Lysate RNase protection method for soft tissues

Current Protocols in Neuroscience, 2001

This unit presents four methods for preparing RNA from eukaryotic cells. For all protocols, the yield of RNA from tissue culture cells is dependent on the health of the cells. It is useful to feed or split cells 12 to 24 hours prior to harvesting them for RNA extraction and then be certain that the cells are in the logarithmic phase of growth.

Analytical Biochemistry, 1976

Investigations of the synthesis of pancreatic RNA from ruminants and rodents, such as the rat, guinea pig, and mouse, are handicapped by the prodigious concentration of pancreatic RNase in these species (1). In developmental studies the difficulty is compounded by the small amount of embryonic tissue available (2,3). During the course of an investigation of ribosomal RNA metabolism in the embryonic rat pancreas, we found that published RNA isolation procedures yielded only degraded preparations from both embryonic and adult tissues. We therefore devised a method for preparing undegraded ribosomal pancreatic RNA from tissues containing 1 to 5 pg RNase/mg homogenate protein. We also increased the sensitivity of an existing method for gel electrophoresis of RNA. This study establishes the feasibility of polyacrylamide gel electrophoresis as an analytical tool in the study of stable RNA in embryonic and adult mammalian tissue containing high levels of RNase. MATERIALS Ammonium persulfate and Cyanogum 41 were purchased from Fischer Scientific Co. Stains-All and 3-dimethylaminopropionitrile were purchased from Eastman Organic Chemicals. Agarose was purchased from Kinman Optical Co. L-arginine from Sigma Chemical Co., formamide from Aldrich Chemical Co. L-methionine from Nutrition Biochemicals Corp. ,, and polyvinyl sulfate from General Biochemicals. Fetal calf serum, Eagle's minimal essential medium (4) containing Hanks' balanced salt solution (5), L-glutamine, and antibiotics were obtained from Grand Island Biological Co. [5,3H]Uridine, 25 Ci/mmol and [4,5-3H]L-leucine, 25 Ci/mmol. were obtained from New England Nuclear Corp. Filters (Ultrathin HA, 0.22 pm) were products of the Millipore Filter Corp. Culturing Procedures METHODS Pregnant rats (Spartan Research, Haslett, Mich.) were killed by cervical dislocation followed by decapitation. Adult tissue and embryonic pancreatic rudiments were dissected in Earle's balanced salt solution (6), then were transferred to Eagle's minimal essential medium and cut into pieces approximately 1 mm in diameter, corresponding to 5-15 pg protein. From 50 to 150 pg of tissue pieces were placed on sterile 33-mm filters.

BioTechniques, 1996

2009

Background: The reliability of gene expression profiling-based technologies to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. It strictly depends upon the technique that has been employed. Hence, the present study aimed at systematically comparing silica-gel column (SGC) and guanidine isothiocyanate (GTC) techniques of RNA isolation to answer the question which technique is preferable when frozen, long-term stored or fresh lung tissues have to be evaluated for the downstream molecular analysis. Methods: Frozen lungs (n = 3) were prepared by long-term storage (2.5 yrs) in-80°C while fresh lungs (n = 3) were harvested and processed immediately. The purity and quantification of RNA was determined with a spectrophotometer whereas the total amounted copy numbers of target sequences were determined with iCycler detection system for assessment of RNA intactness (28S and 18S) and fragment sizes, i.e. short (GAPDH-3' UTR), medium (GAPDH), and long (PBGD) with 200 bp, 700 bp, and 1400 bp distance to the 3'ends of mRNA motif, respectively. Results: Total yield of RNA was higher with GTC than SGC technique in frozen as well as fresh tissues while the purity of RNA remained comparable. The quantitative reverse transcriptase-polymerase chain reaction data revealed that higher mean copy numbers of 28S and a longer fragment (1400 bp) were obtained from RNA isolated with SGC than GTC technique using fresh as well as frozen tissues. Additionally, a high mean copy number of 18S and medium fragment (700 bp) were obtained in RNA isolated with SGC technique from fresh tissues, only. For the shorter fragment, no significant differences between both techniques were noticed. Conclusion: Our data demonstrated that although the GTC technique has yielded a higher amount of RNA, the SGC technique was much more superior with respect to the reliable generation of an intact RNA and effectively amplified longer products in fresh as well as in frozen tissues.

Archives of Biochemistry and Biophysics, 1980

Analytical Biochemistry, 2006

The isolation of RNA can be quite diYcult, particularly in plant tissues that are rich in polyphenolics and polysaccharides . These compounds tend to copurify with RNA because they display physicochemical properties similar to those of nucleic acids. Furthermore, RNA degradation by ribonucleases (RNases) 1 signiWcantly reduces the recovery of intact RNA . RNA isolation methodologies are very diverse and guanidine salts are the most used denaturant reactants for RNase inactivation in total RNA puriWcation protocols . Others procedures use combinations of diVerent denaturant agents in order to inactivate RNases, for example, the combination of RNase-inactivating agents urea and LiCl for total RNA isolation from mouse myeloma cells . Tai et al. , who used the same combination of RNase denaturant agents, added some extra puriWcation steps through Qiashredder columns and the addition of polyvinylpyrrolidone for removal of saccharides and phenolics which coprecipitated with the RNA in the isolation of total RNA from plant tissues. Scott et al. introduced a wash with catrimox-14 in order to separate RNA from other macromolecular contaminants that precipitated together with RNA, after a urea LiCl treatment for RNase inhibition in potato tuber tissues. These extra puriWcation steps are time consuming and expensive and decrease the RNA puriWcation yield.

