Extraction & Purification of
RNA from FFPE Tissue
A Mechanistic Guide to the Silica Spin-Column Protocol
Follow this interactive walkthrough to understand every step of the protocol — what you do, and why it works at the molecular level.
The Protocol Blueprint
Preparation
- 1 Deparaffinization
- 2 Lysis & Crosslink Reversal
DNA Removal
- 3 DNase Digestion
Purification
- 4 RNA Binding
- 5 Stringent Wash
- 6 Desalting Wash
- 7 Dry Spin
Elution
- 8 RNA Elution
Step 1: Deparaffinization (Wax Removal)
Action
What You DoAdd a deparaffinization solvent to the pre-prepared FFPE tissue samples, followed by centrifugation and ethanol washes.
Function
Why It WorksDissolves and completely removes the embedding paraffin wax. This exposes the biological tissue, allowing the subsequent aqueous buffers and enzymes to effectively penetrate the cells.
Step 2: Tissue Lysis & Reverse Crosslinking
Action
What You DoAdd Tissue Lysis Buffer (optimized for RNA) and Proteinase K, then incubate at specific elevated temperatures.
Function
Why It WorksThe buffer and enzyme work together to digest cellular proteins and instantly inactivate destructive RNases. The heat incubation is crucial for reversing formalin-induced cross-links, freeing the fragile RNA molecules from protein complexes without degrading them.
Step 3: DNase Digestion (Genomic DNA Removal)
Action
What You DoApply DNase I enzyme (can be done in-solution or directly on-column depending on the kit).
Function
Why It WorksSpecifically degrades massive genomic DNA strands into tiny, soluble fragments that will later wash away. Without this, gDNA would co-purify and severely contaminate the final RNA yield.
Step 4: RNA Binding to Silica
Action
What You DoAdd Binding Buffer (high chaotropic salt concentration) and pure Ethanol to the sample, then transfer to a silica spin-column. Centrifuge.
Function
Why It WorksEthanol and chaotropic salts dehydrate the RNA and disrupt the hydration shell of the silica membrane. This forces the negatively charged RNA to form a strong "salt bridge" with the silica, anchoring the RNA to the column while contaminants flow through.
Step 5: Stringent Wash (Removing Protein/Salt)
Action
What You DoApply Wash Buffer 1 (contains moderate chaotropic salts and ethanol) to the column. Centrifuge.
Function
Why It WorksWashes away residual proteins, lipids, and some chaotropic salts. The ethanol ensures RNA stays tightly bound to the silica while contaminants are released.
Step 6: Desalting Wash
Action
What You DoApply Wash Buffer 2 (mostly ethanol) to the column. Centrifuge.
Function
Why It WorksRemoves any remaining chaotropic salts from the previous buffers. High ethanol content maintains the RNA-silica bond.
Step 7: Dry Spin
Action
What You DoCentrifuge the column at max speed for 1-2 minutes with the lid open or in a fresh empty tube.
Function
Why It WorksCompletely removes any residual ethanol from the wash buffers. Ethanol carryover can inhibit downstream enzymatic reactions (like PCR or sequencing).
Step 8: Elution (Pure RNA Extraction)
Action
What You DoTransfer the column to a fresh RNase-free tube. Add RNase-free water directly to the center of the silica membrane. Incubate briefly, then centrifuge.
Function
Why It WorksThe water lacks chaotropic salts and ethanol, which rehydrates the silica membrane and breaks the salt bridge. The pure RNA detaches from the membrane and flows into the collection tube.
Protocol Complete!
You have successfully extracted pure, intact RNA from FFPE tissue. The RNA is now ready for downstream applications such as RT-qPCR or RNA-Seq.