Oct 17, 2025

Direct RNA ISS- Phylotypic V.1

 Forked from Home made direct RNA detection
This  protocol  is a draft, published without a DOI.
  • 1Stockholm University
Christina Bekiari
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Protocol Citation: Christina Bekiari, Marco Grillo, Mats Nilsson 2025. Direct RNA ISS- Phylotypic. protocols.io https://dx.doi.org/
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 14, 2025
Last Modified: October 17, 2025
Protocol  Integer ID: 229748
Keywords: multiplexed spatial profiling of mrna, direct rna detection, target mrna, mrna, rna, cellular diversity, multiplexed spatial profiling, direct ligation of padlock probe, situ expression map, situ expression maps of hundred, few hundred gene, gene, situ combinatorial barcoding, direct ligation, efficient detection of mrna, direct rna, situ sequencing, hybridization
Funders Acknowledgements:
EASIgenomics
Abstract
Highly multiplexed spatial profiling of mRNAs has recently gained traction as a tool to explore cellular diversity, allowing to visualize the cell composition of tissues directly in-situ.
This protocol exploits direct ligation of padlock probes on their target mRNAs, coupled with rolling circle amplification and hybridization-based in situ combinatorial barcoding, to achieve high detection efficiency, high throughput and large multiplexing (up to few hundred genes). The protocol borrows elements from SCRINSHOT (see https://pubmed.ncbi.nlm.nih.gov/33216742/) and Hybridization-based In Situ Sequencing (https://pubmed.ncbi.nlm.nih.gov/39099456/) for cheap and efficient detection of mRNAs in situ.
Guidelines
This protocol has been thoroughly tested on a variety of fresh frozen tissue sections coming from various animal models with minimal modification. Different tissues have sometimes slightly different cell or extra-cellular matrix composition and might need specific pre-treatments to make the tissue layers accessible to reagents and enzymes.


Equipment
  • Hydrophobic pen
  • Forceps
  • 30°C, 37°C, and 45°C incubators
  • Humidity chamber for slide incubation
  • SecureSeal hybridization chambers (Grace Bio-Labs)
  • Coverslips
  • Coplin jars or similar for washing of slides
  • Adhesive microscopy slides (e.g., Menzel Gläser SuperFrost Plus)
  • Wide-field epifluorescence microscope (6-channel)
  • Plate rocker


Necessary enzymes:

