Developing a standardized kit and normal reference profile for circular RNAs in the nervous system

描述（由申请人提供）：修饰核糖核酸（RNA）是翻译和其他细胞途径的重要调节因子。非编码RNA如microRNA在神经元发育中起重要作用，并可作为精神疾病的生物标志物。另一种这样的修饰的RNA是环状RNA（circRNA），一种存在于细胞质中的非编码转录物。由于circRNA的丰度低于其他RNA分子，并且与mRNA具有序列同源性，因此难以从总RNA中分离。为了便于识别和检查这些分子，我们建议开发一种简化的方法，使用选择性减少线性和核糖体RNA结合一种新的逆转录酶，以丰富环状RNA。为了优化稳健的减少和扩增，我们将测试一系列条件，并使用对已知circRNA和合成的加标线性RNA特异性的qRT-PCR评估其相对效率。我们将使用我们经过验证的方法来生成多个脑组织的circRNA谱，这将对有兴趣了解circRNA在神经元发育，药物滥用和精神疾病中的作用的研究人员有重大意义。由于市场上没有试剂盒可以满足简单的circRNA富集的需求，因此该方案的开发将促进这一新兴领域的更广泛研究，并进一步了解circRNA的作用。

DESCRIPTION (provided by applicant): Modified ribonucleic acids (RNA) are important regulators of translation and other cellular pathways. Non- coding RNAs like microRNAs play significant roles in neuronal development, and can serve as biomarkers for psychiatric disorders. Another such modified RNA is circular RNA (circRNA), a non-coding transcript present in the cytoplasm. Since circRNAs are present in lower abundance than other RNA molecules, and share sequence homology with mRNA, they are difficult to isolate from total RNA. To facilitate identification and examination of these molecules, we propose to develop a streamlined approach that uses selective reduction of linear and ribosomal RNAs combined with a novel reverse transcriptase to enrich for circular RNAs. To optimize for robust reduction and amplification, we will test a range of conditions and assess their relative efficiencies using qRT-PCR specific for known circRNAs and synthetic spiked-in linear RNAs. We will use our validated approach to generate circRNA profiles of multiple brain tissues, which will be of significant benefit to researchers interested in understanding the role of circRNA in neuronal development, substance abuse, and psychiatric disorders. As there is no kit on the market which addresses the need for easy circRNA enrichment, development of this protocol will foster broader research in this nascent field and further understanding of the role of circRNAs.