Assay Controls for qPCR Gene Expression Analysis

This technical report discusses the various types of assay controls essential for quantitative PCR (qPCR) experiments, focusing on their importance in ensuring reliable gene expression results. It outlines the use of synthetic DNA templates and specific assays such as the DNA Contamination Control Assay, Positive PCR Control Assay, RNA Quality Assay, and Reverse Transcription Control Assay. Each assay is described in terms of its purpose and methodology, emphasizing how they can be utilized to assess factors that may impact PCR results. The report also addresses the selection and validation of reference genes, which are critical for normalizing RT-qPCR results. It details a systematic approach for identifying suitable reference genes, including evaluating their stability across different experimental conditions. The report highlights the pitfalls of using housekeeping genes as reference genes, noting their variability and potential regulation by experimental conditions, which can compromise the accuracy of gene expression analysis.