Why is RNA integrity critical for RT-PCR experiments?

• A. RNA integrity doesn't affect RT-PCR
• B. Degraded RNA leads to unreliable cDNA synthesis and biased gene expression measurements
• C. High integrity RNA decreases PCR efficiency
• D. RNA integrity only matters for sequencing

Answer: (B)

RNA integrity is crucial because RT-PCR depends on faithfully converting RNA into cDNA and then amplifying that cDNA to measure gene expression. When RNA is degraded, it breaks into fragments, so the pool of cDNA that gets produced no longer reflects the original RNA population. This fragmentation can make certain regions of a transcript absent or underrepresented, especially if the primers or the amplified region lie near a breakpoint created by degradation. As a result, the amount of cDNA you detect can be misleading, leading to biased or unreliable measures of expression that aren’t due to true biological differences. To get trustworthy results, you assess RNA quality, design assays that target short regions, and handle samples in RNase-free conditions, using appropriate controls to distinguish technical artifact from real changes in expression.