SplintR ligase
Phi29 polymerase
RNAse Inhibitor
General Guideline and Controls
  1. Enzymes and other reagents included in this protocol can be purchased from several different manufacturers (e.g. New England Biolabs or Thermo Fisher Scientific) and have performed equally well in our hands. Optimization, testing, and benchmarking should be performed whenever new reagents from different vendors are used.
  2. Enzymes can be produced on the cheap by your local protein production facility, if you have one nearby. The patent rights for these enzymes have expired, we provide here links to the primary sequences for your convenience.
  3. Stock concentrations of reagents could vary depending on vendor used. Adjust tables so that final concentration of reagents is the same. Optimization might also be required.
  4. This protocol involves RNA work and special care needs to be taken to prevent RNases. It is recommended to have designated space and equipment for RNA work and should be treated/cleaned with commercially available RNase and DNAse inactivating agents and then wiping with 100% ethanol after treatment.
  5. Using sterile, disposable, RNase-free plasticware (pipette tips, slide boxes, tubes, and flasks) is recommended.
  6. PBS and water can be purchased as RNase free from numerous vendors. It is also possible to treat PBS and water with DEPC (diethyl pyrocarbonate) to make them RNase free.
  7. Synthetic DNA targets can be used to validate specificity of padlock probes. Rolling circle amplification (RCA) can be monitored in vitro by staining rolling circle products (RCPs) with either intercalating dyes (SYBR dyes) or decorator probes and visualized under a microscope or qPCR system.
  8. This protocol assumes correct design of padlock probes (PLPs), L-probes, and detection oligos (DOs) for sequencing. See publications for further details on probe design to target genes of interest.
  9. This protocol does not go into detail on padlock probe design and analysis of data. See publications for further detail and image analysis.
Abbreviations
PLP: padlock probe
RCA: rolling circle amplification
RCP: rolling circle product
SBH: sequence by hybridization
DO: detection oligonucleotide
Safety warnings
See safety data sheets for proper chemical handling, precautionary measures and waste disposal.
Obey all local regulations/guidelines for handling and disposal of used reagents and and solutions containing reagents mixed in.
Formamide:
Handle with proper attire including gloves and eye protection. Work under fume hood when handling solution and dispose of waste appropriately.
Suspected of causing cancer.
May damage fertility or the unborn child.
May cause damage to organs (Blood) through prolonged or repeated exposure if swallowed.
Methanol:
Handle with proper attire including gloves and eye protection. Work under fume hood when handling solution and dispose of waste appropriately.
Highly flammable liquid and vapor.
Toxic if swallowed, inhaled or in contact with skin.
Can cause damage to organs (Eyes, Central nervous system).
Formaldehyde/paraformaldehyde/formalin solution (PFA):
Handle with proper attire including gloves and eye protection. Work under fume hood when handling solution and dispose of waste appropriately.
May cause cancer.
Toxic if swallowed, in contact with skin or if inhaled.
Causes severe skin burns and eye damage.
May cause an allergic skin reaction.
May cause respiratory irritation.
Suspected of causing genetic defects.
Causes damage to organs (Eyes).

Before start
Embed your fresh-frozen sample in a cryo embedding mold of your choice using OCT medium.
Cut sections to a thickness of 10-20 micra and attach the sections to Superfrost slides.
Store the sections at -80 until use.
Tissue Preparation
Fresh frozen tissue samples are embedded in OCT (optimal cutting temperature) compound and stored at -80°C until cryosectioning.

Tissue is cryosectioned at 10 μm thickness and collected on SuperFrost Plus adhesion slides and can be stored at -80°C until used for experiment.

Safety information
Extra precautions should be taken with tissue of human origin. Obey local guidelines/rules and take appropriate precautionary measures when handling tissue, especially fresh tissue to avoid any risks.

Buffer Preparation for the sample processing
Prepare the buffers for the fixation and permeabilisation steps.

ItemStock CFinal CVolume
Formaldehyde37%3,7%2 mL
PBS 0,1M--18 mL
Total--20 mL
3,7% Formaldehyde preparation


ItemStock CFinal CTotal volume
SDS10%1%2 mL
Nuclease-free water--18 mL
Total--20 mL
1% SDS preparation


ItemStock CFinal CVolume
Methanol100%70%14 mL
Nuclease-free water--6 mL
Total--20 mL
70%Methanol preparation
NOTE: Chill the Methanol solution in the freezer (-20) during the next steps and until needed.
Tissue Fixation
36m
Take the slides from -80°C and leave them at room temperature (RT) for 3-5 min to air dry and reach room temperature.
00:03:00 Thaw at RT

3m
Fix with the freshly prepared 3,7% formaldehyde in PBS for 30 min at RT.
00:30:00 Tissue fixation


Note
Formaldehyde solution can be applied directly on top of the section, or the slide can be submerged in a large amount of fixative.

Safety information
Safety precaution: Formaldehyde
Formaldehyde should be handled in a biosafety fume hood due to its hazardous nature.


30m
Discard formaldehyde from tissue section and incubate in PBS at RT for twice.

Note
For the next washing steps, submerge the glass slide in ~20mL PBS in a jar or container that can hold slides.

3m
Tissue Permeabilisation
1h 8m
Immerse the slide in the 1% SDS and incubate for 2 min at room temperature.
00:02:00 SDS Incubation

Note
This can be performed by submerging the glass slide in ~20 mL solution of 1% SDS in a jar or container that can hold slides.


