Validation of internal reference genes for real-time quantitative polymerase chain reaction studies in the tick, Ixodes scapularis (Acari: Ixodidae).
Validation of internal reference genes for real-time quantitative polymerase chain reaction studies in the tick, Ixodes scapularis (Acari: Ixodidae).
复制标题
DOI：
10.1603/me12034
复制
发表时间：
2013-01
影响因子：
2.1
通讯作者：
Park Y
中科院分区：
文献类型：
--
作者：
Koci J;Simo L;Park Y
使用实时定量聚合酶链式反应(QPCR)获得可靠的基因表达数据高度依赖于归一化方法的选择。我们测试了多个候选基因在多个取血阶段在雌性肩部硬蜱的唾液腺(SG)和联神经节(SYN)中的表达稳定性。我们发现，在取食硬蜱的过程中，SG和SYN的总RNA含量都显著增加，分别比未取食的62和7.1 ng增加了34倍和5.8倍。我们测试了从肩关节基因组数据中预测的编码甘油醛3-磷酸脱氢酶(GAPDH)、核糖体蛋白L13A(13a)、TATA盒结合蛋白(TBP)、核糖体蛋白S4(RPS4)、葡萄糖6-磷酸脱氢酶(GPDH)和β-葡萄糖醛酸苷酶(GUSB)的候选基因。GeNorm和NormFinder算法被用来分析不同取食阶段的数据(即，在三个重复实验中，从未进食的雌性到完全充血的雌性在7-d时间段内的每日样本)。我们发现，RPS4和13a基因在SG和SYN的取食持续时间内都表现出高度稳定的表达模式。此外，高表达的RPS4基因使其在我们使用微量的解剖组织进行定量聚合酶链式反应的研究时作为一个标准化因子很有用。我们的结论是，RPS4和13a，无论是单独的还是成对的，都可以作为QRT-PCR研究肩胛鱼SG和SYN的合适的内参基因。
Obtaining reliable gene expression data using real-time quantitative polymerase chain reaction(qPCR)is highly dependent on the choice of normalization method. We tested the expression stability of multiple candidate genes in the salivary glands (SG) and synganglia (SYN) of female Ixodes scapularis (Say) ticks in multiple blood-feeding phases. We found that the amount of total RNA in both the SG and SYN increases dramatically during tick feeding, with 34× and 5.8× increases from 62 and 7.1 ng of unfed tick, respectively. We tested candidate genes that were predicted from I. scapularis genome data to encode glyceraldehyde 3-phosphate dehydrogenase (gapdh), ribosomal protein L13A (l13a), TATA box-binding protein (tbp), ribosomal protein S4 (rps4), glucose 6-phosphate dehydrogenase (gpdh), and beta-glucuronidase (gusb). The geNorm and NormFinder algorithms were used to analyze data from different feeding phases (i.e., daily samples from unfed to fully engorged females over a 7-d period in three replicate experiments). We found that the rps4 and l13a genes showed highly stable expression patterns over the feeding duration in both the SG and SYN. Furthermore, the highly expressed rps4 gene makes it useful as a normalization factor when we perform studies using minute amounts of dissected tissue for qPCR. We conclude that rps4 and l13a, whether individually or as a pair, serve as suitable internal reference genes for qRT-PCR studies in the SG and SYN of I. scapularis.
登录
查看更多内容
影响因子：
3.7
作者：
de Jonge HJ;Fehrmann RS;de Bont ES;Hofstra RM;Gerbens F;Kamps WA;de Vries EG;van der Zee AG;te Meerman GJ;ter Elst A
通讯作者：
ter Elst A
影响因子：
4.1
作者：
Thellin, O;Zorzi, W;Heinen, E
通讯作者：
Heinen, E
影响因子：
3.5
作者：
Bustin, SA
通讯作者：
Bustin, SA
影响因子：
--
作者：
Mehta R;Birerdinc A;Hossain N;Afendy A;Chandhoke V;Younossi Z;Baranova A
通讯作者：
Baranova A
影响因子：
2.1
作者：
Mamidala, Praveen;Rajarapu, Swapna P.;Mittapalli, Omprakash
通讯作者：
Mittapalli, Omprakash