Ioannou Andriani, Eleftheriou Iro, Lubatti Andrea, Charalambous Anna, Skourides Paris A
Department of Biological Sciences, University of Cyprus, 1678 Nicosia, Cyprus.
J Biomed Biotechnol. 2012;2012:627602. doi: 10.1155/2012/627602. Epub 2012 Jan 12.
The photostability and narrow emission spectra of nanometer-scale semiconductor crystallites (QDs) make them desirable candidates for whole-mount fluorescent in situ hybridization to detect mRNA transcripts in morphologically preserved intact embryos. We describe a method for direct QD labeling of modified oligonucleotide probes through streptavidin-biotin and antibody-mediated interactions (anti-FITC and anti-digoxigenin). To overcome permeability issues and allow QD conjugate penetration, embryos were treated with proteinase K. The use of QDs dramatically increased sensitivity of whole-mount in situ hybridization (WISH) in comparison with organic fluorophores and enabled fluorescent detection of specific transcripts within cells without the use of enzymatic amplification. Therefore, this method offers significant advantages both in terms of sensitivity, as well as resolution. Specifically, the use of QDs alleviates issues of photostability and limited brightness plaguing organic fluorophores and allows fluorescent imaging of cleared embryos. It also offers new imaging possibilities, including intracellular localization of mRNAs, simultaneous multiple-transcript detection, and visualization of mRNA expression patterns in 3D.
纳米级半导体微晶（量子点）的光稳定性和窄发射光谱使其成为全胚胎荧光原位杂交检测形态学保存完好的完整胚胎中mRNA转录本的理想候选物。我们描述了一种通过链霉亲和素-生物素和抗体介导的相互作用（抗FITC和抗地高辛）对修饰的寡核苷酸探针进行直接量子点标记的方法。为了克服通透性问题并使量子点缀合物能够穿透，胚胎用蛋白酶K处理。与有机荧光团相比，量子点的使用显著提高了全胚胎原位杂交（WISH）的灵敏度，并能够在不使用酶扩增的情况下对细胞内的特定转录本进行荧光检测。因此，该方法在灵敏度和分辨率方面都具有显著优势。具体而言，量子点的使用缓解了困扰有机荧光团的光稳定性和亮度有限的问题，并允许对透明胚胎进行荧光成像。它还提供了新的成像可能性，包括mRNA的细胞内定位、同时多转录本检测以及三维可视化mRNA表达模式。
