Humes Sara T, Hentschel Shannon, Lavelle Candice M, Smith L Cody, Lednicky John A, Saleh Navid B, Sabo-Attwood Tara
Department of Environmental and Global Health, University of Florida.
Department of Molecular Genetics and Microbiology, University of Florida.
Biotechniques. 2017 Aug 1;63(2):81-84. doi: 10.2144/000114578.
Nanomaterials (NMs) of various types, including carbon nanotubes (CNTs), can interfere with standard quantitative real-time PCR (qRT-PCR) assays, resulting in inaccurate gene expression measurements; however, the precise step in the qRT-PCR pipeline where this interference occurs has not been well described. Here, we investigated where in the process surface-oxidized multi-walled CNTs (oxMWNTs) inhibited qRT-PCR measurement of the expression of the housekeeping gene GAPDH and explored several strategies to minimize such inhibition. We determined that the interference occurred during the reverse transcription (RT) step and found that doubling reaction reagents or adding BSA successfully mitigated the inhibition. We observed assay interference in the presence of CNTs that were surface-oxidized, but pristine CNTs did not cause the same level of interference. These results highlight the importance of monitoring qRT-PCR assays for interference by CNTs that differ by surface chemistry, as these NMs are commonly used in gene expression assays at concentrations that we have shown to be inhibitory.
包括碳纳米管（CNT）在内的各种类型的纳米材料（NM）会干扰标准的定量实时聚合酶链反应（qRT-PCR）检测，导致基因表达测量不准确；然而，qRT-PCR流程中这种干扰发生的确切步骤尚未得到很好的描述。在此，我们研究了表面氧化的多壁碳纳米管（oxMWNT）在该过程中的何处抑制管家基因GAPDH表达的qRT-PCR测量，并探索了几种将这种抑制降至最低的策略。我们确定干扰发生在逆转录（RT）步骤，并发现将反应试剂加倍或添加牛血清白蛋白（BSA）成功减轻了抑制作用。我们观察到在存在表面氧化的碳纳米管时检测受到干扰，但原始碳纳米管不会引起相同程度的干扰。这些结果凸显了监测qRT-PCR检测以防止因表面化学性质不同的碳纳米管产生干扰的重要性，因为这些纳米材料在基因表达检测中常用的浓度已被证明具有抑制作用。
