Foss D L, Baarsch M J, Murtaugh M P
Department of Veterinary PathoBiology, University of Minnesota, St. Paul 55108, USA.
Anim Biotechnol. 1998;9(1):67-78. doi: 10.1080/10495399809525893.
Various "housekeeping" genes are often used as endogenous controls in gene expression experiments. We have cloned from swine, three genes commonly used as endogenous controls in other species and have characterized their relative levels of expression in various porcine tissues and their response to various cell activators. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin were readily detected by northern hybridization in various tissues and in alveolar macrophages. The expression of hypoxanthine phosphoribosyltransferase (HPRT) was detected only by northern hybridization of poly-A+ enriched RNA and by reverse transcriptase-polymerase chain reaction (RT-PCR), making it more suitable for highly sensitive detection methods. Expression of GAPDH varied less among tissues than did beta-actin, making it more useful control for comparisons of gene expression between tissues with northern hybridizations. Various treatments of cultured alveolar macrophages differentially affected levels of beta-actin and GAPDH, while HPRT expression was unchanged in alveolar macrophages or spleen cells similarly treated. Therefore, while HPRT can be used as the endogenous control with sensitive detection methods such as RT-PCR, less sensitive detection methods require a more abundant gene such as GAPDH.
在基因表达实验中，各种“管家”基因常被用作内参。我们从猪身上克隆了在其他物种中常用作内参的三个基因，并对它们在各种猪组织中的相对表达水平以及对各种细胞激活剂的反应进行了表征。通过Northern杂交在各种组织和肺泡巨噬细胞中很容易检测到甘油醛-3-磷酸脱氢酶（GAPDH）和β-肌动蛋白。次黄嘌呤磷酸核糖转移酶（HPRT）的表达仅通过富含poly-A +的RNA的Northern杂交和逆转录聚合酶链反应（RT-PCR）检测到，这使得它更适合用于高灵敏度检测方法。与β-肌动蛋白相比，GAPDH在组织间的表达变化较小，这使得它在通过Northern杂交比较组织间基因表达时作为对照更有用。对培养的肺泡巨噬细胞进行的各种处理对β-肌动蛋白和GAPDH的水平有不同影响，而在同样处理的肺泡巨噬细胞或脾细胞中，HPRT的表达没有变化。因此，虽然HPRT可以与RT-PCR等灵敏检测方法一起用作内参，但不太灵敏的检测方法需要像GAPDH这样更丰富的基因。