2m
Discard SDS solution and incubate in PBS at RT for 1 min 2 times.
3m
Immerse the slide in the pre-chilled 70% Methanol solution.
Incubate for 60 min on ice.
On ice 01:00:00 Methanol permeabilisation

The whole container should be submerged in ice.
Safety information
Safety precaution: Methanol
Methanol should be handled in a biosafety fume hood due to its hazardous nature.

1h
Discard methanol solution and incubate in PBS at RT for 1 min 2 times.
3m
Tissue Dehydration
5m
Dehydrate the sections in order to aid the adhesion of SecureSeal Hybridisation chambers.
Submerge slides in 70% Ethanol for 1 min.
Transfer and submerge in ~100% Ethanol for 1 min.
2m
Remove slide and let air dry (1-5 min)
3m
With a dry surface around tissue section, apply appropriate size SecureSeal Hybridisation chamber over the tissue.

Note
SecureSeal Hybridisation chambers come in different sizes, shapes, and depths. Small chambers have a volume of ~50 µl (round, 9 mm diameter, and 0.8 mm deep: enough for half a coronal section of a mouse brain). For larger tissue specimens, larger chambers and shapes can be used and consequently volumes in protocol should be adjusted.
Chambers have to be mounted on dry surfaces for a proper seal to prevent reagent content loss during incubations.

Sections and chamber are washed by applying PBS-T to chamber inlet and then followed by one wash of PBS.
Note: Let PBS remain in chamber until next reagents are combined.

Note
Apply and remove solutions to chamber by tilting the slide/chamber slightly and pipetting into lower inlet. This will aid in preventing bubble formation within the chamber.

Hybridisation of padlock probes
12h 11m
Combine reagents for PLP hybridisations in table below.

ReagentStockFinalAmount
20x SSC20x2x5 μL
Formamide100%10%5 μL
PLPs1 uM each10 nm each0.5 μL
Nuclease-free water--39.5 μL
Total--50 μL
PLP Hybridisation Mix

Note
Note: H2O is used to get total volume required. Other vendors can supply reagents at various stock concentrations and adjustments need to be made accordingly, including possible optimisation of concentrations.

Note
The amount of padlock probes will vary depending on user/experiment and initial concentration may vary. Final concentration of each padlock probe should be around 10 nM each. This may require optimisation depending on the total amount of padlock probe targets.


PBS from chamber is removed and PLP Hybridisation Mix is added to the chamber.
Incubate at 37oC overnight.
37 °C Overnight PLP Hybridisation

Safety information
Safety precaution: Formamide

12h
Prepare wash buffer.

ReagentStockAmount
Nuclease-free water-400 μL
Formamide100%50 μL
SSC20X50 μL
Total-500 μL

Safety information
Safety precaution: Formamide

Preheat the wash buffer at 37oC for 10 mins.
37 °C 00:10:00 Wash buffer preheat

10m
Remove reagents from chamber after incubation.

Add the wash buffer to the chamber and incubate for 1 min at 37oC twice.
37 °C 00:01:00 Wash buffer incubation

Wash twice in PBS.

Note: Let PBS remain in chamber until next reagents are combined.
1m
Padlock probe ligation
2h
Reagents for PLP ligation are combined as in the table below.

ReagentsStock CFinal CAmount
Nuclease-free water--40,25 μL
SplintR buffer New England Biolabs)10x1x5 μL
RCA primer1 μM0.05 µM2,5 μL
RiboProtect (RNase inhibitor)40 U/μL1 U/µl1,25 μL
SplintR Ligase (New England BioLabs)25 U/μL0,25 U/μL1 μL
Total50 μL
PLP Ligation Mix
Note: H2O is used to get total volume required. Other vendors can supply reagents at various stock concentrations and adjustments need to be made accordingly, including possible optimisation of concentrations.

