sometimes produced if, instead of being heated at 1000 for 10 min., the reaction mixture was allowed to stand overnight at room temperature. As a result of this observation the procedure has been modified, principally by adding acetaldehyde to the reagents and by allowing the solution to stand for about 17 hr. at 30° instead of heating it at 1000. The modified method is 3-5 times as sensitive as Dische's original procedure, and several substances which interfere in the original method do not do so in the modified procedure. Some observations on the mechanism of the reaction have been made; in particular it was discovered that there is a liberation of inorganic orthophosphate from DNA during the early stages of the reaction. This finding has a bearing on the structure of DNA. The modified method has already been used in an investigation of nucleic acid metabolism during bacteriophage multiplication (Burton, 1955).
13,649 citations
TL;DR: The results show similarity between the patterns of solubilizing effects of guanidine hydrochloride and urea, although Guanidine Hydrochloride is 2 to 3 times more effective than urea at the same concentrations.
334 citations
TL;DR: Two translocations of the human ets gene were found to translocate from chromosome 11 to 4 in the t(4;11)(q21;23), a translocation characteristic of a subtype of leukemia that represents the expansion of a myeloid/lymphoid precursor cell.
Abstract: Human probes identifying the cellular homologs of the v-ets gene, Hu-ets-1 and Hu-ets-2, and two panels of rodent-human cell hybrids were used to study specific translocations occurring in acute leukemias. The human ets-1 gene was found to translocate from chromosome 11 to 4 in the t(4;11)(q21;23), a translocation characteristic of a subtype of leukemia that represents the expansion of a myeloid/lymphoid precursor cell. Similarly, the human ets-2 gene was found to translocate from chromosome 21 to chromosome 8 in the t(8;21)(q22;q22), a nonrandom translocation commonly found in patients with acute myeloid leukemia with morphology M2 (AML-M2). Both translocations are associated with expression different from the expression in normal lymphoid cells of ets genes, raising the possibility that these genes play a role in the pathogenesis of these leukemias.
173 citations
62 citations