Hydrolysis with lead perchlorate: A new method used to estimate RNA in sugarcane tissues

The precipitation and hydrolysis of RNA by lead perchlorate has been studied and the results have been applied to estimate RNA in three widely different tissues from Saccharum species (sugarcane). This new method makes it possible to estimate RNA in highly lignified tissue for which existing methods using cold perchloric acid or warm alkali are unsuitable. As far as comparisons are possible, lead perchlorate gives the same results as extraction with acid and alkali, except that values are slightly lower in rapidly dividing tissue. Acid-soluble materials are removed by a single extraction with 0.01 m Pb(ClO₄)₂ at pH 2, where RNA and DNA are quantitatively precipitated but not hydrolyzed. Specific hydrolysis of RNA is then carried out at neutral pH with a solution containing 3 m Pb(ClO₄)₂ and 2.5 n NaOH, and the products are extracted at pH 2. DNA and most interfering materials remain insoluble. RNA in the extract is measured by uv absorption versus a combined tissue/reagent blank. The entire procedure can be carried out at room temperature. DNA in the tissue can be estimated as usual (1–3).     

Detection of Mycobacterium avium subsp. paratuberculosis in formalin- fixed,paraffin embedded tissues of goats by IS900 polymerase chain reaction

The objective of the present study was to optimise and evaluate a procedure for detection of Mycobacterium avium subsp. paratuberculosis in archived formalin-fixed, paraffin embedded tissue sections from cases of naturally occurring paratuberculosis in goats. A pilot study assessed 3 procedures for extraction of DNA for detection by PCR. The procedure that gave the most consistent results involved removal of paraffin by treatment with xylene and ethanol, disruption of tissue pellets by beating with zirconium/silica beads, extraction of DNA using a DNeasy kit (Qiagen) with overnight proteinase K digestion and final ethanol precipitation. This procedure was used to analyse 82 paraffin embedded tissues (44 small intestine, 38 mesenteric lymph node) with various grades of histological lesions of paratuberculosis and acid-fast bacilli (AFB) loads. The overall sensitivity of the PCR was about 72% of all samples including both paucibacillary and multibacillary lesions. The sensitivity of the assay was 87.5% (42/48) in all paraffin sections having clearly and easily demonstrable AFB. Fifty percent of the tissue sections with rarely detectable AFB were positive by PCR. There was no significant difference (<0.05) between the sensitivity of the PCR analyses carried out on intestinal and mesenteric lymph node tissues. The results of this study suggest that IS900 PCR on formalin-fixed, paraffin embedded histological sections is a practical and important tool for confirming diagnosis of paratuberculosis in goats, where fresh tissues for bacterial culture or PCR are not available due to problem of maintaining a cold chain system.     

Analysis of folate cofactor levels in tissues using high-performance liquid chromatography

A method for the isolation and concentration of the monoglutamate forms of folate cofactors from tissues and for their subsequent separation and quantitation using HPLC coupled with uv detection at 284 nm is described. A chromatographic procedure utilizing Dowex 50 has been developed for the separation of the folate monoglutamates from a large portion of the nonfolate-related material following digestion of the polyglutamated froms with a highly purified preparation of rat liver conjugase. This chromatographic procedure combined with concentration of the Dowex eluate by lyophilization eliminates uv-absorbing material, which interferes with the detection and quantitation of the folate cofactors and makes possible uv measurement of the individual folates. Reverse-phase paired-ion chromatography on μBondapak C₁₈ coupled with uv detection allows direct quantitation of the folates in the nanogram range.     

High-quality plant DNA extraction for PCR: an easy approach

Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies. For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires maceration of plant tissue of about 1.0 cm² (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes, followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20–30 μg cm⁻² for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5–1.8. The DNA is quite suitable for PCR using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive.     

