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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, ligating on a DNA:DNA substrate gives better junction specificity and lower self-ligation, at the cost of detection efficiency (~⅕ of dRNA). See Library Construction Strategies for a full comparison.

This protocol is not sufficient for SNP genotyping

T4 DNA Ligase at 37°C is the right general-purpose choice here, but it is not accurate enough to call single-nucleotide variants -- in a 256-junction benchmark it mis-ligated 78 of the single-mismatch substrates at >50% yield.3 Allele discrimination needs a thermostable high-fidelity ligase run at elevated temperature (Tth DNA Ligase, or NEB HiFi Taq DNA Ligase). See Ligase Fidelity.

  • Applicable samples: Fresh Frozen (FF) tissue sections
  • Version: v3.0
  • Last modified: 2026-08-18

Materials

Ordering: Bill of Materials → RCA (Library Construction) — see the Enzymes — cDNA route only table for the RT enzyme and ligase. Prepared ahead of time: PDMS Chambers · Pepsin Solution. Strategy choice: Library Construction Strategies. Buffer chemistry: Reaction Buffers & Stringency.

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 priming, supplemented with target-specific primers for low-abundance transcripts.

Reagent Stock Final 100 μl
RNase Free H₂O 63.5 μl
5X RT Buffer 5X 1X 20 μl
dNTPs 25 mM 500 μM 2 μl
Random Decamers 100 μM 5 μM 5 μl
Custom RT Primer(s) 10 μM 0.1 μM each 1 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 (Maxima H Minus) 200 U/μl 10 U/μl 5 μl
  • Incubate: 50°C for 6 h to overnight (sealed / humid chamber).

Why Maxima H Minus

Maxima H Minus is an engineered M-MuLV reverse transcriptase that is RNase H-minus, thermostable and highly processive.2 The RNase H-minus property is the reason it is the right enzyme for this route: the RNA strand of the RNA:cDNA hybrid must survive intact through a long incubation and be removed deliberately, by RNase H in Step 3. An RT carrying residual RNase H activity nicks its own template as it goes and costs full-length cDNA yield. The enzyme also incorporates modified nucleotides and is 90% active after 60 min at 50°C.

Buffer strength -- 5X, not 10X

Maxima H Minus ships with a 5X RT Buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl₂, 50 mM DTT), hence 20 μl per 100 μl. If you substitute a 10X RT buffer, use 10 μl and make up the difference with water.

Primer strategy -- random decamers plus custom primers

Random decamers prime the transcriptome without bias, but leave low-abundance transcripts under-sampled. Spiking in target-specific RT primers at 100 nM each on top of the decamers raises local cDNA yield at those loci and measurably improves detection of low-abundance mRNAs.

Design them to anneal 3' of (downstream of) the padlock target site on the mRNA, so reverse transcription reads through the region the padlock arms need to bind. For a pool of several custom primers, keep each at 100 nM and scale the volume accordingly (taking it out of the water).

Incubation temperature -- 50°C, long

The vendor in-tube protocol runs 50°C for 15--30 min.2 In situ we keep the temperature but stretch the time: 50°C, 6 h to overnight. 50°C is the enzyme's working temperature (90% active after 60 min there) and, just as importantly, it melts mRNA secondary structure so the RT reads through structured regions and past the padlock target site. The long incubation is what covers diffusion into fixed tissue -- that is the limiting factor, not enzyme turnover. Dropping the temperature to buy gentleness costs read-through instead, and on this route read-through is the padlock template. Seal properly: evaporation over a long 50°C incubation is the real failure mode -- use a humid chamber and check the seal before walking away.

Earlier v1.0 of this protocol ran 37°C for the same 6 h to overnight; 50°C is the current bench condition.

Warning

Do not wash between RT and the next fixation step. Proceed directly to post-RT fixation.

2. Post-RT Fixation (~30 min)

Goal: Fix cDNA in place before subsequent enzymatic steps.

  • Remove RT reagents carefully from chamber.
  • Fix: Apply 4% PFA in PBS directly (no wash in between), incubate at RT for 30 min.
  • Wash: PBS wash 2 x 2 min.

