Departament de Ciència Animal i dels Aliments, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.
BMC Biotechnol. 2011 Jun 25;11:70. doi: 10.1186/1472-6750-11-70.
MicroRNAs are important regulators of gene expression at the post-transcriptional level and play an important role in many biological processes. Due to the important biological role it is of great interest to quantitatively determine their expression level in different biological settings.
We describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision in microRNA quantification.
MiR-specific quantitative RT-PCR with DNA primers is a highly specific, sensitive and accurate method for microRNA quantification.
MicroRNAs 是在后转录水平上对基因表达进行重要调控的因子，在许多生物过程中发挥着重要作用。由于其重要的生物学作用，定量确定其在不同生物环境中的表达水平具有重要意义。
我们描述了一种基于单个逆转录反应的用于 microRNAs 定量的 PCR 方法，该方法结合了实时 PCR 和两个 microRNA 特异性 DNA 引物。通过在引物上添加 DNA 尾巴来优化引物退火温度，并且可以以 94%的成功率进行设计。该方法能够定量检测超过 8 个数量级的合成模板，并且可以轻松区分具有单个核苷酸差异的 microRNAs。重要的是，与基于锁核酸（LNA）加标引物的类似方法相比，DNA 引物的 PCR 具有更高的生物样本扩增效率，这与 LNA 干扰短模板有效扩增的观察结果一致。DNA 引物的更高扩增效率转化为 microRNA 定量的更高灵敏度和精度。
MiR 特异性定量 RT-PCR 与 DNA 引物结合使用是一种高度特异、敏感和准确的 microRNA 定量方法。
