Home › Cell Biology › How to Isolate RNA: Total RNA Extraction Protocol for qPCR
Steps
  1. 1 Introduction to Total RNA Extraction --:--
  2. 2 Manual RNA Extraction Workflow 00:40
  3. 3 Reagent Preparation and Bead Binding 02:00
  4. 4 Washing and Elution Steps 03:15
  5. 5 Automated RNA Isolation with KingFisher Flex 04:30
  6. 6 RNA Purification for qPCR and Storage 06:00
Cell Biology Thermo Fisher Scientific

How to Isolate RNA: Total RNA Extraction Protocol for qPCR

Protocol

Total RNA extraction protocol suitable for downstream qPCR: cell/tissue lysis, organic-phase separation (or spin column), aqueous phase recovery, precipitation, wash, and elution.

Difficulty
intermediate
Total time
45-60 min
Biosafety
BSL-2

Steps

1
Introduction to Total RNA Extraction

Why total RNA extraction matters for downstream qPCR and sequencing; overview of magnetic-bead-based approaches that work both manually and on automated platforms.

▶ --:--
2
Manual RNA Extraction Workflow

Step-by-step manual workflow: starting from lysed cells or tissue, mix with magnetic-bead binding buffer, then capture RNA onto beads using a magnetic stand.

▶ 00:40
3
Reagent Preparation and Bead Binding

Prepare lysis, binding, wash, and elution buffers; pipette magnetic beads into the lysate; vortex to ensure RNA binds completely to bead surface.

▶ 02:00
4
Washing and Elution Steps

Use magnetic stand to hold beads while aspirating supernatant; wash twice with ethanol-based wash buffer to remove salts, proteins, and DNA; elute purified RNA in RNase-free water.

▶ 03:15
5
Automated RNA Isolation with KingFisher Flex

Switch to the KingFisher Flex platform for high-throughput, hands-free extraction: load plates, select the program, and let the instrument run binding, washes, and elution automatically.

▶ 04:30
6
RNA Purification for qPCR and Storage

Quantify the extracted RNA on a NanoDrop or Qubit; check 260/280 ratio (>1.8 = pure); aliquot and store at -80 C to preserve integrity for downstream qPCR.

▶ 06:00

🚨 Failure Case Library (7) + Submit your own case

critical
No amplification — Ct value missing or > 40
Amplification plot is flat for both target and reference; software reports "no Ct" or Ct > 40 even for positive controls.
💡 5 · ✓ 5
severe
Poor Ct reproducibility — high within-group variability
Technical replicates of the same sample have SD > 0.5 cycles; standard curve linearity is poor; loading order influences results.
💡 5 · ✓ 5
severe
Reference gene is unstable — normalization fails
Reference gene Ct varies > 1 cycle between samples; trend of reference vs target gene disagrees; normalization gives strange results.
💡 4 · ✓ 4
severe
Poor RNA Performance in Downstream Applications
Purified RNA fails to perform as expected in RT-PCR, RT-qPCR, RNA-seq library preparation, or other downstream assays despite acceptable concentration and A260/280 ratio.
💡 4 · ✓ 5
severe
Abnormal melt curve (multiple peaks or shoulder)
Melt curve shows multiple peaks or a shoulder, Tm does not match expectation, gel shows multiple bands.
💡 5 · ✓ 5
severe
Low RNA Yield After Cleanup
Recovered RNA concentration is significantly lower than expected based on input amount. Spectrophotometric measurement shows inadequate RNA quantity for downstream applications.
💡 5 · ✓ 5
moderate
Atypical amplification curves (not the canonical S-shape)
Curves cross threshold but are not the canonical sigmoidal shape — may have weird plateaus, early plateaus, or noisy baselines.
💡 5 · ✓ 5
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