Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, China.
Key Laboratory of Diagnostic Medicine Designated by the Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, China.
Lab Invest. 2021 Dec;101(12):1618-1626. doi: 10.1038/s41374-021-00648-9. Epub 2021 Aug 10.
Due to the short length and differences in abundance of microRNAs, microRNA profile screening and quantification is challenging. In this study, we found that size selection magnetic beads could be employed to easily and efficiently remove long RNA transcripts. After removing the long transcripts, the remaining small RNAs could be concentrated and then reverse-transcribed using universal stem-loop primers (USLP), with six randomized nucleotides at the 3' end region. The efficiency of reverse transcription decreased when the number of randomized nucleotides was reduced. In addition, we found that touchdown qPCR improved microRNA profile detection, with lower C values and better detection efficiency than the regular qPCR protocol, especially for those low-abundance microRNAs. Finally, we incorporated these observations to create a new protocol we named long transcripts minus touchdown qPCR (LTMT-qPCR). We performed a side-by-side comparison of LTMT with USLP and traditional stem-loop primer (TSLP) protocols. We found that LTMT has higher detection efficiency than USLP, especially for the detection of low-abundance microRNAs. Although LTMT was equivalent to TSLP in terms of microRNA profile detection, LTMT is more convenient, user-friendly, and cost-effective. Taken together, the present data indicate that LTMT is a simple, rapid, and user-friendly approach that has higher precision, accuracy, and sensitivity than the previously described methods, making it more suitable for microRNA profile screening and quantification.
由于 microRNA 长度较短且丰度存在差异，因此 microRNA 谱筛选和定量具有一定挑战性。在本研究中，我们发现大小选择磁珠可用于轻松高效地去除长 RNA 转录本。去除长转录本后，剩余的小 RNA 可以使用通用茎环引物 (USLP) 进行浓缩，随后在 3' 端区域使用六个随机核苷酸进行逆转录。当随机核苷酸的数量减少时，逆转录效率降低。此外，我们发现降落式 qPCR 可提高 microRNA 谱检测效率，与常规 qPCR 方案相比，C 值更低，检测效率更好，尤其是对于那些低丰度的 microRNA。最后，我们将这些观察结果纳入一个新方案中，命名为长转录物减去降落式 qPCR (LTMT-qPCR)。我们将 LTMT 与 USLP 和传统茎环引物 (TSLP) 方案进行了并排比较。我们发现 LTMT 的检测效率高于 USLP，尤其是对低丰度 microRNA 的检测。尽管 LTMT 在 microRNA 谱检测方面与 TSLP 等效，但 LTMT 更方便、用户友好且具有成本效益。综上所述，本研究数据表明 LTMT 是一种简单、快速且用户友好的方法，与先前描述的方法相比，具有更高的精度、准确性和灵敏度，更适合 microRNA 谱筛选和定量。
