Cardiac Metabolism Research Group, Department of Physiology, Anatomy & Genetics, University of Oxford, Sherrington Building, Parks Road, Oxford, OX1 3PT, UK.
Mol Biol Rep. 2012 Apr;39(4):4857-67. doi: 10.1007/s11033-011-1281-5. Epub 2011 Nov 9.
Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs may be isolated in vitro, via the formation of cardiospheres, to give cardiosphere-derived cells (CDCs). Although qRT-PCR analyses of CDCs have been performed, no justification for the selection of the housekeeping gene has been published. Here, we evaluated the most suitable housekeeping gene for RNA expression analysis in CDCs cultured under normoxia, hypoxia or with prolyl-4-hydroxylase inhibitors (PHDIs), from both neonatal and adult rats, to determine the effects of ageing and different culture conditions on the stability of the housekeeping gene for CDCs. Six candidate housekeeping genes, [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), beta-2-microtubulin (β2M), 60S acidic ribosomal protein large P1 (RPLP-1) and TATA box binding protein (Tbp)] were evaluated in this study. Analysis using geNorm and NormFinder revealed that GAPDH was the most constant housekeeping gene among all genes tested under normoxia for both neonatal and adult CDCs, whereas Actb was the most stable housekeeping gene under hypoxia. For the PHDI-treated CDCs, overall, GADPH, Actb and β2M were more consistently expressed, whereas HPRT-1, RPLP-1 and Tbp showed unstable expression. The ranking for β2M, HPRT-1 and RPLP-1 stability was different for neonatal and adult cells, indicating that expression of these genes was age-dependent. Lastly, independent of age or culture conditions, Tbp was the least stable housekeeping gene. In conclusion, a combination of Actb and GADPH gave the most reliable normalization for comparative analyses of gene transcription in neonatal and adult rat CDCs preconditioned by hypoxia or PHDIs.
梗死不可逆地损伤心脏，形成无运动的瘢痕，可能导致心力衰竭。内源性心脏干细胞（CSC）是恢复丢失组织从而预防心力衰竭的有前途的候选细胞来源。CSC 可以在体外通过形成心肌球体来分离，从而获得心肌球体衍生细胞（CDC）。尽管已经对 CDC 进行了 qRT-PCR 分析，但尚未发表选择管家基因的依据。在这里，我们评估了最适合用于在常氧、低氧或脯氨酰-4-羟化酶抑制剂（PHDIs）下培养的新生和成年大鼠的 CDC 的 RNA 表达分析的管家基因，以确定老化和不同培养条件对 CDC 的管家基因稳定性的影响。本研究评估了 6 个候选管家基因，[甘油醛-3-磷酸脱氢酶（GAPDH）、β-肌动蛋白（Actb）、次黄嘌呤磷酸核糖基转移酶 1（HPRT-1）、β-2-微管蛋白（β2M）、60S 酸性核糖体蛋白 P1（RPLP-1）和 TATA 框结合蛋白（Tbp）]。使用 geNorm 和 NormFinder 分析表明，在常氧条件下，GAPDH 是所有测试基因中新生和成年 CDC 最稳定的管家基因，而 Actb 是低氧条件下最稳定的管家基因。对于 PHDI 处理的 CDC，总体而言，GADPH、Actb 和β2M 表达更稳定，而 HPRT-1、RPLP-1 和 Tbp 表达不稳定。β2M、HPRT-1 和 RPLP-1 稳定性的排名在新生和成年细胞之间有所不同，表明这些基因的表达依赖于年龄。最后，无论年龄或培养条件如何，Tbp 都是最不稳定的管家基因。总之，在低氧或 PHDIs 预处理的新生和成年大鼠 CDC 中，Actb 和 GADPH 的组合为基因转录的比较分析提供了最可靠的归一化。