Why 30 min, not 15

30 min is the condition the ISS lineage this protocol comes from uses (3.7% PFA in DEPC-PBS, RT, 30 min1) and it is not over-fixation. Formaldehyde acts in two stages: methylol addition is fast (minutes), but the methylene bridges that actually crosslink take hours -- complete crosslinking is a 24--48 h process. At 15--30 min you are still on the rising part of that curve, so the difference between 15 and 30 min is real, and both are two orders of magnitude away from the prolonged-formalin regime that is known to block nucleic-acid detection. Err long, not short: this fixation has to hold the cDNA through the 45°C overnight hybridization and 3 x 10 min 10% formamide stringent wash -- the harshest stretch of the whole workflow. Under-anchored cDNA washes away and costs signal.

3. Probe Hybridization with RNase H Digestion (~2.5 h, or overnight)

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 concentration and hybridization time -- two working points

Preferred: 10 nM per probe, overnight (~16 h) at 45°C. The low probe concentration substantially reduces nonspecific probe deposition, and the long incubation recovers on-target yield -- worth more here than on the direct-RNA route, since the cDNA route starts from a smaller pool of targets.

Same-day: 50 nM per probe, 120 min at 45°C. Use when the run has to finish in one day; expect more nonspecific background.

Probe stock concentration and pooled-panel volume are panel-dependent -- compute the volume that reaches the target per-probe final concentration and 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) 10 nM (overnight) / 50 nM (2 h) (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 (~16 h overnight at 10 nM, or 120 min at 50 nM). Seal to prevent evaporation -- for the overnight condition use a humid chamber.
  • Stringent Wash: 10% formamide in 2X SSC, 3 x 10 min, at room temperature. Washing buffer: 100 µL 20X SSC + 100 µL formamide + 800 µL NFW.
  • Wash: PBS-Tween 0.05% wash 3 x 2 min (10% formamide is ~2.5 M, and 2X SSC carries 390 mM Na⁺ — both cost ligase activity on carry-over. See Reaction Buffers & Stringency).

Pin the temperature, and note this route is the more formamide-sensitive one

Room temperature, not 45 °C — the two are ~20 °C apart in effective stringency, against ~5 °C for the 10%-vs-20% formamide difference.

On this route the padlock binds cDNA, so the duplex is DNA:DNA and formamide destabilizes it more per percent than the DNA:RNA duplex of the direct-RNA route — roughly 0.6--0.7 °C/% against 0.5. The wash also lands while the padlock is still linear, before ligation locks it into a circle. Both reasons point the same way: do not raise this wash without a control arm.

20% formamide is the high-stringency arm, not the default

Available as a deliberate comparison — washing buffer: 100 µL 20X SSC + 200 µL formamide + 700 µL NFW. Record its use in the run notes; yield and background both move, so data from the two conditions are not directly comparable.

Yes, keep the tRNA -- even though RNase H is in the same mix

tRNA is a carrier / blocking nucleic acid. It saturates the nonspecific nucleic-acid-binding sites in fixed tissue (charged proteins, matrix) so padlock probes adsorb to the target rather than to the section. That job is unchanged on the cDNA route -- the probes are still DNA oligos going onto fixed tissue -- so tRNA stays at 0.2 μg/μl.

The obvious worry is that RNase H, in this same mix, will simply eat it. It does not: RNase H cleaves RNA only within an RNA:DNA heteroduplex. Free tRNA is folded on its own intramolecular RNA:RNA stems, which is not a substrate. It survives and keeps blocking. (In principle tRNA could form a short chance RNA:DNA hybrid with a probe and consume a little enzyme — negligible at 0.2 μg/μl against 0.4 U/μl RNase H.)

Where tRNA is genuinely wrong is the RT step (Section 1) — there it would give the reverse transcriptase an alternative template/primer pool. Note it is absent from that mix, and keep it that way.

