RCA Protocol: cDNA Strategy¶
This protocol uses the cDNA ligation strategy: mRNA is first reverse-transcribed to cDNA, then padlock probes hybridize to the cDNA strand and are ligated by T4 DNA Ligase. Compared to the direct RNA strategy, this approach offers SNP specificity at the cost of lower detection efficiency (~⅕ of dRNA). See Library Construction Strategies for a full comparison.
- Applicable samples: Fresh Frozen (FF) tissue sections
- Version: v1.0
- Last modified: 2026-04-09
Pre-treatment
Complete sample pre-treatment (Fresh Frozen) before starting this protocol. Pre-treatment steps (sectioning, fixation, permeabilization, dehydration) are identical to the direct RNA workflow.
1. Reverse Transcription (~6 h to overnight)¶
Goal: Convert mRNA to cDNA using random-primed reverse transcription.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| RNase Free H₂O | 69.5 μl | ||
| RT Buffer | 10X | 1X | 10 μl |
| dNTPs | 25 mM | 500 μM | 2 μl |
| Random Decamers | 100 μM | 5 μM | 5 μl |
| BSA | 20 μg/μl | 0.2 μg/μl | 1 μl |
| RNase Inhibitor (RiboLock) | 40 U/μl | 1 U/μl | 2.5 μl |
| Reverse Transcriptase (TranscriptME) | 200 U/μl | 20 U/μl | 10 μl |
- Incubate: 37°C for 6 h to overnight (sealed / humid chamber).
Primer choice
Random decamers provide unbiased priming across the transcriptome. Target-specific LNA primers can also be used for higher efficiency on selected targets; in that case, 1--2 h at 42°C is typically sufficient.
Warning
Do not wash between RT and the next fixation step. Proceed directly to post-RT fixation.
2. Post-RT Fixation (~40 min)¶
Goal: Fix cDNA in place before subsequent enzymatic steps.
- Remove RT reagents carefully from chamber.
- Fix: Apply 3% formaldehyde in PBS directly (no wash in between), incubate at RT for 40 min.
- Wash: PBS wash 2 x 2 min.
Pause point
After post-RT fixation, samples can be stored at 4°C in PBS for a few days.
3. Probe Hybridization with RNase H Digestion (~2.5 h)¶
Goal: RNase H digests the RNA strand of the RNA:cDNA hybrid, exposing single-stranded cDNA. Padlock probes then hybridize to the cDNA template. Both reactions proceed in the same mix.
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 H₂O to 100 μl total.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| RNase Free H₂O | to 100 μl | ||
| Ampligase Buffer | 10X | 1X | 10 μl |
| KCl | 1 M | 0.05 M | 5 μl |
| Formamide | 100% | 20% | 20 μl |
| Probes | (panel-dependent) | 0.05--0.2 μM | (variable) |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| tRNA | 10 μg/μl | 0.2 μg/μl | 2 μl |
| RNase H | 5 U/μl | 0.4 U/μl | 8 μl |
- Incubate: 37°C for 30 min (RNase H digestion) → 55°C (15--20 min denaturation) → 45°C (120 min hybridization). Seal to prevent evaporation.
- Stringent Wash: 10% Formamide in 2X SSC, 3 x 10 min (45°C optimal, or RT). Washing buffer: 100 µL 20X SSC + 100 µL formamide + 800 µL NFW.
- Wash: PBS-Tween 0.05% wash 2--3 x 1 min (remove residual formamide to avoid deactivating ligase).
4. Ligation (~2 h)¶
Goal: Ligation of hybridized padlock probes into circles; T4 DNA Ligase works on DNA:DNA hybrids (cDNA template), providing SNP discrimination at the ligation junction.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| RNase Free H₂O | 77 μl | ||
| T4 DNA Ligase Buffer | 10X | 1X | 10 μl |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| ATP | 10 mM | 0.1 mM | 1 μl |
| T4 DNA Ligase | 5 U/μl | 0.5 U/μl | 10 μl |
- Incubate: 37°C for 2 hours (sealed).
- Wash: PBS-Tween 0.05% wash 2 x 1 min.
ATP
T4 DNA Ligase requires ATP as a cofactor. Most commercial T4 DNA Ligase buffers already contain ATP; check your buffer composition and add ATP only if not included.
Why T4 DNA Ligase?
Unlike SplintR Ligase (used in the direct RNA protocol for DNA:RNA hybrids), T4 DNA Ligase operates on DNA:DNA substrates and provides single-nucleotide mismatch discrimination at the ligation junction, enabling SNP-level specificity.
5. Rolling Circle Amplification (~12--16 h)¶
Goal: Amplify circularized probes into DNA nanoballs (rolonies).
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| RNase Free H₂O | 63.5 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 |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| RCA Primer | 10 μM | 0.3 μM | 3 μl |
| Phi29 Polymerase | 10 U/μl | 0.25 U/μl | 2.5 μl |
| RiboLock | 40 U/μl | 1 U/μl | 2.5 μl |
- Incubate: 30°C for 12--16 h overnight (sealed / humid chamber).
- Wash: PBS-Tween 0.05% wash 2 x 1 min.
6. Post-amplification Fix & Strip¶
Goal: Fix RCA products and strip unbound probes.
- Fix: Add 4% PFA, mix by gentle pipetting (3--5 times), incubate 15--30 min at RT.
- 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.
Autofluorescence Quenching (Optional)¶
When to use
This step is optional and depends on sample autofluorescence. FFPE samples almost always require it. Some FF samples (e.g. tissues with high lipofuscin content) may also benefit. Perform before signal readout (PRISM imaging or SPRINTseq sequencing).
Sudan Black B:
Stock solution: 0.1% (wt/vol) Sudan Black B in 70% EtOH (e.g. 10 mg in 10 ml). Shake at RT for days, filter before use.
- Incubate: RT for 10 min.
- Wash: 70% EtOH wash 10 x 3 min.
- Wash: PBS-Tween 0.05% wash 2 x 1 min.
Warning
Sudan Black B may reduce signal intensity. Test on control samples first.
Next step: Signal readout -- PRISM Imaging or SPRINTseq Sequencing