Song Hao, Dang Xin, He Yuan-Qiu, Zhang Tao, Wang Hai-Yan
CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.
University of Chinese Academy of Sciences, Beijing, China.
PeerJ. 2017 May 31;5:e3398. doi: 10.7717/peerj.3398. eCollection 2017.
The veined rapa whelk is an important commercial shellfish in China and quantitative real-time PCR (qRT-PCR) has become the standard method to study gene expression in . For accurate and reliable gene expression results, qRT-PCR assays require housekeeping genes as internal controls, which display highly uniform expression in different tissues or stages of development. However, to date no studies have validated housekeeping genes in for use as internal controls for qRT-PCR.
In this study, we selected the following 13 candidate genes for suitability as internal controls: elongation factor-1 (), -actin (), cytochrome c oxidase subunit 1 (), nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) 1 subcomplex subunit 7 (), 60S ribosomal protein L5 (), 60S ribosomal protein L28 (), glyceraldehyde 3-phosphate dehydrogenase (), -tubulin (), 40S ribosomal protein S25 (), 40S ribosomal protein S8 (), ubiquitin-conjugating enzyme E2 (), histone H3 (), and peptidyl-prolyl cis-trans isomerase A (). We measured the expression levels of these 13 candidate internal controls in eight different tissues and twelve larvae developmental stages by qRT-PCR. Further analysis of the expression stability of the tested genes was performed using GeNorm and RefFinder algorithms.
Of the 13 candidate genes tested, we found that was the most stable internal control gene in almost all adult tissue samples investigated with and as secondary choices. For the normalization of a single specific tissue, we suggested that and are the best combination in gonad, as well as and for intestine, and for kidney, and for gill, and for Leiblein and mantle, , , and for liver, , and for hemocyte. From a developmental perspective, we found that was the most stable gene in all developmental stages measured, and and were appropriate secondary choices. For the specific developmental stage, we recommended the following combination for normalization, , , and for stage 1, and for stage 2 and 5, and for stage 3, and and for stage 4.
Our results are instrumental for the selection of appropriately validated housekeeping genes for use as internal controls for gene expression studies in adult tissues or larval development of in the future.
脉红螺是中国一种重要的商业贝类，定量实时荧光定量聚合酶链反应（qRT-PCR）已成为研究其基因表达的标准方法。为获得准确可靠的基因表达结果，qRT-PCR检测需要管家基因作为内参，这些管家基因在不同组织或发育阶段表现出高度一致的表达。然而，迄今为止，尚无研究验证脉红螺中可作为qRT-PCR内参的管家基因。
在本研究中，我们选择了以下13个候选基因作为内参：延伸因子-1（EF-1）、β-肌动蛋白（β-actin）、细胞色素c氧化酶亚基1（COX1）、烟酰胺腺嘌呤二核苷酸脱氢酶（泛醌）1亚复合体亚基7（NDUFS7）、60S核糖体蛋白L5（RPL5）、60S核糖体蛋白L28（RPL28）、甘油醛-3-磷酸脱氢酶（GAPDH）、α-微管蛋白（α-tubulin）、40S核糖体蛋白S25（RPS25）、40S核糖体蛋白S8（RPS8）、泛素结合酶E2（UBE2）、组蛋白H3（H3）和肽基脯氨酰顺反异构酶A（PPIA）。我们通过qRT-PCR测量了这13个候选内参基因在8种不同组织和12个幼虫发育阶段的表达水平。使用GeNorm和RefFinder算法对测试基因的表达稳定性进行了进一步分析。
在测试的13个候选基因中，我们发现EF-1在几乎所有研究的成体组织样本中是最稳定的内参基因，RPL5和RPL28为次选。对于单个特定组织的标准化，我们建议在性腺中EF-1和RPL5是最佳组合，在肠中GAPDH和β-actin是最佳组合，在肾中COX1和RPS8是最佳组合，在鳃中NDUFS7和UBE2是最佳组合，在肝胰脏和外套膜中RPL28、RPS25和PPIA是最佳组合，在血细胞中H3和RPS8是最佳组合。从发育角度来看，我们发现EF-1在所有测量的发育阶段中是最稳定的基因，RPL5和RPL28是合适的次选。对于特定的发育阶段，我们推荐以下标准化组合：在第1阶段为EF-1、RPL5和RPL28，在第2和5阶段为GAPDH和β-actin，在第3阶段为COX1和RPS8，在第4阶段为NDUFS7和UBE2。
我们的结果有助于未来为脉红螺成体组织或幼虫发育的基因表达研究选择经过适当验证的管家基因作为内参。
