Lipoxygenases in the Rat Spinal Cord: Effect of Different Extraction Methods on the Yield of RNA and Protein
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- Category: Vol. 79, March 2011
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Lipoxygenases in the Rat Spinal Cord: Effect of Different Extraction Methods on the Yield of RNA and Protein,SALLY A.M. ABD EL-MOATY, YASSER M. MOUSTAFA, EL-SAYED E. EL-AWADY, ALAA EL-DIN S. ABD EL-HAMID, CAMILLA SVENSSON and ERNST BRODIN
Abstract
Introduction and Aim of the Work: Lipoxygenases play an important role in the synthesis of pro-inflammatory and anti-inflammatory lipid mediators; it is important therefore to find the optimal methods for the detection of their protein and RNA expression. Several techniques are employed to isolate protein and RNA from biological samples; these techniques might affect the quality of the isolated target. Accordingly, this study compared the effect of different extraction methods on the yield of protein and RNA from the rat spinal cord and on the ultimate detection of lipoxygenases.
Material and Methods: Lumbar spinal cords were collected from rats following the induction of peripheral inflammation with carrageenan. Spinal cord samples were thereafter sub-jected to simultaneous RNA and protein extraction using a commercial kit made for this purpose, the PARIS kit, or they were individually isolated using specific reagents, Trizol for RNA and a standard protein extraction buffer for protein. Levels of RNA and protein were measured using spectropho-tometry. Lipoxygenase mRNA and protein levels in the samples were measured using real time quantitative polymerase chain reaction and Western blotting respectively.
Results and Conclusions: While a higher protein yield was obtained using the PARIS kit the final quality of the lipoxygenases bands was not different following the different isolation techniques. However, although the RNA levels were not significantly different between the two different techniques, the quality indices of RNA isolated by the PARIS kit was significantly lower leading to compromised mRNA measure-ments. Accordingly, this study does not recommend the use of the PARIS kit for the isolation of protein and RNA aiming at determination of lipoxygenases levels.