TITLE: Detection of single nucleotide polymorphisms with padlock probes
AUTHORS: Tomasz Krzywkowski, Mats Nilsson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification. In the accompanying publication, we take advantage of a single nucleotide variant within conserved ACTB mRNA to successfully differentiate human and mice co-cultured cells and apply following protocol to genotype PCDH X and Y homologs in human brain tissue sections. These are used as examples in the accompanying publication and custom padlock probes can be designed to allow for the targeting of own desired mRNA. We provide a method for automated characterization and quantitation of target mRNA in single cells or chosen tissue area. </div><div class = "text-block">mRNA of interest, harboring a polymorphism, is first reverse-transcribed to cDNA. Allele specific padlock probes are hybridized to the cDNA target and enzymatically circularized maintaining a physical link with the parent mRNA molecule. Lastly, circularized probes are replicated in situ, using rolling circle amplification mechanism to facilitate detection.</div><div class = "text-block">This protocol is taken directly from the accompanying publication. Further details and background information can be found in the cited published article.</div></div>

[STEPS]
?. [Specimen Pretreatment: Adherent Cells]
Culture cells in a flask until 80-90% confluence.
?. [Specimen Pretreatment: Adherent Cells]
Wash cells twice with PBS, and dislodge cells by adding 1 mL/25 cm2 of 0.25% Trypsin-EDTA for 1-2 min.
[Trypsin]
?. [Specimen Pretreatment: Adherent Cells]
Resuspend the cells in appropriate culturing medium (FBS in a medium inactivates the trypsin).
?. [Specimen Pretreatment: Adherent Cells]
Place sterile microscope slides in a 150 mm X 25 mm culture dish and add ~22 mL of medium to cover the slides. Cells adhere to most type of slides.Note: You could also use specialized chambered culture slides that contain cell cultures to small volume directly on the slide.
?. [Specimen Pretreatment: Adherent Cells]
Carefully, seed a total 3 mL of suspended cells directly on the slides (see Note 9).
Note 9: Unlike in many in situ hybridization methods, where cells are grown or coverslips, we grow cells on the microscope slides directly. We found slides to be more resistant to breaking (especially when applying and pealing the silicone chambers off) and on the slides, multiple experiments can be run in parallel (up to eight), making the work more convenient. Special microscope culture slides can also be used in growing cells on slides. Also, multiple consecutive tissue section can be placed on a single slide to facilitate fast experimenting and imaging.
?. [Specimen Pretreatment: Adherent Cells]
Carefully transfer the closed dish into the incubator and incubate the cells under appropriate conditions to allow them to adhere to the microscope slides (see Note 10).
Note 10: Optimal seeding conditions should be identified experimentally for every cell line. For cells with large cytoplasm, 3 mL suspension is usually enough to create homogenous cell layer on each slide. Cells with smaller cytoplasm can be seeded at higher density. In our experience, overnight incubation allows cells to adhere to slides efficiently. Extended incubation can result in cell proliferation on-slide (too dense or clumped cells are difficult to analyze by image analysis software).
?. [Specimen Pretreatment: Adherent Cells]
Wash the slides twice with cold PBS and transfer the slides to a sterile Coplin jar or sterile slides transport box.
?. [Specimen Pretreatment: Adherent Cells]
Fix the cells with 3.7% formaldehyde at room temperature (RT) for 20 min.
[Fixation]
Safety precautions: Formaldehyde
?. [Specimen Pretreatment: Adherent Cells]
Discard formaldehyde appropriately and wash the slides twice with PBS.
?. [Specimen Pretreatment: Adherent Cells]
Gradually dehydrate and pre-permeabilize the cells in ethanol series (70, 85, and 99.5% each step for 3 min).
[70% EtOH]
[85% EtOH]
[99.5% EtOH]
?. [Specimen Pretreatment: Adherent Cells]
Air dry the slides.For future use, slides can be stored at -80°C (long-term storage) or -20°C (up to 2 weeks). Make sure slides are labeled appropriately.For stored frozen slides, allow to thaw to RT before continuing.
?. [Specimen Pretreatment: Adherent Cells]
Attach Secure-Seal chamber of appropriate size and rehydrate the cells by adding PBS-T into the chamber (see Note 11).