PBS from chamber is removed and PLP Ligation Mix is added to the chamber.
Incubate for 2 hours at 37oC.
37 °C 02:00:00 PLP Ligation

2h
Amplification of the padlock probes - Rolling Circular Amplification (RCA)
12h 2m
Remove reagents from chamber after incubation.
Wash twice with PBS.

Note: Let PBS remain in chamber until next reagents are combined.
2m
Reagents for RCA are combined as in the table below.
ReagentsStock CFinal CAmount
Nuclease-free water--34 μL
phi29 buffer10X1X5 μL
glycerol (Placed at RT)50%5%5 μL
dNTPs25 mM0,25 mM0,5 μL
BSA20 μg/uL0.2 µg/mL0,5 μL
phi29 polymerase10 U/μL1 U/µL5 μL
Total50 μL
RCA Mix
Note: H2O is used to get total volume required. Other vendors can supply reagents at various stock concentrations and adjustments need to be made accordingly, including possible optimization of concentrations.
12h
PBS from chamber is removed and RCA mix is applied to the chamber.

Incubate at 30oC overnight.
30 °C Overnight RCA
Removal of Hybridisation Chamber
After overnight incubation, remove reagents from chamber.
Wash chamber twice with PBS

After washing steps, carefully remove SecureSeal chamber.

Note
Remove SecureSeal chamber with the aid of forceps.

Wash the section with PBS one more time.
Then carefully remove as much liquid as possible from area surrounding the tissue section. Use paper towel to dry area if needed. Then, with a hydrophobic pen, draw a barrier surrounding tissue section.

After the barrier has been drawn around tissue and dried slightly, apply additional PBS to section to prevent it from drying out and proceed to next step.

Note
We use ImmEdge Hydrophobic Barrier PAP Pen by Vector Laboratories (Cat. No. H-4000). Other hydrophobic pens have shown to impede the in situ sequencing visualisation.

It is also possible to reapply SecureSeal hybridisation chambers instead of using hydrophobic pen for subsequent hybridisation steps. We find it easier to continue all steps without the use of the chamber.
Note that the same volume should be used on tissue sections as what the chamber volume was.

Sequence By Hybridisation: L-probes
1h
Prepare 4X Hybridisation Buffer according to the table below.

ReagentsStockAmount
Nuclease-free water-100 μL
Formamide100%100 μL
SSC20X50 μL
Total-250 μL
4X Hybridisation Buffer

NOTE: Keep the hybridisation buffer when not using.
Combine reagents for L-Probe hybridisation as in table below.

ReagentsStock CFinal CAmount
Nuclease-free water--22,6 μL
4X Hybridisation buffer4Χ1Χ25 μL
L-Probe mix2,083 μΜ0,1 μΜ2,4 μL
Total--50 μL
L-Probe Mix

Note: H2O is used to get total volume required.
Various stock concentrations are possible and adjustments need to be made accordingly, including possible optimisation of concentrations.
PBS is removed from section and is washed once with 2X SSC.

L-probe hybridisation mix is added to the section.
Incubate L-probes Mix for 1h at RT in dark, on a rocker.
Room temperature 01:00:00 L-probe incubation

1h
Sequence By Hybridisation: Detection Oligo
1h
Discard reagents and wash 2 times with 2XSSC.
Let 2XSSC remain until next step reagents are prepared.
Combine reagents for detection oligo hybridisation as in table below.

ReagentsStock CFinal CAmount
Nuclease-free water--22,25 μ
4X Hybridisation Buffer4X1X25 μL
DO_seq110 μg/μL0,1 μg/μL1 μL
DO_seq210 μg/μL0,1 μg/μL1 μL
DO_seq310 μg/μL0,1 μg/μL1 μL
DO_seq410 μg/μL0,1 μg/μL1 μL
DO_seq510 μg/μL0,1 μg/μL1 μL
DAPI100 μg/μL0,5 μg/μL0,25 μL
Total--50 μL
Detection Oligo Mix

Note: H2O is used to get total volume required.
Various stock concentrations are possible and adjustments need to be made accordingly, including possible optimisation of concentrations.
2XSSC is removed from the tissue.

