RNASwift is an inexpensive, versatile method for the rapid extraction of RNA. Existing RNA extraction methods typically use hazardous chemicals including phenol, chloroform and formamide which are often difficult to completely remove from the extracted RNA. RNASwift uses sodium chloride and sodium dodecyl sulphate to lyse the cells and isolate the RNA from the abundant cellular components in conjunction with solid phase extraction or isopropanol precipitation to rapidly purify the RNA. Moreover, the purified RNA is directly compatible with downstream analysis. Using spectrophotometry in conjunction with ion pair reverse phase chromatography to analyse the extracted RNA, we show that RNASwift extracts and purifies RNA of higher quality and purity in comparison to alternative RNA extraction methods. The RNASwift method yields approximately 25 µg of RNA from only 10 8 Escherichia coli cells. Furthermore, RNASwift is versatile; the same simple reagents can be used to rapidly extract RNA from a variety of different cells including bacterial, yeast and mammalian cells. In addition to the extraction of total RNA, the RNASwift method can also be used to extract double stranded RNA from genetically modified E. coli in higher yields compared to alternative methods.
UV absorbance spectrophotometry is widely used for the quantification of nucleic acids. For accurate quantification, it is important to determine the hypochromicity of the oligonucleotide or complex nucleic acid structure. The use of thermal denaturation studies in conjunction with UV spectrophotometry to determine hypochromicity requires prolonged, elevated temperatures, which may cause partial hydrolysis of RNA. In addition, dsRNA is difficult to denature even at elevated temperature, and the extinction coefficients of nucleic acids are also affected by temperature, which makes it difficult to accurately determine the nucleic acid concentration. To overcome these caveats, we have utilized the chemical denaturant dimethyl sulfoxide which, in conjunction with a short thermal denaturation, prevents renaturation of the duplex nucleic acids (dsDNA/RNA). Using this approach, we have measured the absorbance of both the unstructured and structured nucleic acids to accurately measure their hypochromicity and determine their extinction coefficients. For a range of different dsRNA, we have for the first time determined values of 46.18-47.29 μg/mL/A for the quantification of dsRNA using UV spectrophotometry. Moreover, this approach enables the accurate determination of the relative proportion of duplex nucleic acids in mixed ds/ss nucleic acid solutions, demonstrating significant advantages over current methods.
Highlightsrapid purification of dsRNA in a single step protocol.high throughput purification and analysis of a wide range of dsRNAs.developed IP RP HPLC for the rapid, high resolution analysis of the dsRNA.developed a novel method utilising RNase T1 for RNase mass mapping of dsRNA.
Spinosad is a natural product with biological activity against a range of insects including lepidoptera. It is comprised of two major components namely spinosyns A and D. The degradation of spinosad in soil under aerobic conditions was investigated using two U.S. soils (a silt loam and a sandy loam) which were treated with either 14 C-spinosyn A or -spinosyn D at a 2X use rate of 0.4mg/kg soil for spinosyn A and 0.1mg/kg for spinosyn D. Further samples of soil were pre-sterilised prior to treatment in order to establish whether spinosyns A and D degrade abiotically. Flasks of treated soil were incubated in the dark at 25°C for up to one year after treatment. 477 478 HALE AND PORTWOOD HPLC and LC-MS of soil extracts confirmed that the major degradation product of spinosyn A was spinosyn B, resulting from demethylation on the forosamine sugar. Other dégradâtes were hydroxylation products of spinosyns A and B, with hydroxylation probably taking place on the aglycone portion of the molecule. Half lives were similar for both spinosyns and were in the range 9-17 days, with longer half lives in the pre-sterilised soils (128 -240 days) suggesting that degradation was largely microbial.
The effect of extrusion cooking of a bran-flour mixture on iron and zinc retention was measured in normal adults. The stable isotopes 58Fe (1.253 mg) and 67Zn (5.13 mg) were administered with 40 g nonextruded or extruded cereal with milk and isotopic retention was measured from fecal excretion over the next 4-7 d by neutron-activation analysis (Fe) and fast-atom-bombardment mass spectrometry (Zn). 58Fe retention was 15.1 +/- 2.4% (means +/- SEM) with the nonextruded meal and 16.5 +/- 2.7% with the extruded meal. 67Zn retention was 18.9 +/- 1.7% with the nonextruded meal and 18.3 +/- 1.5% with the extruded meal. Extrusion cooking had no effect on 58Fe or 67Zn retention.
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