Spectrophotometric analysis of RNA and DNA using cetyltrimethylammonium bromide

A versatile procedure is described for the analysis of RNA and DNA in brain using cetyltrimethylammonium bromide as the initial precipitant. Optimal conditions are described for the precipitation, hydrolysis, and effective separation of the RNA and DNA fractions from contaminating protein. The RNA and DNA fraction can now be accurately estimated by uv absorbance without a two wavelength correction. This method has also been used for the analysis of other mammalian organs and for mammalian cells obtained from tissue culture. The method may also be used for the simultaneous determination of radioactivity in nucleic acids. The orcinol reaction is shown to give high values for brain RNA.     

Simultaneous determination of tocopherols,ubiquinols, and ubiquinones in blood,plasma, tissue homogenates,and subcellular fractions

A fast single-step lipid extraction procedure and high-performance liquid chromatography with in-line uv and electrochemical detection are used for the simultaneous quantitative determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. The compounds of interest can be quantitatively extracted into hexane from a sodium dodecyl sulfate-treated aqueous homogenate after precipitation of protein by addition of an equal volume of ethanol. α-, γ-, and δ-Tocopherol, ubiquinol 9, ubiquinol 10, and ubiquinones 9 and 10 can be well separated on a reversed phase column. Ubiquinones are detected at 275 nm by the uv detector, and ubiquinols and tocopherols by the electrochemical detector in the oxidative mode. Quantitation is done by comparing chromatographic peak heights to those of a standard solution containing known amounts of tocopherols, ubiquinols 9 and 10, and ubiquinones 9 and 10, analyzed under identical conditions. The high sensitivity of the electrochemical detection allows operation at low potentials (+0.5 V) with low detector response, but high selectivity for the easily oxidizable tocopherols and ubiquinols and decreased baseline noise. The uv detection limits the overall sensitivity of the procedure to 2 pmol ubiquinone, corresponding to 0.1 μm ubiquinone in the lipid extract. The ranges of values obtained for rat and guinea pig tissues, for rat liver mitochondria, and for blood and plasma from rats and humans are given.     

Kinetic characterization of unspecific alkaline phosphatase at different villus sites of rat jejunum

Summary A quantitative histochemical method to determine the apparent Km and V _max values of rat intestinal unspecific alkaline phosphatase at different sites of the villi is described. Naphthol-As-Bi-phosphate (0.025–1.5 mM) is employed as substrate and Fast Blue B as coupling reagent, and the resulting azo-dye in the brush border membrane has an absorbance maximum at 550 nm. The ratio between the absorbance at 550 and 500 nm is constant as calculated from automatically recorded spectra at different intense dye deposits. Its absorbance is a linear function of incubation time up to 3 min and thickness of the slices up to 10 m both with medium (0.5 mM) and high (1.5 mM) substrate concentrations. Using the histochemical assay under comparable conditions in test tube experiments with homogenates of intestinal mucosa an app. Km of 0.26±0.081 mM (weighted regression analysis) and 0.28–0.084 mM (direct linear plotting) is determined, demonstrating an affinity to the histochemical substrate, which is about 10 times higher than for p-Nitro-phenyl-phosphate with the purified enzyme.The results obtained by scanning the total dye deposits along jejunal villi show considerable differences in enzymatic activity between single villi and an increase from the villus base up to the transition between medium and apical villus third. As well in the apical region as at the villus base saturation curves are obtained by determining the relationship between the absorbance and the substrate concentration under standard conditions (slice thickness 10 m, incubation time 3 min, 37°C, pH 8.3). Calculated by weighted regression analysis and direct linear plotting from the absorbance data of six female rats the medium app. kinetic data ±SD from the jejunal villi read as follows. Apical: Km=0.81±0.438 mM, V _max=3.99±1.217 absorbance units (A) and Km=0.87±0.428 mM, V _max=4.02±1.191 A, respectively. Basal: Km=0.82±0.261 mM, V _max=3.26±0.719 A and Km=0.77±0.184 mM, V _max=3.04±0.518 AU, respectively. As demonstrated by factorial analysis of variance only V _max is influenced by the villus position.Supported by the Deutsche Forschungsgemeinschaft GU 184/1     