L-probe hybridisation mix is added to the section.
Incubate L-probes Mix for 1h at RT in dark, on a rocker.
Room temperature 01:00:00 Detection Oligo incubation
1h
TrueView tissue Quenching
1m
Discard reagents and wash with PBS 2 times.
Let PBS remain until TrueView reagents are prepared.
Mix equal parts of TrueView reagents, by first mixing reagents A and B together and then adding reagent C.
Vortex well in between each addition and spin down the mix.

Discard the PBS from the tissue.
Add the TrueView mix on the tissue and incubate for 1 min at RT in dark.
Room temperature 00:01:00 TrueView incubation

1m
Discard the TrueView mix and wash the tissue with PBS three times.
Mounting the Section
Remove as much PBS as possible and then add small drop of mounting media and apply cover slip to slide section.


Note
We use SlowFade Gold Antifade Mountant as it has shown to perform well with our conditions and also allows for removal of cover slip in future steps.

10 μl of mounting media should be enough for the area of one mouse brain coronal section.


Note
Slides at this point can be stored at +4°C with mounted coverslips in the dark for several weeks. It is recommended to image sections soon after detection oligos are added.

Imaging
Section can now be imaged.

Note
Labs will have different microscope set ups and identical setups are not needed. Fluorophore dyes can be interchanged depending on your own microscopes capabilities. The number of detection oligo sequences presented here is 5, but this is only limited by your microscopes ability to distinguish fluorophores and can be adjusted to be more or less which would also require adjustments to your combinatorial decoding scheme.
We use a Leica DMI6000 epifluorescence microscope. It is equipped with 6-channel LED light source and sCMOS camera. A good filter setup is essential to provide good wavelength separation and minimal crosstalk between different channels. A typical imaging run includes DAPI for nuclei and 5 separate fluorophores for the different detection oligo sequences.

Imaging is set up to do repeated cycles of the same region every round. Tile region outlines are set up for the first cycle and saved. After every round/cycle, slides are placed back in stage slide holder to the same position for proper alignment. This is aided with the use of an automatic stage that can calibrate to a reference point every time it is used so tile regions have the same coordinates.
Stripping L-Probes and Detection Oligos
30m
Submerge slide with coverslip in clean PBS in a coplin jar and let sit for a few minutes. Proceed to next step during this time and prepare reagents.
While slide is submerged, prepare stripping solution according to the table below.

ReagentsStock CFinal CAmount
Nuclease-free water--25 μL
Formamide100%65%65 μL
SSC20X2X10 μL
Total--100 μL
Stripping solution

Safety information
Safety precaution: Formamide


Pre-incubate combined reagents and humidity chamber at 30°C until used.
30 °C Stripping solution pre-incubation

Note: After incubation of section with stripping solution, section is washed once with additional stripping solution, therefore prepare twice as much needed.
Prepare 2XSSC washing solution.
For 5 ml: 500 μl 20XSSC + 4.5 ml Nuclease-free water
Remove slide from PBS and if cover slip does not slide off by itself, either let it sit longer in PBS or carefully slide it off with the aid of forceps.
Wash section 2 times with PBS to remove residual mounting media.
Place slide in prewarmed humidity chamber. Apply prewarmed stripping solution on top of section and place in 30°C incubator for 30 min.
30 °C 00:30:00 Stripping incubation

Place any remaining stripping solution in the incubator as well.
30m
After 30 min, remove solution from section and wash 1 time with remaining stripping solution.
Wash 5 times with 2XSSC.
Repeated Cycles
Now the L-Probes and Detection Oligos has been stripped from the tissue.

From this point, the next cycle can be performed as before with next cycle of L-probes.