Note 11: Tween 20, as a surfactant, coats the chambers, facilitates buffer exchange and prevents formation of "dead spaces” inside the chamber. As a detergent, it can provoke bubble formation. We recommend adding a buffer into a chamber when slide is slightly tilted.
?. [Specimen Pretreatment: Adherent Cells]
Remove PBS-T and permeabilize the cells by adding 0.1 M HCl for 5 min.
[0.1 M HCl]
Safety precautions: Hydrochloric acid
?. [Specimen Pretreatment: Adherent Cells]
Remove HCl and wash the cells twice with PBS-T
?. [Specimen Pretreatment: Freshly Frozen Tissue]
Tissue is cryosectioned and mounted on microscope slides (see Note 12) and can be stored at -80°C until use.
Note 12: Any slides that enhance adhesion of tissue sections can be used. (SuperFrost Plus® from Menzel-Glaser work very well in our hands). Sections should be as thin as possible (preferably few cell layer). Thinner sections are more prone to break and fold during cutting. We commonly use 10 μm thick sections.
?. [Specimen Pretreatment: Freshly Frozen Tissue]
Thaw slides at RT for a few minutes.
[RT Thaw]
?. [Specimen Pretreatment: Freshly Frozen Tissue]
Fix the tissue in 3.7% formaldehyde for 45 min. Use a sterile Coplin Jar or perform fixation directly on tissue sections on flat laying slides.
[Fixation]
Safety precautions: Formaldehyde
?. [Specimen Pretreatment: Freshly Frozen Tissue]
Wash once with PBS for 5 min.
[PBS wash]
?. [Specimen Pretreatment: Freshly Frozen Tissue]
(Optional) Permeabilize the tissue by incubating with prewarmed pepsin in 0.1 M HCl at 37°C. Digestion with 250 U/mL (0.1 mg/mL) pepsin for 5 min is a good starting point in our experience. Optimal conditions need to be identified for the respective specimen (see Notes 4 and 13).
[HCl]
Safety precautions: Hydrochloric acid
Note 4: Lyophilized pepsin batches may vary in activity, even from the same supplier. Every new batch of pepsin should be tested on established model. We typically detect housekeeping gene (ACTB or GAPDH) in the same tissue type to evaluate detection reproducibility.Note 13: The fixation time needs to compromise optimal fixative diffusion and minimize loss of tissue content. We recommend pragmatic evaluation of fixation parameters (consecutive sections should be used for each condition). Fixation time may vary for different tissues and different specimen thicknesses. Housekeeping gene is a good candidate for such optimization studies. Use conditions showing maximal signal amount.
?. [Specimen Pretreatment: Freshly Frozen Tissue]
Wash once with PBS for 5 min.
[Wash]
?. [Specimen Pretreatment: Freshly Frozen Tissue]
Dehydrate the tissue section in the ethanol series (70, 85, and 99.5% ethanol, 3 min each).
[70% EtOH]
[85% EtOH]
[99.5% EtOH]
?. [Specimen Pretreatment: Freshly Frozen Tissue]
Let slide air dry and mount appropriate size Secure-Seal chamber.
?. [Specimen Pretreatment: Freshly Frozen Tissue]
Rehydrate the tissue with PBS-T.
?. [SNP Detection Protocol: Reverse Transcription]
Combine reagents in table below and then remove PBS-T from chamber and apply the RT mix to the chamber.µ  ABC1ReagentConc.Volume (µl)2RT-reverse transcriptase buffer10X53RNase Inhibitor40 U/µl14BSA20 µg/µl0.55dNTP mix10 mM2.56LNA primer/random decamers100 µM0.5/2.57TRANSCRIPTME reverse transcriptase200 U/µl5 U/µl for cell lines and 20 U/µl for tissue sections8DEPC-ddH2OUp to 50
ABC1ReagentConc.Volume (µl)2RT-reverse transcriptase buffer10X53RNase Inhibitor40 U/µl14BSA20 µg/µl0.55dNTP mix10 mM2.56LNA primer/random decamers100 µM0.5/2.57TRANSCRIPTME reverse transcriptase200 U/µl5 U/µl for cell lines and 20 U/µl for tissue sections8DEPC-ddH2OUp to 50
?. [SNP Detection Protocol: Reverse Transcription]
Cover the chamber inlets and incubate the slides at 37°C. The optimal incubation time needs to be determined experimentally. With LNA-modified target-specific primers, 1 hour is sufficient.With random decamers, reverse transcription is typically conducted overnight.Decamers overnight.
