Introducing undergraduate students to real-time PCR

Abstract

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 globin, 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.