多鳞鱚不同组织荧光定量PCR内参基因筛选

Screening of Reference Genes for Real-time PCR in Different Tissues from Sillago sihama

【目的】筛选多鳞鱚(Sillago sihama)雌雄不同组织中稳定表达的内参基因。【方法】应用实时荧光定量PCR(qRT-PCR)技术定量分析多鳞鱚snrpd1、rps27、rpl7a、rpl7、cnpb、rps4、rps20、ef1a、ube2、rplp2等10个候选内参基因m RNA在雌、雄个体脑、鳃、性腺、心、肠、肾、肝、肌肉、皮肤、脾、胃等22个组织中的表达水平,通过BestKeeper、NormFinder、GeNorm工具测评10个内参基因表达的稳定性。【结果】10个候选内参基因均可获得特异性的扩增产物和理想的扩增效率,其中Bestkeeper软件计算候选内参基因稳定性由高到低的顺序为:snrpd1=rps27> rpl7a> rpl7>cnpb=rps4> rps20> ef1a> ube2> rplp2;Norm Finder软件分析结果为:rpl7>rpl7a> rps27> rps4> cnpb> ef1a> rps20> ube2> rplp2> snrpd1;Ge Norm软件分析结果为:rpl7/rpl7a> rps4>ef1a> rps27> rps20> cnpb> ube2> rplp2> snprd1。【结论】rpl7、rpl7a、rps27基因的表达稳定性较高,建议选择rpl7和rpl7a共同作为多鳞鱚q RT-PCR研究的内参基因。

【Objective】To screen for stable reference genes in different tissues of Sillago sihama.【Method】Quantitative Real-time PCR （qRT-PCR） was employed to analyze the expression of ten candidate reference genes （snrpd1, rps27, rpl7a, rpl7, cpnb, rps4, rps20, ef1a, ube2, rplp2） in different tissues （brain, pupae, gonad, heart, intestine, kidney, muscle, skin, spleen, and stomach） from S. sihama. Using a combination of software including BestKeeper, NormFinder and geNorm software,the expression stability of 10 reference genes was analyzed.【Result】 Specific amplification products were obtained for these candidate genes with optimal amplification efficiency. Bestkeeper analysis result showed that the stability of these reference genes was as follows： snrpd1 = rps27 〉 rpl7a〉 rpl7 〉 cnpb = rps4 〉 rps20 〉 ef1a〉 ube2 〉 rplp2; NormFinder analysis result showed that the reference genes stability was as follows： rpl7 〉 rpl7a〉 rps27 〉 rps4 〉cnpb 〉 ef1a 〉 rps20 〉 ube2 〉 rplp2 〉 snrpd1. GeNorm analysis results show that the stability was as follow： rpl7/rpl7a 〉 rps4 〉 ef1a〉 rps27 〉 rps20 〉cnpb 〉 ube2 〉 rplp2 〉 snprd1. 【Conclusion】Since the expression stability of rpl7, rpl7a, and rps27 was higher we recommended to use rpl7 and rpl7a as coreference gene in qRT-PCR gene expression study in S. sihama.