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Introducing Undergraduate Students to Real-Time PCR

机译：向大学生介绍实时PCR

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An experiment is conducted, which in four 3 h laboratory sessions, introduces third year undergraduate Biochemistry students to the technique of real-time PCR in a biological context. The model used is a murine erythroleukemia cell line (MEL cells). These continuously cycling, immature red blood cells, arrested at an early stage in erythropoiesis, can be induced to progress further through the process by 72 h exposure to 1.8% DMSO. This gives a control cell sample and a DMSO-treated preparation. Students isolate RNA from both cell cultures, check its purity, yield and integrity by UV spectrophotometry and denaturing gel electrophoresis, then synthesized cDNA. The relative levels of three sequences: β glo-bin, amino levulinate synthase, and carbonic anhydrase-1 are estimated by real-time PCR, using 18S rRNA as the reference sequence. The changes in gene expression are robust and reproducible, enabling students to experience a "cutting edge" research technique in an undergraduate lab setting. While the undergraduate student experience in practical classes with such sensitive techniques is often mixed, the changes in gene expression in this model are sufficiently great that students can gain the satisfaction of consistent results. In addition they gain experience at setting up checks and controls at stages throughout a multistep process and an appreciation of the difference between a reaction which has gone to completion with one that is measured as a rate. This experiment would also complement cell biology projects involving red cell development. It could also be extended to more thoroughly investigate the technique of real-time PCR.
机译：进行了一个实验，在四个3小时的实验室会议中，向三年级生物化学专业的本科生介绍了在生物环境中进行实时PCR的技术。使用的模型是鼠类红细胞白血病细胞系（MEL细胞）。这些持续循环的，未成熟的红细胞，在红细胞生成的早期阶段就被阻滞，在暴露于1.8％DMSO的72小时中，可以诱导其进一步进展。这给出了对照细胞样品和DMSO处理的制剂。学生从两种细胞培养物中分离RNA，通过紫外分光光度法和变性凝胶电泳检查其纯度，产量和完整性，然后合成cDNA。使用18S rRNA作为参考序列，通过实时PCR估算了3个序列的相对水平：βglo-bin，氨基乙酰丙酸合酶和碳酸酐酶-1。基因表达的变化是健壮且可重现的，使学生能够在本科实验室环境中体验“前沿”研究技术。尽管本科生在使用此类敏感技术的实践课上的经历常常是混杂的，但是该模型中基因表达的变化足够大，以至于学生可以获得满意的一致结果。此外，他们在整个多步骤过程的各个阶段都建立检查和控制的过程中积累了经验，并且对以速率进行测量的反应完成之间的差异有所了解。该实验还将补充涉及红细胞发育的细胞生物学项目。它也可以扩展到更全面地研究实时PCR技术。

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