2010-Pena_et_al - Technical note Efficient protocol for...

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R. N. Pena, A. Cánovas and J. Estany lyophilized fat and muscle pig samples Technical note: Efficient protocol for isolation of total ribonucleic acid from 2009-10-09T07:35:01-07:00; 2010, 88:442-445.doi: 10.2527/jas.2009-2298 originally published online J ANIM SCI http://jas.fass.org/content/88/2/442 the World Wide Web at: The online version of this article, along with updated information and services, is located on www.asas.org by guest on May 21, 2011 jas.fass.org Downloaded from
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ABSTRACT: Isolation of total RNA from frozen muscle and fat samples typically results in small yields due to the presence of connective tissue between muscle fibers, which impairs complete tissue homogenization, and the excess of fat and relatively small cellularity of adipose tissue. Meat quality studies involve deter- mination of fatty acid composition and content from muscle and subcutaneous fat samples, a process that may produce an excess of lyophilized tissue samples. The purpose of this work was to investigate the stabil- ity of total RNA in lyophilized tissue samples generated during the routine detection of fatty acid content of pig muscle and fat tissues, stored at room temperature or at −20°C. The protocol described here results in in- creased yields of total RNA from freeze-dried samples stored at −20°C, which facilitates the homogenization step. The isolated RNA is suitable for common gene expression techniques such as final point and quantita- tive reverse transcription-PCR. Key words: freeze-dry, gene expression, lyophilization, meat quality, pig ©2010 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2010. 88:442–445 doi:10.2527/jas.2009-2298 INTRODUCTION Fat composition and content are important traits in- fluencing sensory and nutritional meat quality prop- erties. Thus, there is a growing interest in the study of the molecular mechanisms underlying subcutaneous and intramuscular fat deposition events. Many of these studies focus on global or candidate gene expression characterization in muscle and fat tissues. Isolation of RNA from muscle and fat samples is largely inefficient because connective tissue within the muscle samples is difficult to homogenize, even with mechanical rotors, and the large fat content of adipose tissue interferes with the isolation process, whereas its relatively small cellularity results in decreased RNA concentrations. Meat quality studies often rely also on muscle and fat chemical composition analysis, some of which in- volve previous sample lyophilization. Freeze-drying or lyophilization is a widely used technique for sample preservation commonly applied to store vaccines, mi- croorganisms, and plant (Jaiprakash et al., 2003) and virus (Vaughan et al., 2006) samples. Although RNA is an unstable molecule, easily degraded by enzymes and at alkaline environments, viral RNA has been detected from a wide range of freeze-dried biological samples. The RNA is rapidly degraded in shelf-preserved freeze-
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2010-Pena_et_al - Technical note Efficient protocol for...

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