qPCR for second year undergraduates: A short, structured inquiry to illustrate differential gene expression

Abstract

We describe a structured inquiry laboratory exercise that examines transcriptional regulation of the NOS2 gene under conditions that simulate the inflammatory response in macrophages. Using quantitative PCR and the comparative C T method, students are able determine whether transcriptional activation of NOS2 occurs and to what degree. The exercise is aimed at second year undergraduates who possess basic knowledge of gene expression events. It requires only 4-5 hr of dedicated laboratory time and focuses on use of the primary literature, data analysis, and interpretation. Importantly, this exercise provides a mechanism to introduce the concept of differential gene expression and provides a starting point for development of more complex guided or open inquiry projects for students moving into upper level molecular biology, immunology, and biochemistry course work. V C 2015 by the International Union of Biochemistry and Molecular Biology, 43(4): 273-282, 2015.

An undergraduate laboratory experiment with real‐world applications: Utilizing templateless polymerase chain reaction and real‐time polymerase chain reaction to test for SARS‐CoV‐2 RNA

“…27 Positive controls could be generated for other genes (Supporting Information). The qPCR can be done quantitatively, as demonstrated by Hancock et al 13 or McCauslin et al 15 This would require better quantitation of the positive control and could include a spike-in of a small amount of the positive control in sample duplicates to rule out interfering substances and verify quantitation. In future years, SARS-CoV-2 will be less of a health concern, but these methods could be easily adapted to coronaviruses such as HCoV-OC43 that cause the common cold, 28 or other mild viral pathogens by simply changing the sequences for the positive control and for the primers.…”

“…While some of these sources use the acronym RT-PCR to stand for real-time PCR, 12,13 others use RT-PCR to stand for reverse transcription PCR 14 and some just refer to the process as qPCR. 15 Some COVID testing protocols use the term RT-qPCR, 16 but our procedure is not a truly quantitative PCR (qPCR) because we did not calculate starting RNA calculations nor use a housekeeping gene for comparison of gene expression; thus, we will refer to the technique as RT-PCR. In our experiment, students investigated the presence of SARS-CoV-2 RNA on high-touch surfaces by swabbing them and performing RT-PCR on the samples.…”

Stressing Escherichia coli to educate students about research: A CURE to investigate multiple levels of gene regulation

“…Many laboratory modules center on central dogma of biology , one type of regulation of gene expression or even multiple types of regulation . However, CURES that simultaneously illustrate different types of regulation mechanisms and the process of discovering new regulatory factors are scarce.…”