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qPCR (RT-qPCR) critical

No amplification — Ct value missing or > 40

Symptom
Amplification plot is flat for both target and reference; software reports "no Ct" or Ct > 40 even for positive controls.
Common Causes
  1. 1 Template (RNA / cDNA) degraded or concentration too low
  2. 2 Reverse transcription failed (no cDNA generated)
  3. 3 Primer design problem or primer degraded
  4. 4 Reaction mix composition wrong (missing component, wrong dilution)
  5. 5 Instrument or program settings wrong
Solutions
  1. 1 Re-extract RNA, check RIN ≥ 7 by Bioanalyzer, requantify
  2. 2 Optimize RT conditions; use a positive control RNA to validate the RT step
  3. 3 Re-design primers or use validated published primers; check primer stock concentration
  4. 4 Re-prepare the master mix and double-check the cycling program
  5. 5 Verify the fluorescence channel (FAM / SYBR) is correctly selected
Related Video (3)
Thermo Fisher Scientific ★ 95
How to Isolate RNA: Total RNA Extraction Protocol for qPCR
"Directly addresses RNA extraction protocol—the primary cause of template degradation/low concentration that leads to missing Ct values and failed amplification."
Bilibili (China-Accessible Mirrors) ★ 78
qPCR Principles, Experimental Workflow and Results Analysis
"Comprehensive qPCR workflow and results analysis; shows how proper template preparation and positive controls should perform, enabling diagnosis of amplification failures."
Thermo Fisher Scientific ★ 72
How to Optimize qPCR using SYBR Green Assays - Ask TaqMan #38
"Covers qPCR optimization and troubleshooting including design and assay conditions that affect amplification sensitivity, relevant context for diagnosing no-amplification cases."
Source: xiaohongshu.com ↗
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