4. Ligation (~2 h)

Goal: Ligation of hybridized padlock probes into circles; T4 DNA Ligase works on DNA:DNA hybrids (cDNA template), giving a well-defined, template-dependent junction.

Reagent Stock Final 100 μl
RNase Free H₂O 78 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 2 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 -- and where it stops

Unlike SplintR Ligase (used in the direct RNA protocol for DNA:RNA hybrids), T4 DNA Ligase operates on DNA:DNA substrates. That buys a cleaner, template-dependent junction and much lower self-ligation, and for ordinary targeted panels -- where the probe arms are the specificity element -- T4 at 37°C is entirely adequate and remains the default here.

What it does not buy is single-base discrimination. As a mesophilic ligase working at 37°C, T4 tolerates mismatches at the junction readily.3 If the readout has to distinguish alleles, switch to a thermostable high-fidelity ligase at 45--65°C -- see Ligase Fidelity.

5. Rolling Circle Amplification (~12--16 h)

Goal: Amplify circularized probes into DNA nanoballs (rolonies).

Reagent Stock Final 100 μl
RNase Free H₂O 70.5 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 0.1 μM 1 μl
Phi29 Polymerase 10 U/μl 0.25 U/μl 2.5 μl
  • Incubate: 30°C for 12--16 h overnight (sealed / humid chamber).
  • Wash: PBS-Tween 0.05% wash 2 x 2 min.

Glycerol -- reaction speed and rolony uniformity

Glycerol raises the viscosity and lowers the water activity of the mix, which slows phi29 elongation and restricts how far the growing concatemer can spread. In practice this keeps rolonies compact and more even in size, which is what the readout needs: signal calling thresholds and counts individual rolonies, so oversized products that sprawl and merge with their neighbours are lost to segmentation. Glycerol also helps stabilize phi29 across the long incubation.

If rolonies come out too small or too dim, lower the glycerol (10% → ~5%) or leave it out entirely. The trade is a wider size distribution. This is worth checking on the cDNA route in particular, where fewer circles are formed to begin with.

Nucleotide load is sample-dependent

Some tissues give under-sized rolonies at the standard nucleotide load; TNBC is the known case in-house. For these, double both nucleotide components -- dNTPs to 0.5 mM (5 μl) and amino-dUTP to 100 μM (5 μl) -- taking the extra 5 μl out of the water (70.5 → 65.5 μl). Scale the two together so the amino-dUTP : dTTP ratio, and therefore the amine density available for labelling, stays constant.

No RNase inhibitor in the RCA mix

RiboLock is not used at this step -- by the time RCA starts the template is the ligated DNA circle, so an RNase inhibitor adds nothing.

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 30 min at RT.
  • Wash: PBS-Tween 0.05% wash 3 x 2 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 2 min.

Next step: Signal readout -- PRISM Imaging or SPRINTseq Sequencing

Changelog

  • v3.0 -- 2026-08-18 -- Stringent wash returns to 10% formamide in 2X SSC at room temperature; the wash temperature is pinned. MAJOR, and applied in step with RCA Protocol v7.0 — see that entry for the full reasoning, which is shared. In short: the 20% default and the "45 °C optimal, or RT" clause both arrived on 2026-07-29 with no recorded reason; every lab record that produced data used 10%; our hybridization mix is SCRINSHOT's component for component and SCRINSHOT washes at 10%; and no published method washes above its own hybridization stringency.

    Two things are specific to this route and are now stated on the page. First, the padlock here binds cDNA, so the duplex is DNA:DNA and formamide destabilizes it more per percent than the DNA:RNA duplex of the direct-RNA route — roughly 0.6--0.7 °C/% against 0.5. The same 20% wash was therefore costing this route more than it cost the other one. Second, this route already runs at ~⅕ the detection efficiency of direct RNA, so it has the least yield to spare.

    The post-RT fixation rationale in §2, which cited the "3 x 10 min 20% formamide stringent wash" as the condition the cDNA has to survive, is corrected to 10%. The 3 x PBS-T chase is unchanged but its stated reason now includes the salt term — 2X SSC is 390 mM Na⁺, above the point where ligases lose activity.