Parameters affecting substance P measurement in heart, lung, and skin

Substance P (SP), a neuropeptide that is widely distributed both peripherally and centrally, mediates several pathophysiological processes. Among current assays for SP, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) have been most widely used. Several previous studies, mostly performed with nerve extracts or organ perfusates, determined that acidity of the extraction buffer as well as the number extractions performed constitute factors influencing accurate measurements. We used an ELISA protocol in this study to analyze methodological aspects of SP measurement in extracts from heart, skin, and lung. The extraction procedure had two steps, an acid extraction followed by a column extraction. We could effectively measure SP with extract from as little as 10 mg of tissue. For each tissue examined, different variables influenced the SP measured. For all tissues, the weight of tissue extracted was critical; the more tissue extracted, the lower the sensitivity of the assay. This problem could be overcome in skin by omitting the column extraction. When mechanical loses were considered (e.g., loss during extraction and SP retained by the column after elution), column extraction improved SP measurements only with lung tissue. The amount of SP remaining in the sample after the first extraction also varied among tissues. The first acid extraction effectively isolated 80% of total SP from skin. In contrast, the first extraction with lung tissue recovered only 58%. Because both acid and heat effectively release SP from nerve endings, this could reflect the presence of non-neuronal SP, especially in lung. High-dose capsaicin treatment, which depletes SP in nerve endings, caused 42% loss of SP in skin independent of amount of tissue extracted Our results suggest that a second acid extraction of tissue should be performed and that column extraction is clearly detrimental with skin samples.     

A rapid high-performance liquid chromatographic procedure for the simultaneous determination of methionine, ethionine, S-adenosylmethionine, S-adenosylethionine, and the natural polyamines in rat tissues

A method for the analysis of S-adenosyl-L-methionine (SAM) and S-adenosyl-L-ethionine (SAE) and their major metabolites by high-performance liquid chromatography is described. The procedure allows the simultaneous analysis of the natural polyamines, putrescine, spermidine, and spermine, and some of the major amino acids, methionine, tyrosine, and tryptophan. The uv absorbance at 254 nm is used for the determination of the SAM and SAE analogs, whereas the polyamines and amino acids are analyzed by fluorescence detection after postcolumn derivatization with o-phthalaldehyde. The method allows SAM and polyamine determinations by direct injection of the tissue extracts without prepurification. The procedure is applied to study the effects of DL-ethionine treatment on the SAM, SAE, methionine, and polyamine levels in various tissues of rats.     

A sensitive and precise isotopic assay of ATPase activity

A manual ATPase assay which measures the release of ³²P_i from [γ-³²P]ATP is described. Sodium dodecyl sulfate is used to terminate the enzyme reaction and extraction of the phophomolybdate complex into xylene: isobutanol is used to separate ³²P_i from [γ-³²P]ATP for quantitation by scintillation counting. The three-step assay is rapid (75–90 samples/h) and minimizes hydrolysis of ATP due to exposure to acidie conditions. The extraction procedure separates 10⁻¹⁵ to 10⁻⁷ mol of ³²P_i from aqueous solution with an efficiency of 100,7 ± 0.62%. Less than 1% of unhydrolyzed [γ-³²P]ATP is extracted. Extraction efficiency is not affected by protein or salts commonly present in enzyme incubation mixtures. Results obtained with this assay are precise, with an intraassay coefficient of variation of 0.6% and an interassay coefficient of variation of 1.8%. The results are comparable to results obtained with a spectrophotometric assay, with a correlation coefficient of 0,996, though assay performance and sensitivity are greatly improved with the isotopic assay.     

