No amplification — Ct value missing or 40 (qPCR (RT-qPCR))

Question

No amplification — Ct value missing or > 40 (qPCR (RT-qPCR))

Accepted Answer

Amplification plot is flat for both target and reference; software reports "no Ct" or Ct > 40 even for positive controls. Common causes: 1) Template (RNA / cDNA) degraded or concentration too low 2) Reverse transcription failed (no cDNA generated) 3) Primer design problem or primer degraded 4) Reaction mix composition wrong (missing component, wrong dilution) 5) Instrument or program settings wrong Solutions: 1) Re-extract RNA, check RIN ≥ 7 by Bioanalyzer, requantify 2) Optimize RT conditions; use a positive control RNA to validate the RT step 3) Re-design primers or use validated published primers; check primer stock concentration 4) Re-prepare the master mix and double-check the cycling program 5) Verify the fluorescence channel (FAM / SYBR) is correctly selected