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1989

Porcine spleen DNase Ill (EC 3.1.22.|), one of the best-characterized DNases ll, is subee|lulariy |ocated in |ysosomes because the enzyme is co-sedimented with two of the |ysosoma| market enzymes, eatheps|n D and acid phosphatase. The physicochemica| properties, ine|ud|ng the subunit s~memre, sensitivity to ledoaeetate inactivation, native mo|ecular weight and ehromatoFaphle behavior, of the DNase || purified from the iso|ated |ysosomes of porcine spleen are indistinguishable from those of the same enzyme purified from the whole porcine spleen homogenate. DNase |! can also be extracted from porcine |iver with 0.05 M H2SO 4 or 0.1 M NaC| and purified from either extract by a series of column ehromatographies. The purified fiver DNase || from either extract has the same subunit structure (a-chain, Mr 35000 and ~-chain, M r 10000) as the purified DNase |l of porcine spleen. The two fiver extracts as well as the extracts of spleen and gastric mucosa contain DNase || with very. sim||ar properties on Sephadex G-I~ go| fi|tration, on ac|d polyaerylamide gel e|ectrophoresis under non-denatur|ng conditions, and on isoe|ectric focusing. The data strongly suggest that, for the same species of animal the DNase i| activities in various tissues are associated with protein molecules of identical structure.

The Journal of biological chemistry, 1991

RNase L activated by 2-5A (a series of 2'-5'-linked adenylic oligoribonucleotides) is a key enzyme of the interferon system. To study RNase L (endonuclease L) in intact cells independently of intracellular 2-5A and of its activity, we have developed polyclonal antibodies against RNase L. RNase L from mouse spleen was purified on a column of 2-5A-Sepharose and used to immunize rabbits in co-injection with polyadenylic-polyuridylic acid as adjuvant. Antibodies were purified by chromatography on Affi-Gel blue and 2-5A-Sepharose-immobilized RNase L. These polyclonal antibodies immunoprecipitate the 80- and 40-kDa forms of RNase L in mouse spleen. In Western blot, only the 80-kDa form of RNase L is recognized by these antibodies. These purified antibodies were used to localize RNase L in the cytoplasm of intact mouse NIH 3T3 cells by immunofluorescence. The cytoplasmic localization of RNase L was confirmed by its 2-5A binding activity after cellular fractionation.

Cancer research, 1974

for nondividing and dividing normal and tumor cells have not been performed. We suggest that different sensitivity of cells to the impairing action of alkylating agents and nitrosoureas might result from their differing ability to degrade damaged RNA, proteins, and lipids. Preliminary results of this work have been reported (15). MATERIALS AND METHODS Materials. MNU-l-'4C was synthesized from methyla mine'4C and urea using the method cited by Semenow et a!. (26) and had a specific activity of 445 mCi/mmole. MNU obtained by this method was of high purity (100%); according to nuclear magnetic resonance and polarographic (I I) analyses, it contained no impurities. Millipore filters (0.3-.tm pore diameter) were obtained from Chemapol, Prague, Czechoslovakia. Precipitates were filtered through a filtering device (Millipore Corporation, Bedford, Mass.). Methods. Hepatoma 22a (8) was transplanted s.c. to adult C3HA mice from our colony and the animals were killed 12 to 13 days after the transplantation. MNU was adminis tered i.p., 80 mg/kg body weight, to groups of 4 to 5 mice. The animals were sacrificed I, 5, 19, and 48 hr after the administration. Tumor, liver, and spleen were rapidly removed and frozen in liquid nitrogen. Tissues were homog enized in 3 volumes of Buffer 2, consisting of 0.05 M Tris-HCI, pH 7.6; 0.025 M KCI; 0.005 M MgCl2; and 0.25 M sucrose (1); they were fractionated according to the scheme presented in Chart 1 (see also Refs. 2 and 16). Radioactivity in RNA, DNA, proteins, and lipids of the homogenate and subcellular fractions was determined according to the method of Kennel (12). As shown in special experiments, contamination of lipid fraction by RNA is possible when using this method. Therefore, special corrections were introduced for the determination of RNA and lipid radioac tivity. Chemical determinations of DNA, RNA, and protein in homogenates and subcellular fractions were conducted according to the method of Blobel and Potter (3). Radioac tivity of cellular fractions was usually calculated per mg of DNA in homogenate. Radioactivity was measured using a Mark II scintillation counter (Nuclear Chicago, Chicago, Ill.

Biochemical and Biophysical Research Communications, 1972