Practice question

Quantification of mRNA requires conversion of labile RNA into stable complementary DNA followed by exponential amplification and real-time detection. Reverse transcription using oligo-dT, random hexamers or gene-specific primers with reverse transcriptase generates cDNA proportional to original transcript abundance. Subsequent quantitative PCR measures Ct values inversely related to initial copy number, allowing relative or absolute quantification via standard curves and reference genes. Southern blotting detects DNA, FISH visualizes localization, and microarrays provide hybridization profiles with lower dynamic range. RT-qPCR remains gold standard for sensitivity, specificity and wide dynamic range in gene expression studies.