PO.MCB10.01 · 分子与细胞生物学
使用数字PCR同时定量microRNA155和let-7a
Simultaneous quantification of microRNA155 and let-7a using digital PCR
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
microRNA（miRNA）是一类在转录后调控基因表达的小的非编码RNA。由于其长度短且家族成员间序列高度相似，miRNA的检测在技术上仍具挑战性。因此，临床研究需要准确、灵敏的定量方法。数字PCR（dPCR）无需标准品即可提供绝对定量，具有高灵敏度和对抑制剂的耐受性。这些特性使dPCR成为miRNA分析的强大工具。在本研究中，采用先进的类TaqMan探针设计，开发了一种单孔多重检测方法，用于同时检测miR155、let-7a和作为内参的U6 snRNA。使用靶标特异性反向引物进行cDNA合成，其5'端带或不带10-12 nt人工序列，随后使用配备28,000个分区的通用纳米孔板的Roche Digital LightCycler® dPCR系统。每个靶标用四条引物以优化比例进行扩增。类TaqMan探针整合了一条靶标特异性反向引物，其延伸了额外的20-22 nt序列并在5'端标记荧光基团，与一条含3'淬灭基团以阻断延伸的互补引物配对。使用合成的成熟miR155和let-7a miRNA以及商品化细胞系RNA测试了检测性能。成熟miRNA的十倍系列稀释表明，在单重和多重检测中，两个靶标均可稳健检测至每反应4-6个拷贝。通过加标样本，当一个靶标以相对另一个1000倍过量存在时，未观察到交叉反应或竞争性抑制。在Raji细胞系的对照RNA中，该检测定量let-7a为165 ± 21个拷贝、miR155为1,185 ± 141个拷贝；而在含混合细胞系的通用人类参考RNA中，在25 pg总RNA里测得let-7a为120 ± 14个拷贝、miR155为33 ± 8个拷贝。此外，该检测成功地将let-7a与密切相关的家族成员let-7b（相差两个核苷酸）和let-7c（相差单个核苷酸）区分开来。中间荧光（rain）是使用dPCR进行准确定量的主要挑战之一。为在本研究中获得可靠的定量结果，该检测需要每反应补充20-37.5 mM dNTP，以及优化的热循环参数。此外，为解决意外聚簇问题，多重检测需要为每个靶标使用不同的类TaqMan探针序列。总之，这些发现表明，先进的类TaqMan多重dPCR检测能够对临床相关miRNA进行灵敏、特异且可靠的定量，在生物标志物发现和癌症研究中具有潜在应用。*数据存档于Roche Diagnostics，Wilmington，MA，USA
查看英文原文 English abstract
MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression post transcriptionally. Detection of miRNAs remains technically challenging due to the short length and high sequence similarity among family members. Accurate and sensitive quantification methods are therefore desired for clinical research. Digital PCR (dPCR) provides absolute quantification without the need for standards, offering high sensitivity and tolerance to inhibitors. These features make dPCR a powerful tool for miRNA analysis. In this study, a single well multiplex assay for the simultaneous detection of miR155, let-7a, and U6 snRNA as an internal control was developed with an advanced TaqMan-like probe design. Target specific reverse primers were used for cDNA synthesis, with or without 10-12 nt artificial sequence at the 5' end, followed by using Roche Digital LightCycler® dPCR system with universal nanowell plates equipped with 28,000 partitions. Each target was amplified with four primers in optimized ratios. The TaqMan-like probe incorporated a target specific reverse primer extended with an additional 20-22 nt sequence labeled with a 5' fluorophore, paired with a complementary primer containing a 3' quencher to block extension. Synthetic mature miR155 and let-7a miRNAs, along with commercial cell line RNAs were used to test assay performance. Ten-fold serial dilutions of mature miRNAs demonstrated robust detection down to 4-6 copies per reaction in singleplex and multiplex for both targets. No cross reactivity or competitive inhibition was observed when one target was present at 1000-fold excess relative to the other through spiked samples. In control RNA from Raji cell line, the assay quantified let-7a at 165 ± 21 copies and miR155 at 1,185 ± 141 copies, while in universal human reference RNA with pooled cell lines, it measured let-7a at 120 ± 14 copies and miR155 at 33 ± 8 copies in 25 pg total RNA. Furthermore, the assay successfully discriminated let-7a from closely related family members let-7b (two nucleotides difference) and let-7c (single nucleotide difference). Intermediate fluorescence (rain) is one of the major challenges for accurate quantification by using dPCR. To achieve reliable quantification results in this study, assay required supplement with 20-37.5 mM dNTPs per reaction, as well as optimized thermocycling parameters. In addition, to address unexpected clusters, multiplexing required distinct TaqMan-like probe sequences for each target. Together, these findings demonstrate that advanced TaqMan-like multiplex dPCR assay provides sensitive, specific, and reliable quantification of clinically relevant miRNAs, with potential applications in biomarker discovery and cancer research.*Data on file at Roche Diagnostics, Wilmington, MA, USA
利益披露 Disclosure
Y. Zhao, None..
L. Cambria, None.