    Evidence: the internal evidence review Stringency, Nonspecific Deposition and FFPE Background (lab site → Research).

  • v2.1 -- 2026-08-18 -- Post-stringency wash tightened from "2--3 x 2 min" to a firm 3 x 2 min, matching RCA Protocol v5.6. MINOR: same method, narrower spec. Rationale and the general wash convention: Reaction Buffers & Stringency.

  • v2.0 -- 2026-08-10 -- Major: runs before and after this version are not comparable. The reverse-transcription temperature change below alters how much template reaches the padlock, so cDNA-route data generated under v1.x should not be pooled with data generated under v2.0, and anyone trained on v1.x needs to be told the condition moved. (Recorded as v1.2 when first written; renumbered to v2.0 under the versioning rule in CLAUDE.md, which keys MAJOR to data comparability rather than diff size. No step text changed in the renumbering.) Reverse transcription: incubation temperature corrected 37°C → 50°C. 50°C is Maxima H Minus's working temperature and melts mRNA secondary structure so the RT reads through to the padlock target site; the 6 h--overnight duration (not a reduced temperature) is what covers diffusion into fixed tissue. Post-RT fixation: standardised to 4% PFA, RT, 30 min (was 3% formaldehyde / 40 min) -- same crosslinking load, now matching the ISS source condition1 and every other PFA step in the lab's protocols; a note explains why 30 min is neither over-fixation nor safely shortened to 15.

  • v1.1 -- 2026-07-29 -- Reverse transcription reworked: enzyme switched to Maxima H Minus RT at 10 U/μl (5 μl of 200 U/μl) with its 5X buffer, and target-specific custom RT primers at 100 nM each are now spiked in alongside the random decamers to improve detection of low-abundance transcripts. Hybridization: 10 nM per probe overnight is now preferred, 50 nM / 2 h kept as the same-day option. Stringent wash: 20% formamide in 2X SSC is the new default. RCA: primer lowered 0.3 → 0.1 μM, RiboLock removed, glycerol rationale documented, and a sample-dependent option to double dNTPs + amino-dUTP (e.g. TNBC) added. All PBS-T washes standardised to 2 min per change (previously 1 min). Documented why tRNA stays in the hybridization mix despite RNase H being present, and why it must stay out of the RT mix. Arithmetic corrections: the RCA table previously summed to 97.5 μl and the T4 ligation table to 99 μl; both now sum to 100 μl. Specificity claim corrected: T4 DNA Ligase remains the default for ordinary panels, but the protocol no longer claims SNP-level discrimination -- see the new Ligase Fidelity section.
  • v1.0 -- 2026-04-09 -- First cDNA-strategy release (TranscriptME RT, RNase H digestion, T4 DNA Ligase).

References

  1. Thermo Fisher Scientific. Maxima H Minus Reverse Transcriptase user guide, Pub. No. MAN0012047 Rev. C.00 (2024).
  2. Thermo Fisher Scientific. Detection of single-nucleotide polymorphisms using a ligation--rolling circle amplification approach. Application note APN-9503200 (2025).
  3. Yokota C, Gyllborg D, Nilsson M. In situ sequencing for RNA analysis in tissue sections. protocols.io (2020). DOI: 10.17504/protocols.io.bb2giqbw.

  1. Yokota C, Gyllborg D, Nilsson M. In situ sequencing for RNA analysis in tissue sections. protocols.io (2020). DOI: 10.17504/protocols.io.bb2giqbw. Post-RT fixation: 3.7% PFA in DEPC-PBS, RT, 30 min. ↩↩

  2. Thermo Fisher Scientific. Maxima H Minus Reverse Transcriptase user guide, Pub. No. MAN0012047 Rev. C.00 (2024). ↩↩

  3. Thermo Fisher Scientific. Detection of single-nucleotide polymorphisms using a ligation--rolling circle amplification approach. Application note APN-9503200 (2025). Ligase fidelity benchmark across 256 ligation-junction combinations. ↩↩

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