Real-Time PCR And Gene Expression Specificity

Real-time PCR is a technique used to quantify gene-specific RNA transcripts, but it faces challenges such as unreliable gene expression predictions, reverse transcription vulnerabilities, and assay specificity issues. Specificity, the degree to which assay results reflect the target signal while excluding nontarget contributions, is critical. Nontarget PCR products, including nonspecific products and homologs (genes with sequence relationships to the target gene), can compromise specificity.

While high target transcript abundance can lower assay specificity requirements, low target transcript abundance necessitates higher specificity. TaqMan probe-based assays are often preferred over SYBR Green dye-based assays due to their higher specificity in gene expression analysis. This application note explores strategies to overcome specificity challenges and optimize gene expression analysis.