RCA Protocol: FFPE Tissue¶
- Applicable samples: FFPE
- Version: v1.0
- Last modified: 2026-01-29
Convention
In the following steps, "Jar" denotes a step performed in a separate EasyDip™ Slide Staining Jar previously decontaminated with RNaseZap®. Unless otherwise indicated, all steps are at room temperature (RT).
1. Baking (~16 h)¶
Goal: Adhere section to slide and stabilize tissue before downstream steps.
- Attach: One coverslip with a mounted FFPE section onto a microscope slide, taping the edge close to the sample area where the sample name is written.
- Bake: 56°C for 16 h.
2. Deparaffinization (~1 h)¶
Goal: Remove paraffin and hydrate tissue through graded solvents.
- Jar #1: Dewaxing agent (or xylene), 10 min x 2.
- Jar #2: 100% Ethanol, 10 min.
- Jar #3: Methanol--acetic acid 3:1 (v/v), 5 min.
- Jar #2: 100% Ethanol, 5 min.
- Jar #4: 85% Ethanol, 5 min.
- Jar #5: 70% Ethanol, 5 min.
- Jar #6: NFW, 3 min.
3. RNA Retrieval (~1 h)¶
Goal: Antigen/epitope retrieval to restore RNA accessibility.
- Jar #7: Sodium citrate 0.01 M pH 6 pre-warmed at 80°C; incubate 45 min at 80°C in a water bath.
- Jar #6: NFW, 5 min.
- Jar #5: 70% Ethanol, 5 min.
- Jar #4: 85% Ethanol, 5 min.
- Jar #2: 100% Ethanol, 5 min.
- Air-dry tissue sections at RT.
Note: At this point, sections can be stored dry for several days at +4°C.
- Assemble Chamber: Cover each tissue section with a PDMS chamber.
4. Rehydration (~20 min)¶
Goal: Rehydrate tissue for aqueous-based steps.
- Incubate: 100% Ethanol, 5 min.
- Incubate: 85% Ethanol, 5 min.
- Incubate: 70% Ethanol, 5 min.
- Incubate: NFW, 5 min.
5. Autofluorescence Quenching (~40 min)¶
Goal: Reduce autofluorescence with NaBH₄.
- Incubate: 1% (wt/vol) NaBH₄ (in 1X PBS) for 30 min.
- Wash: PBS-Tween 0.05% wash 3 x 2 min.
NaBH₄ Safety
Prepare 10 ml of 1% (wt/vol) NaBH₄ in 1X PBS on ice freshly each time. Use extreme caution when weighing; accidental introduction of liquid into the stock bottle can cause an explosive reaction. Gas bubbles form continuously in the chamber -- aspirate and replenish with fresh solution every 1--2 min during the incubation.
6. Digestion (~20 min)¶
Goal: Enhance probe access.
- Incubate: 0.04% Pepsin (in 0.1 M HCl) at 37°C for 10 min.
- Wash: PBS-Tween 0.05% wash 3 x 2 min.
7. Blocking (~30 min)¶
Goal: Block nonspecific sites.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| NFW | 56.5 μl | ||
| Ampligase Buffer | 10X | 1X | 10 μl |
| KCl | 1 M | 0.05 M | 5 μl |
| Formamide (deionized) | 100% | 20% | 20 μl |
| Oligo dT | 100 μM | 1 μM | 1 μl |
| tRNA (e.g. Ambion AM7119) | 10 μg/μl | 0.2 μg/μl | 2 μl |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| RiboLock (Thermo) | 40 U/μl | 1 U/μl | 2.5 μl |
| RNase H | 5 U/μl | 0.1 U/μl | 2 μl |
- Add blocking solution into the chamber, mix by gentle pipetting (3--5 times).
- Incubate: RT for 30 min.
8. Hybridization of Padlock Probes (~3 h)¶
Goal: Probe-specific binding to target mRNA.
Probe volume
Probe stock concentration and volume depend on the specific probe panel. Adjust to achieve the desired final concentration (typically 0.05--0.2 μM per probe). Fill NFW to 100 μl total.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| NFW | to 100 μl | ||
| Ampligase Buffer | 10X | 1X | 10 μl |
| KCl | 1 M | 0.05 M | 5 μl |
| Formamide (deionized) | 100% | 20% | 20 μl |
| Probes | (panel-dependent) | 0.05--0.2 μM | (variable) |
| tRNA (e.g. Ambion AM7119) | 10 μg/μl | 0.2 μg/μl | 2 μl |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| RiboLock (Thermo) | 40 U/μl | 1 U/μl | 2.5 μl |
- Add solution into the chamber, mix by gentle pipetting (3--5 times) and seal.
- Denaturation: 55°C for 20 min; Hybridization: 45°C for 120 min.
- Stringent Wash: washing buffer 3 x 10 min to remove unhybridized probes. Washing buffer: 100 µL 20X SSC + 100 µL formamide + 800 µL NFW.
- Wash: PBS-Tween 0.05% wash 3 x 1 min to remove residual formamide (to avoid deactivating the enzyme in the next step).
9. Ligation of Padlock Probes (~2 h)¶
Goal: Ligation of hybridized padlock probes; SplintR ligase (PBCV-1 DNA Ligase) works on DNA:RNA hybrids.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| NFW | 76.5 μl | ||
| SplintR Buffer | 10X | 1X | 10 μl |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| SplintR Ligase (NEB) | 25 U/μl | 2.5 U/μl | 10 μl |
| RiboLock (Thermo) | 40 U/μl | 1 U/μl | 2.5 μl |
- Add solution into the chamber, mix by gentle pipetting (3--5 times) and seal.
- Incubate: 37°C for 2 h (sealed).
- Wash: PBS-Tween 0.05% wash 2 x 1 min.
10. Rolling Circle Amplification (RCA, ~3--17 h)¶
Goal: Amplify signal.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| NFW | 65.5 μl | ||
| Phi29 Buffer | 10X | 1X | 10 μl |
| Glycerol | 100% | ~10% | 10 μl |
| dNTPs | 10 mM | 0.25 mM | 2.5 μl |
| Amino-dUTP | 2 mM | 50 μM | 2.5 μl |
| RCA Primer | 10 μM | 0.3 μM | 3 μl |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| Phi29 Polymerase | 10 U/μl | 0.25 U/μl | 2.5 μl |
| RiboLock (Thermo) | 40 U/μl | 1 U/μl | 2.5 μl |
- Add solution into the chamber, mix by gentle pipetting (3--5 times) and seal.
- Incubate: 30°C for 12--16 h (sealed / humid chamber).
- Wash: PBS-Tween 0.05% wash 2 x 1 min.
11. Post-amplification Fix¶
Goal: Fix RCA products and strip unbound probes.
- Add 4% PFA, mix by gentle pipetting (3--5 times); Incubate: RT for 15--30 min to fix RCA products.
- Wash: PBS-Tween 0.05% wash 3 x 1 min.
- Strip: 65% formamide wash 3 x 2--10 min at 30°C (e.g. on a PCR block or Eppendorf Thermostat). Washing buffer: 650 µL formamide + 350 µL NFW.
- Wash: PBS-Tween 0.05% wash 2 x 1 min.
12. Autofluorescence Bleach¶
Goal: Quench autofluorescence (optional; may reduce signal intensity).
Sudan Black B (storage): 0.1% (wt/vol) in 70% EtOH (e.g. 10 mg in 10 ml), shake at RT for days and filter before use.
- Incubate: 0.1% Sudan Black B (in 70% EtOH, filtered) at RT for 10 min.
- Wash: 70% EtOH wash 10 x 3 min.