Guidelines for RNA Analysis by Reverse Transcription Quantitative Polymerase Chain Reaction

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) stands as an essential molecular biology tool for analyzing gene expression, offering a precise and accurate measure of gene expression levels. Its significance is critical in unraveling cellular processes and deciphering the complexities of disease mechanisms. This chapter details a comprehensive methodology for obtaining high-quality RNA from cell culture, followed by RNA extraction and column-based purification. After these steps, reverse transcription (RT) converts the RNA into complementary DNA (cDNA) before the quantitative PCR (qPCR) is performed. Protocols utilizing oligo(dT) and random hexamers for cDNA synthesis are described. Furthermore, we employed two different assays for qPCR: TaqMan and SYBR Green, to ensure a thorough examination of gene expression. These techniques are a valuable resource for researchers who want to study gene expression in a wide range of biological contexts.