Kunwar Arzu, Ablordeppey Kenny Kwabena, Mireskandari Alidad, Sheinerman Kira, Kiefer Michael, Umansky Samuil, Kumar Gyanendra
DiamiR Biosciences Laboratory, 2 Church Street South, Suite B05, New Haven, CT 06519, USA.
Diagnostics (Basel). 2023 Jun 26;13(13):2170. doi: 10.3390/diagnostics13132170.
We have been developing a novel approach to identify cognitive impairment-related biomarkers by profiling brain-enriched and inflammation-associated microRNA (miRNA) in plasma specimens of cognitively unimpaired and cognitively impaired patients. Here, we present an analytical validation of the novel miRNA panel, CogniMIR, using two competing quantitative PCR technologies for the expression analysis of 24 target miRNAs. Total RNA from the plasma specimens was isolated using the MagMAX mirVana Kit, and RT-qPCR was performed using stem-loop-based TaqMan and LNA-based qPCR assays. Evaluation of RNA dilution series for our target 24 miRNAs, performed by two operators on two different days, demonstrated that all CogniMIR panel miRNAs can be reliably and consistently detected by both qPCR technologies, with sample input as low as 20 copies in a qPCR reaction. Intra-run and inter-run repeatability and reproducibility analyses using RNA specimens demonstrated that both operators generated repeatable and consistent Cts, with R values of 0.94 to 0.99 and 0.96 to 0.97, respectively. The study results clearly indicate the suitability of miRNA profiling of plasma specimens using either of the qPCR technologies. However, the LNA-based qPCR technology appears to be more operationally friendly and better suited for a CAP/CLIA-certified clinical laboratory.
我们一直在开发一种新方法，通过对认知功能正常和认知功能受损患者的血浆样本中脑富集且与炎症相关的微小RNA（miRNA）进行分析，来识别与认知障碍相关的生物标志物。在此，我们使用两种竞争性定量PCR技术对24种靶标miRNA进行表达分析，对新型miRNA检测板CogniMIR进行了分析验证。使用MagMAX mirVana试剂盒从血浆样本中分离总RNA，并使用基于茎环的TaqMan和基于锁核酸（LNA）的qPCR检测方法进行逆转录定量PCR（RT-qPCR）。由两名操作人员在两天内对我们的24种靶标miRNA的RNA稀释系列进行评估，结果表明，两种qPCR技术均可可靠且一致地检测到所有CogniMIR检测板miRNA，qPCR反应中的样本输入低至20个拷贝。使用RNA样本进行的批内和批间重复性及再现性分析表明，两名操作人员均产生了可重复且一致的Ct值，R值分别为0.94至0.99和0.96至0.97。研究结果清楚地表明，使用任何一种qPCR技术对血浆样本进行miRNA分析都是合适的。然而，基于LNA的qPCR技术在操作上似乎更简便，更适合经美国病理学家协会（CAP）/临床实验室改进修正案（CLIA）认证的临床实验室。