Rapid High Quality RNA Preparation from Pine Seedlings

Conventional RNA extraction methods have been shown to produce poor quality RNA when applied to pine and other gymnosperms. We present a protocol for extracting highly pure RNA from pine. Modifications to conventional procedures include: 1) the use of seedlings, 2) the use of phenol and PVP to rapidly remove DNA, proteins and pigments, and 3) the use of salt precipitation to remove other contaminating compounds. The procedure can be completed in less than eight hours. Yield and purity were monitored by denaturing gel electrophoresis and by UV absorbance (A₂₆₀ /A₂₈₀ and A₂₆₀/A₂₃₀). These ratios were over 2, indicating an absence of contaminating metabolites. Additionally, a new absorbance ratio (A₂₆₀/A₂₁₀) was introduced to monitor the RNA purity in each step (it indicates the ratio of covalent links in the solution belonging to RNA). The yield was around 300 µg total RNA per gram of tissue of Pinus sylvestris and over 400 µg of total RNA per gram of P. pinaster tissue, which is a high recovery (more than 63%) for gymnosperms. The RNA was of sufficient quality for use in a RT-PCR reaction that amplified 1 kb of the pine GS gene. This protocol has been applied with success to other woody plants like Populus species.Abbreviations: DEPC, diethyl pyrocarbonate; AMV, avian myeloblastosis virus; FW, fresh weight.     

A rapid ion exchange procedure that facilitates spectrophotometric assays of phosphorylated metabolites in potato extracts

A variety of metabolites are routinely assayed after perchloric acid extraction of plant tissues. A common technique uses coupled enzyme assays that produce or consume pyridine nucleotides together with spectrophotometric detection at 340 nm. Because of the presence of pigments in plant tissues, the high absorbance of such extracts usually limits the amount of extract that can be assayed spectrophotometrically. Here, we show that after batch adsorption with AG50WX8 (H⁺ form), the absorbance of potato root perchloric acid extracts at 340 nm is significantly reduced. This clean up procedure does not interfere with the assay or the recovery of anionic metabolites such as hexose phosphates. It therefore facilitates spectrophotometric assays of metabolites in plant extracts with high absorbance.     

Isolation of functional RNA from plant tissues rich in phenolic compounds.

A method for the isolation of RNA from different tissues of trees (seedlings, saplings, and adult trees) is described. Using this procedure it is possible to remove large amounts of disturbing polyphenolic compounds from nucleic acids. The method involves an acetone treatment of the freeze-dried and powdered plant material, the use of high salt concentrations in the extraction buffer and an aqueous two-phase system. These steps were combined with the conventional phenol/chloroform extraction and CsCl centrifugation. The method has been successfully applied to the isolation and purification of RNA from pine (Pinus sylvestris L. and Pinus mugo Turr.), Norway spruce (Picea abies L.), and beech (Fagus sylvatica L.). The functional quality of RNA extracted by this procedure has been characterized by its uv spectrum, by agarose gel electrophoresis with ethidium bromide staining, Northern blot hybridization, and in vitro translation.     

Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.     

Quantitation of biological retinoids by high-pressure liquid chromatography: primary internal standardization using tritiated retinoids

A single method is described for quantitation of 14 retinoids found in biological material. The method consists of reversed-phase HPLC, internal standardization, and carrier extraction procedures with three synthetic retinoids. Primary standardization of HPLC uv detector is achieved using tritiated all-trans-retinoic acid, all-trans-retinol, all-trans-retinyl palmitate, and all-trans-retinyl acetate. Extraction methods are standardized by correlating the uv absorbance of retinoids at 340 nm with radioactivity of tritiated retinoids of known specific activity. Quantitation of 10 pg of tritiated or 5 ng of nonradioactive retinoid per 0.1 g sample in a polarity range from 4-oxo-retinoic acid to retinyl stearate can be achieved in a single, 50-min chromatographic run. A single HPLC pump, a C18 reversed-phase analytical column, a multistep three-solvent gradient, and inexpensive solvents based on methanol, water, and chloroform comprise this cost-effective chromatographic system. Our primary standardization method allows investigators employing different procedures to compare results between laboratories by standardizing the HPLC uv detector with commercially available tritiated retinoids. With this method we were able to quantitate nanomolar amounts of endogenous retinoic acids and retinyl esters, that "HPLC uv only" conditions usually would not detect in the circulation and liver of rats under physiological conditions.     

