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Quantitative mRNA Imaging with Dual Channel qFIT Probes to Monitor Distribution and Degree of Hybridization
ACS Chemical Biology ( IF 4 ) Pub Date : 2018-01-29 00:00:00 , DOI: 10.1021/acschembio.7b01007
Imre Gaspar 1 , Felix Hövelmann 2 , Jasmine Chamiolo 2 , Anne Ephrussi 1 , Oliver Seitz 2
Affiliation  

Fluorogenic oligonucleotide probes facilitate the detection and localization of RNA targets within cells. However, quantitative measurements of mRNA abundance are difficult when fluorescence signaling is based on intensity changes because a high concentration of unbound probes cannot be distinguished from a low concentration of target-bound probes. Here, we introduce qFIT (quantitative forced intercalation) probes that allow the detection both of probe–target complexes and of unbound probes on separate, independent channels. A surrogate nucleobase based on thiazole orange (TO) probes the hybridization status. The second channel involves a nonresponsive near-IR dye, which serves as a reporter of concentration. We show that the undesirable perturbation of the hybridization reporter TO is avoided when the near-IR dye Cy7 is connected by means of short triazole linkages in an ≥18 nucleotides distance. We used the qFIT probes to localize and quantify oskar mRNA in fixed egg chambers of wild-type and mutant Drosophila melanogaster by wash-free fluorescence in situ hybridization. The measurements revealed a relative 400-fold enrichment of oskar within a 3000 μm3 large volume at the posterior pole of stage 8–9 oocytes, which peaked at a remarkably high 1.8 μM local concentration inside 0.075 μm3 volume units. We discuss detection limits and show that the number of oskar mRNA molecules per oocyte is independent of the oocyte size, which suggests that the final levels are attained already during the onset of oskar localization at stage 8.

中文翻译:

使用双通道qFIT探针定量mRNA成像以监测杂交的分布和程度

荧光寡核苷酸探针可促进细胞内RNA靶标的检测和定位。但是,当荧光信号基于强度变化时,很难定量测量mRNA的丰度,因为无法将高浓度的未结合探针与低浓度的目标结合探针区分开。在这里,我们介绍qFIT(q的定量的˚F orcedñ ŧ直立)探针,可在单独,独立的通道上同时检测探针-靶标复合物和未结合的探针。基于噻唑橙(TO)的替代核碱基可检测杂交状态。第二个通道涉及一种无响应的近红外染料,它是浓度的报告分子。我们表明,当近红外染料Cy7通过短的三唑键以≥18个核苷酸的距离连接时,可以避免杂交报告子TO的不良干扰。我们使用qFIT探针通过免洗荧光原位杂交来定位和定量野生型和突变果蝇果蝇的固定卵室中的oskar mRNA 。测量结果表明,奥斯卡浓缩的相对含量为400倍3000微米内3在阶段8-9的卵母细胞,其峰值在显着高的1.8μM局部浓度内部0.075微米的后极大体积3体积单位。我们讨论了检测极限,并显示每个卵母细胞的oskar mRNA分子的数量与卵母细胞大小无关,这表明在阶段8的oskar定位过程中已经达到了最终水平。
更新日期:2018-01-29
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