Introducing undergraduate students to real-time PCR

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Real-time PCR (polymerase chain reaction) is a powerful technique for quantifying gene expression.
• Undergraduate practical classes often face challenges in providing hands-on experience with sensitive molecular techniques.
• Murine erythroleukemia (MEL) cells offer a tractable model for studying cellular differentiation and gene regulation.

Purpose of the Study:

• To introduce third-year undergraduate Biochemistry students to real-time PCR within a biological context.
• To provide students with practical experience in RNA isolation, cDNA synthesis, and gene expression analysis.
• To demonstrate robust and reproducible changes in gene expression using a DMSO-induced differentiation model in MEL cells.

Main Methods:

• Utilized a murine erythroleukemia cell line (MEL cells) induced to differentiate with DMSO.
• Performed RNA isolation, purity, yield, and integrity checks (UV spectrophotometry, gel electrophoresis).
• Synthesized cDNA and performed real-time PCR to quantify relative gene expression of β globin, amino levulinate synthase, and carbonic anhydrase-1, using 18S rRNA as a reference.

Main Results:

• Observed robust and reproducible changes in the expression levels of target genes following DMSO treatment.
• Students successfully applied real-time PCR techniques, gaining experience with controls and reaction kinetics.
• The experiment provided consistent results, enhancing the undergraduate student experience with sensitive molecular techniques.

Conclusions:

• The described experiment effectively introduces undergraduate students to real-time PCR and gene expression analysis.
• The MEL cell differentiation model provides a suitable system for demonstrating significant and reproducible gene expression changes.
• This practical exercise enhances student understanding of molecular techniques and their application in biological research.