Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
J Mol Neurosci. 2012 Mar;46(3):497-504. doi: 10.1007/s12031-011-9628-x. Epub 2011 Aug 24.
Stably expressed housekeeping genes (HKGs) are necessary for standardization of transcript measurement by quantitative real-time polymerase chain reaction (qRT-PCR). Peripheral nerve injury disrupts expression of numerous genes in sensory neurons, but the stability of conventional HKGs has not been tested in this context. We examined the stability of candidate HKGs during nerve injury, including the commonly used 18S ribosomal RNA, β-tubulin I and β-tubulin III, actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase 1 (HPRT1), and mitogen-activated protein kinase 6 (MAPK6). Total RNA for cDNA synthesis was isolated from dorsal root ganglia of rats at 3, 7, and 21 days following either skin incision alone or spinal nerve ligation, after which the axotomized and adjacent ganglia were analyzed separately. Relative stability of HKGs was determined using statistical algorithms geNorm and NormFinder. Both analyses identified MAPK6 and GAPDH as the two most stable HKGs for normalizing gene expression for qRT-PCR analysis in the context of peripheral nerve injury. Our findings indicate that a prior analysis of HKG expression levels is important for accurate normalization of gene expression in models of nerve injury.
稳定表达的管家基因（HKGs）是通过实时定量聚合酶链反应（qRT-PCR）标准化转录物测量所必需的。周围神经损伤会破坏感觉神经元中许多基因的表达，但在这种情况下尚未测试常规 HKG 的稳定性。我们在神经损伤过程中检查了候选 HKG 的稳定性，包括常用的 18S 核糖体 RNA、β-微管蛋白 I 和 β-微管蛋白 III、肌动蛋白、甘油醛 3-磷酸脱氢酶（GAPDH）和次黄嘌呤磷酸核糖基转移酶 1（HPRT1）以及丝裂原活化蛋白激酶 6（MAPK6）。在单独进行皮肤切口或脊神经结扎后 3、7 和 21 天，从大鼠背根神经节中分离用于 cDNA 合成的总 RNA，然后分别分析轴突切断和相邻神经节。使用统计算法 geNorm 和 NormFinder 确定 HKG 的相对稳定性。这两种分析均将 MAPK6 和 GAPDH 鉴定为周围神经损伤情况下 qRT-PCR 分析中用于基因表达归一化的最稳定的两个 HKG。我们的研究结果表明，在神经损伤模型中，对 HKG 表达水平进行预先分析对于准确归一化基因表达很重要。