High-pressure liquid chromatographic determination of 13-cis-retinoic acid and all-trans-retinoic acid in human plasma

A simple and selective method is described for the determination of 13-cis-retinoic acid and all-trans-retinoic acid in plasma. The assay involves lyophilization of the sample, extraction of the residue with methanol, and separation of the retinoids by reverse-phase high-pressure liquid chromatography. The procedure can detect as little as 0.05 μg of either compound. It is capable of being readily modified to determine the amount of retinoic acid in other tissues as well as tissue concentrations of other retinoids. Using this assay, the absorption and disappearance from human plasma of an oral dose of 13-cis-retinoic acid have been followed.     

A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with alpha-ketoglutarate results in formation of alpha-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between alpha-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (alpha-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure.     

1-Acyl-2-[N-(2,4-dinitrophenyl)aminopropionyl]-sn-glycero-3-phosphocholine as a chromogenic substrate for phospholipase A2 assay

We have developed a spectrophotometric assay for phospholipase A₂ activity using 2,4-dinitrophenyl-labeled phosphatidylcholine as substrate. The assay allows quite simple quantification of phospholipase A₂ activity by measuring the absorbance of the aqueous phase after extraction of the reaction mixture and requires neither chromatographic separation of the reaction products nor the addition of auxiliary coloring reagents.     

A method to reduce interference by sucrose in the detection of thiobarbituric acid-reactive substances

A thiobarbituric acid (TBA) reaction for measuring lipid peroxidation products was evaluated for interference by several ingredients commonly used in solutions to prepare or analyze tissue homogenates or subcellular organelles. These included sucrose (up to 100 mm final concentration in the assay medium), Tris-maleate (up to 40 mm), imidazole (up to 20 mm), inorganic phosphate (up to 10 mm), and 4-morpholinepropanesulfonic acid (up to 20 mm). When the samples were heated at 95°C as recommended in some procedures, only sucrose significantly affected color development. Sucrose concentrations as low as 10 mm significantly increased absorbance at 532 nm of aqueous tetramethoxypropane (TMP) standards, and so the assay could not be applied reliably to tissue samples prepared in sucrose. Sucrose interference was only partially reduced by subsequent organic extraction (n-butanol plus pyridine), with measured absorbances remaining significantly greater (50–100%) than sucrose-free controls at sucrose concentrations of 20 mm or more. Modifying the assay to include sucrose in blanks and TMP standards failed to adequately correct for interference when the absorbance of unextracted (aqueous) solutions was measured. Further modification by adding sucrose to blanks and TMP standards, followed by butanolpyridine extraction, gave standard curves that were linear, through the origin, and had slopes equivalent to those of sucrose-free standards. This modification enabled almost complete recovery (average 2% error) of known amounts of TMP added to aliquots of tissue homogenates containing amounts of sucrose that otherwise significantly interfered. Also, with the modified method the content of TBA-reactive substances in tissues homogenized in sucrose was found to be not significantly different from that measured in tissues homogenized in a noninterfering substance, KCl.     

A fluorometric method for the kinetic assay of indole-3-acetic acid oxidase.

The oxidation of IAA by peroxidase (1) and by more specific oxidases (2) leads to the formation of products which may have physiological activity (3, 4). The colorimetric estimation of IAA oxidation products involving reaction with p-dimethylaminocinnamaldehyde (DMACA) is reported to be more sensitive than other end point determinations such as the Salkowski and Ehrlich procedures which monitor the disappearance of IAA (5). These methods are end point procedures and, as such, are awkward and time consuming and present difficulties in obtaining kinetic data and measuring lag times. IAA oxidation has also been monitored by measuring ¹⁴CO₂ released from [1-¹⁴C] IAA (6) and uv spectral shifts during oxidation of IAA were reported by Meudt (3). The present paper reports a new procedure for the assay of horseradish peroxidase catalyzed oxidation of IAA. The assay procedure is based on the continuous measurement of a fluorescent product of the reaction.