Brannan Jaime L, Holman Patricia J, Olafson Pia U, Pruett John H, Riggs Penny K
Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas 77843-4467, USA.
J Parasitol. 2013 Feb;99(1):19-23. doi: 10.1645/GE-3132.1. Epub 2012 Aug 27.
Molecular investigations of the ruminant response to ectoparasites at the parasite-host interface are critically dependent upon the quality of RNA. The complexity of ruminant skin decreases the capacity to obtain high quality RNA from biopsy samples, which directly affects the reliability of data produced by gene expression experiments. Two methods for isolating total RNA from skin were compared and the use of 4M guanidinium isothiocyanate (GITC) during frozen storage of the specimens was evaluated. In addition, the best procedure for RNA isolation from bovine skin punch biopsies was also tested on white-tailed deer skin biopsies. Skin biopsy punches were collected and frozen prior to pulverization for RNA isolation. Total RNA quantity and integrity were determined by spectrophotometry and capillary electrophoresis technology, respectively. Significantly increased total RNA yield (P < 0.05) and higher integrity (P < 0.05) were obtained with a TRI Reagent® isolation method. Freezing and subsequent storage of bovine skin punch biopsies in 4 M GITC did not affect the amount or integrity of total RNA recovered by either RNA isolation method. However, quantity and integrity of total RNA extracted with the TRI Reagent method were again significantly higher than with the alternate technique, confirming it as the superior method. The TRI Reagent isolation method also yielded high quality total RNA from white-tailed deer skin punch biopsies, suggesting the usefulness of this method for obtaining RNA of a quality suitable for gene expression studies in other ruminant species.
在寄生虫-宿主界面处对反刍动物对外寄生虫反应进行分子研究，严重依赖于RNA的质量。反刍动物皮肤的复杂性降低了从活检样本中获取高质量RNA的能力，这直接影响了基因表达实验所产生数据的可靠性。比较了两种从皮肤中分离总RNA的方法，并评估了在标本冷冻保存期间使用4M异硫氰酸胍（GITC）的情况。此外，还在白尾鹿皮肤活检样本上测试了从牛皮肤打孔活检样本中分离RNA的最佳程序。在粉碎用于RNA分离之前，收集皮肤活检打孔样本并冷冻。分别通过分光光度法和毛细管电泳技术测定总RNA的量和完整性。使用TRI Reagent®分离方法可显著提高总RNA产量（P < 0.05）并具有更高的完整性（P < 0.05）。将牛皮肤打孔活检样本冷冻并随后保存在4M GITC中，并不影响通过任何一种RNA分离方法回收的总RNA的量或完整性。然而，用TRI Reagent方法提取的总RNA的量和完整性再次显著高于另一种技术，证实它是更优的方法。TRI Reagent分离方法也从白尾鹿皮肤打孔活检样本中获得了高质量的总RNA，表明该方法对于获取适合其他反刍动物物种基因表达研究的高质量RNA是有用的。
