Hematopathology Laboratory, ACTREC, Tata Memorial Centre, Homi Bhabha National Institute, Kharghar, Navi Mumbai, India.
Hasan Laboratory, ACTREC, Tata Memorial Centre, Homi Bhabha National Institute, Kharghar, Navi Mumbai, India.
Biochem Med (Zagreb). 2021 Oct 15;31(3):030705. doi: 10.11613/BM.2021.030705.
MicroRNAs are small, non-coding RNA molecules that are becoming popular biomarkers in several diseases. However, their low abundance in serum/plasma poses a challenge in exploiting their potential in clinics. Several commercial kits are available for rapid isolation of microRNA from plasma. However, reports guiding the selection of appropriate kits to study downstream assays are scarce. Hence, we compared four commercial kits to evaluate microRNA-extraction from plasma and provided a modified protocol that further improved the superior kit's performance.
We compared four kits (miRNeasy Serum/Plasma, miRNeasy Mini Kit from Qiagen; RNA-isolation, and Absolutely-RNA MicroRNA Kit from Agilent technologies) for quality and quantity of microRNA isolated, extraction efficiency, and cost-effectiveness. Bioanalyzer-based Agilent Small RNA kit was used to evaluate quality and quantity of microRNA. Extraction efficiency was evaluated by detection of four endogenous control microRNA using real-time-PCR. Further, we modified the manufacturer's protocol for miRNeasy Serum/Plasma kit to improve yield.
miRNeasy Serum/Plasma kit outperformed the other three kits in microRNA-quality (P < 0.005) and yielded maximum microRNA-quantity. Recovery of endogenous control microRNA hsa-miR-24-3p, hsa-miR-191-5p, hsa-miR-423-5p and hsa-miR-484 was higher as well. Modification with the inclusion of a double elution step enhanced yield of microRNA extracted with miRNeasy Serum/Plasma kit significantly (P < 0.001).
We demonstrated that miRNeasy Serum/Plasma kit outperforms other kits and can be reliably used with a limited plasma quantity. We have provided a modified microRNA-extraction protocol with improved microRNA output for downstream analyses.
MicroRNAs 是小的非编码 RNA 分子，在几种疾病中成为流行的生物标志物。然而，它们在血清/血浆中的低丰度给利用其在临床上的潜力带来了挑战。有几种商业试剂盒可用于从血浆中快速分离 microRNA。然而，指导选择合适试剂盒进行下游分析的报告很少。因此，我们比较了四种商业试剂盒，以评估从血浆中提取 microRNA 的效果，并提供了一种改良的方案，进一步提高了最佳试剂盒的性能。
我们比较了四种试剂盒（Qiagen 的 miRNeasy Serum/Plasma 和 miRNeasy Mini Kit；Agilent technologies 的 RNA 分离和 Absolutely-RNA MicroRNA Kit）在提取的 microRNA 质量和数量、提取效率和成本效益方面的表现。使用基于 Bioanalyzer 的 Agilent Small RNA 试剂盒评估 microRNA 的质量和数量。通过实时 PCR 检测四种内源性对照 microRNA 来评估提取效率。此外，我们修改了 miRNeasy Serum/Plasma 试剂盒制造商的方案，以提高产量。
miRNeasy Serum/Plasma 试剂盒在 microRNA 质量（P < 0.005）和提取的 microRNA 数量方面优于其他三种试剂盒。内源性对照 microRNA hsa-miR-24-3p、hsa-miR-191-5p、hsa-miR-423-5p 和 hsa-miR-484 的回收率也更高。通过包括双洗脱步骤的修改，显著提高了 miRNeasy Serum/Plasma 试剂盒提取的 microRNA 产量（P < 0.001）。
我们证明 miRNeasy Serum/Plasma 试剂盒优于其他试剂盒，并且可以在有限的血浆量下可靠地使用。我们提供了一种改良的 microRNA 提取方案，可提高下游分析的 microRNA 产量。
