McCurley Amy T, Callard Gloria V
Department of Biology, Boston University, Boston, MA 02215, USA.
BMC Mol Biol. 2008 Nov 12;9:102. doi: 10.1186/1471-2199-9-102.
Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping genes are known to vary with experimental conditions. To identify housekeeping genes that are stably expressed under different experimental conditions, and thus suitable as normalizers for QPCR in zebrafish, the present study evaluated the expression of eight commonly used housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish.
QPCR analysis was used to quantify mRNA levels of bactin1, tubulin alpha 1(tuba1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), TATA-box binding protein (tbp), beta-2-microglobulin (b2m), elongation factor 1 alpha (elfa), and 18s ribosomal RNA (18s) during development (2 - 120 hr postfertilization, hpf); in different tissue types (brain, eye, liver, heart, muscle, gonads) of adult males and females; and after treatment of embryos/larvae (24 - 96 hpf) with commonly used vehicles for administration and agents that represent known environmental endocrine disruptors. All genes were found to have some degree of variability under the conditions tested here. Rank ordering of expression stability using geNorm analysis identified 18s, b2m, and elfa as most stable during development and across tissue types, while gapdh, tuba1, and tpb were the most variable. Following chemical treatment, tuba1, bactin1, and elfa were the most stably expressed whereas tbp, 18s, and b2m were the least stable. Data also revealed sex differences that are gene- and tissue-specific, and treatment effects that are gene-, vehicle- and ligand-specific. When the accuracy of QPCR analysis was tested using different reference genes to measure suppression of cyp19a1b by an estrogen receptor antagonist and induction of cyp1a by an arylhydrocarbon receptor agonist, the direction and magnitude of effects with stable and unstable genes differed.
This study provides data that can be expected to aid zebrafish researchers in their initial choice of housekeeping genes for future studies, but underlines the importance of further validating housekeeping genes for each new experimental paradigm and fish species.
近年来，使用斑马鱼模型的研究迅速发展。尽管以内部参照（“看家”）基因标准化的实时逆转录PCR（QPCR）是用于定量斑马鱼基因表达变化的常用方法，但许多常用的看家基因已知会随实验条件而变化。为了鉴定在不同实验条件下稳定表达、因而适合作为斑马鱼QPCR标准化参照的看家基因，本研究评估了八个常用看家基因在发育阶段、激素/毒物暴露过程中的表达情况，以及成年鱼不同组织类型和性别的表达情况。
使用QPCR分析定量发育过程中（受精后2至120小时，hpf）、成年雄性和雌性不同组织类型（脑、眼、肝、心、肌肉、性腺）以及用常用给药载体和代表已知环境内分泌干扰物的试剂处理胚胎/幼体（24至96 hpf）后β-肌动蛋白1（bactin1）、微管蛋白α1（tuba1）、甘油醛-3-磷酸脱氢酶（gapdh）、葡萄糖-6-磷酸脱氢酶（g6pd）、TATA框结合蛋白（tbp）、β2-微球蛋白（b2m）、延伸因子1α（elfa）和18S核糖体RNA（18s）的mRNA水平。在所测试的条件下，所有基因均表现出一定程度的变异性。使用geNorm分析对表达稳定性进行排序，结果显示18s、b2m和elfa在发育过程中和不同组织类型中最为稳定，而gapdh、tuba1和tbp变异性最大。化学处理后，tuba1、bactin1和elfa表达最稳定，而tbp、18s和b2m最不稳定。数据还揭示了基因和组织特异性的性别差异以及基因、载体和配体特异性的处理效应。当使用不同的参照基因测试QPCR分析的准确性，以测量雌激素受体拮抗剂对cyp19a1b的抑制作用和芳烃受体激动剂对cyp1a的诱导作用时，使用稳定和不稳定基因时效应的方向和幅度有所不同。
本研究提供的数据有望帮助斑马鱼研究人员为未来研究初步选择看家基因，但强调了针对每种新的实验范式和鱼类物种进一步验证看家基因的重要性。
