RCA Protocol: Direct RNA Strategy¶
This is the direct RNA library construction protocol shared by all sample types (FF, FFPE) and readout technologies (PRISM, SPRINTseq). Padlock probes hybridize directly to mRNA, and SplintR ligase ligates the DNA nick on the RNA template. Start here after completing sample pre-treatment (Fresh Frozen or FFPE).
cDNA strategy
For the cDNA ligation strategy (reverse transcription before hybridization, with T4 DNA Ligase for SNP specificity), see RCA Protocol: cDNA.
- Version: v5.3
- Last modified: 2026-01-29
1. Blocking (~30 min)¶
Goal: Block nonspecific sites.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| RNase Free H₂O | 58.5 μl | ||
| Ampligase Buffer | 10X | 1X | 10 μl |
| KCl | 1 M | 0.05 M | 5 μl |
| Formamide | 100% | 20% | 20 μl |
| Oligo dT | 100 μM | 1 μM | 1 μl |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| tRNA | 10 μg/μl | 0.2 μg/μl | 2 μl |
| RiboLock | 40 U/μl | 1 U/μl | 2.5 μl |
- Incubate: Add Blocking Mix, incubate at RT for 30 min.
2. Hybridization (~2.5 h)¶
Goal: Probe-specific binding.
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 |
| RiboLock | 40 U/μl | 1 U/μl | 2.5 μl |
- Incubate: 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).
3. Ligation (~2 h)¶
Goal: Ligation of hybridized padlock probes into circles; SplintR ligase (PBCV-1 DNA Ligase) works on DNA:RNA hybrids.
| Reagent | Stock | Final | 100 μl |
|---|---|---|---|
| RNase Free H₂O | 76.5 μl | ||
| SplintR Buffer | 10X | 1X | 10 μl |
| Recombinant Albumin | 20 μg/μl | 0.2 μg/μl | 1 μl |
| SplintR Ligase | 25 U/μl | 2.5 U/μl | 10 μl |
| RiboLock | 40 U/μl | 1 U/μl | 2.5 μl |
- Incubate: 37°C for 2 hours (sealed).
- Wash: PBS-Tween 0.05% wash 2 x 1 min.
4. 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.
5. 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