Langnaese Kristina, John Robin, Schweizer Hannes, Ebmeyer Uwe, Keilhoff Gerburg
Institute of Medical Neurobiology, University of Magdeburg, Leipziger Str, 44, D-39120 Magdeburg, Germany.
BMC Mol Biol. 2008 May 28;9:53. doi: 10.1186/1471-2199-9-53.
Cardiac arrest, and the associated arrest of blood circulation, immediately leads to permanent brain damage because of the exhaustion of oxygen, glucose and energy resources in the brain. Most hippocampal CA1 neurons die during the first week post the insult. Molecular data concerning the recovery after resuscitation are sparse and limited to the early time period. Expression analysis of marker genes via quantitative real-time RT-PCR enables to follow up the remodeling process. However, proper validation of the applied normalization strategy is a crucial prerequisite for reliable conclusions.Therefore, the present study aimed to determine the expression stability of ten commonly used reference genes (Actb, actin, beta; B2m, beta-2 microglobulin;CypA, cyclophilin A; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Hprt, hypoxanthine guanine phosphoribosyl transferase; Pgk1, phosphoglycerate kinase 1; Rpl13a, ribosomal protein L13A; Sdha, succinat dehydrogenase complex, subunit a, flavoprotein (Fp); Tbp, TATA box binding protein; Ywhaz, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) in the rat hippocampus four, seven and twenty-one days after cardiac arrest. Moreover, experimental groups treated with the anti-inflammatory and anti-apoptotic drug minocycline have been included in the study as well.
The microglial marker Mac-1, used as a target gene to validate the experimental model, was found to be upregulated about 10- to 20-fold after cardiac arrest. Expression stability of candidate reference genes was analyzed using geNorm and NormFinder software tools. Several of these genes behave rather stable. CypA and Pgk1 were identified by geNorm as the two most stable genes 4 and 21 days after asphyxial cardiac arrest, CypA and Gapdh at 7 days post treatment. B2m turned out to be the most variable candidate reference gene, being about 2-fold upregulated in the cardiac arrest treatment groups.
We have validated endogenous control genes for qRT-PCR analysis of gene expression in rat hippocampus after resuscitation from cardiac arrest. For normalization purposes in gene profiling studies a combination of CypA and Pgk1 should be considered 4 and 21 days post injury, whereas CypA and Gapdh is the best combination at 7 days. CypA is most favorable if restriction to a single reference gene for all time points is required.
心脏骤停以及相关的血液循环停止会立即导致永久性脑损伤，这是由于大脑中的氧气、葡萄糖和能量资源耗尽所致。大多数海马CA1神经元在损伤后的第一周内死亡。关于复苏后恢复的分子数据稀少，且仅限于早期阶段。通过定量实时RT-PCR对标记基因进行表达分析能够追踪重塑过程。然而，对所应用的标准化策略进行适当验证是得出可靠结论的关键前提。因此，本研究旨在确定心脏骤停后4天、7天和21天大鼠海马中十个常用参考基因（Actb，肌动蛋白，β；B2m，β-2微球蛋白；CypA，亲环素A；Gapdh，甘油醛-3-磷酸脱氢酶；Hprt，次黄嘌呤鸟嘌呤磷酸核糖基转移酶；Pgk1，磷酸甘油酸激酶1；Rpl13a，核糖体蛋白L13A；Sdha，琥珀酸脱氢酶复合物，亚基a，黄素蛋白（Fp）；Tbp，TATA盒结合蛋白；Ywhaz，酪氨酸3-单加氧酶/色氨酸5-单加氧酶激活蛋白，ζ多肽）的表达稳定性。此外，用抗炎和抗凋亡药物米诺环素治疗的实验组也被纳入了研究。
用作验证实验模型的靶基因的小胶质细胞标志物Mac-1在心脏骤停后被发现上调了约10至20倍。使用geNorm和NormFinder软件工具分析了候选参考基因的表达稳定性。这些基因中有几个表现得相当稳定。geNorm将CypA和Pgk1鉴定为窒息性心脏骤停后4天和21天最稳定的两个基因，治疗后7天为CypA和Gapdh。结果表明B2m是最不稳定的候选参考基因，在心脏骤停治疗组中上调了约2倍。
我们已经验证了用于心脏骤停复苏后大鼠海马中基因表达qRT-PCR分析的内参基因。在基因谱研究中进行标准化时，损伤后4天和21天应考虑将CypA和Pgk1组合使用，而损伤后7天CypA和Gapdh是最佳组合。如果在所有时间点都需要限制使用单个参考基因，CypA是最适宜的。
