Marchal Elisabeth, Hult Ekaterina F, Huang Juan, Tobe Stephen S
Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Canada.
BMC Res Notes. 2013 Jun 19;6:237. doi: 10.1186/1756-0500-6-237.
Quantitative RT-PCR (q-RT-PCR) is a powerful tool that allows for the large scale analysis of small changes in gene expression. Accurate and reliable results depend on the use of stable reference genes for normalization. However, the expression of some widely used housekeeping genes can vary under different experimental setups. To our knowledge, no validation studies have been reported for reference genes in cockroaches. The aim of the current study is the identification and validation of a set of eight housekeeping genes during the first gonadotrophic cycle of the cockroach, Diploptera punctata. This study made use of two different algorithms (geNorm and Normfinder) to evaluate the stability of gene expression.
Candidate housekeeping genes were sequenced: β-actin (Actin), elongation factor 1 alpha (EF1a), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), armadillo (Arm), ribosomal protein L32 (RpL32), succinate dehydrogenase (SDHa), annexin IX (AnnIX) and α-tubulin (Tub). The expression of these eight genes was analyzed in corpora allata (CA) and ovaries of adult female D. punctata. Both geNorm, as well as Normfinder characterized SDHa, EF1a and Arm as being the most stably expressed in the corpora allata. In the ovary, the geNorm calculation showed Tub, EF1a and RpL32 to be most stable, whereas Normfinder identified Tub, EF1a and Arm as the best. In ovary, the least stable gene was Actin, challenging its usefulness in normalization. As a proof of principle, the expression of follicle cell protein 3c and CYP15A1 was monitored during the first gonadotrophic cycle.
Arm and EF1a form the most stably expressed combination of two reference genes out of the eight candidates that were tested in the corpora allata. Our results show that the combined use of Tub, EF1a and RpL32 ensures an accurate normalization of gene expression levels in ovary of D. punctata. Our study has indicated that neither Actin nor AnnIX should be used for normalization of transcript levels when studying the first gonadotrophic cycle in CA or ovary of D. punctata. The results stress the necessity for validation of reference genes in q-RT-PCR studies in cockroaches.
定量逆转录聚合酶链反应（q-RT-PCR）是一种强大的工具，可用于对基因表达的微小变化进行大规模分析。准确可靠的结果取决于使用稳定的内参基因进行标准化。然而，一些广泛使用的管家基因的表达在不同的实验设置下可能会有所不同。据我们所知，尚未有关于蟑螂内参基因的验证研究报道。本研究的目的是鉴定和验证一组八个管家基因在蜚蠊（Diploptera punctata）第一个促性腺周期中的表达情况。本研究使用了两种不同的算法（geNorm和Normfinder）来评估基因表达的稳定性。
对候选管家基因进行了测序：β-肌动蛋白（Actin）、延伸因子1α（EF1a）、甘油醛-3-磷酸脱氢酶（GAPDH）、犰狳蛋白（Arm）、核糖体蛋白L32（RpL32）、琥珀酸脱氢酶（SDHa）、膜联蛋白IX（AnnIX）和α-微管蛋白（Tub）。分析了这八个基因在成年雌性D. punctata的咽侧体（CA）和卵巢中的表达。geNorm和Normfinder都将SDHa、EF1a和Arm鉴定为在咽侧体中表达最稳定的基因。在卵巢中，geNorm计算显示Tub、EF1a和RpL32最稳定，而Normfinder则将Tub、EF1a和Arm鉴定为最佳。在卵巢中，最不稳定的基因是Actin，这对其在标准化中的有用性提出了挑战。作为原理验证，在第一个促性腺周期中监测了卵泡细胞蛋白3c和CYP15A1的表达。
在测试的八个候选基因中，Arm和EF1a是在咽侧体中表达最稳定的两个内参基因组合。我们的结果表明，联合使用Tub、EF1a和RpL32可确保准确标准化D. punctata卵巢中的基因表达水平。我们的研究表明，在研究D. punctata的咽侧体或卵巢的第一个促性腺周期时，Actin和AnnIX均不应用于转录水平的标准化。结果强调了在蟑螂的q-RT-PCR研究中验证内参基因的必要性。