[LNA primers]
?. [SNP Detection Protocol: Postfixation]
Wash the chamber twice with PBS-T.
?. [SNP Detection Protocol: mRNA Degradation, Padlock Probe Hybridization and Ligation]
Combine reagents in the table below for the padlock probe ligation mix.  ABC1ReagentConc.Volume (μl)2Tth Ligase Buffer10X53Padlock Probe(s)2 μM2.54RNase H5 U/μl45BSA20 μg/μl0.56KCl1 M2.57Formamide100%108Tth Ligase200 U/μl1.259DEPC-H2Oup to 50
ABC1ReagentConc.Volume (μl)2Tth Ligase Buffer10X53Padlock Probe(s)2 μM2.54RNase H5 U/μl45BSA20 μg/μl0.56KCl1 M2.57Formamide100%108Tth Ligase200 U/μl1.259DEPC-H2Oup to 50
Safety precautions: Formamide
?. [SNP Detection Protocol: mRNA Degradation, Padlock Probe Hybridization and Ligation]
Remove PBS-T and add combined ligation mix reagents to the chamber. Cover the chamber inlets and incubate the slides in a humidity chamber at 37°C for 30 min.
[PLP hybridization (1)]
?. [SNP Detection Protocol: mRNA Degradation, Padlock Probe Hybridization and Ligation]
Transfer humidity chamber to 45°C incubator for 45 min. (see Note 14)
[PLP hybridization (2)]
Note 14: RNase H has the highest activity at 37°C. It degrades RNA from mRNA/cDNA heteroduplex during the first 37°C incubation. After 30 min, sample is transferred to 45°C which is the optimal temperature for the Tth ligase. Addition of formamide into the mix lowers dsDNA stability (Tm of PLP arms/cDNAduplex). This enables extension of PLP arms that strengthens probe “locking” on cDNA and gives a good balance between arms melting and specific binding.
?. [SNP Detection Protocol: mRNA Degradation, Padlock Probe Hybridization and Ligation]
Remove reagents and wash the chamber twice with PBS-T.
?. [SNP Detection Protocol: Rolling Circle Amplification]
Combine the reagents in the table below for the RCA mix.  ABC1ReagentsConc.Volume (µl)2Phi29 DNA polymerase buffer10X53dNTP mix10 mM1.254BSA20 µg/µl0.55Glycerol50%56Phi29 DNA polymerase10 U/µl57DEPC-ddH2OUp to 50
ABC1ReagentsConc.Volume (µl)2Phi29 DNA polymerase buffer10X53dNTP mix10 mM1.254BSA20 µg/µl0.55Glycerol50%56Phi29 DNA polymerase10 U/µl57DEPC-ddH2OUp to 50
?. [SNP Detection Protocol: Rolling Circle Amplification]
Remove PBS-T and add combined ligation mix reagents to the chamber. Cover the chamber inlets and incubate the slides in a humidity chamber at 37°C for ≥1 hour. (see Note 15)
[RCA]
Note 15: The optimal temperature for phi29 polymerase is 37°C. We typically conduct RCA for 1 hour. If RCA is performed for several hours (or overnight) at 37°C, RCPs may start to fragment what could interfere with accurate signal counting. If large RCPs are desired (thick tissue sections or those with high autofluorescence), we advise doing RCA at RT overnight. Such approach will generate very large but compact RCPs.
?. [SNP Detection Protocol: Rolling Circle Amplification]
Remove RCA reagents and wash the chamber twice with PBS-T.
?. [SNP Detection Protocol: Decorator Probe Hybridization and Nuclei Counterstaining]
Combine the reagents in the table below for the hybridization mix.  ABC1ReagentConc.Volume (µl)2SSC20X53Formamide100%104Decorator probes(s)10 µM0.55DAPI100 µg/ml0.256DEPC-ddH2Oup to 50
ABC1ReagentConc.Volume (µl)2SSC20X53Formamide100%104Decorator probes(s)10 µM0.55DAPI100 µg/ml0.256DEPC-ddH2Oup to 50
Safety precautions: Formamide
?. [SNP Detection Protocol: Decorator Probe Hybridization and Nuclei Counterstaining]
Remove PBS-T and apply combined reagents to chamber and incubate the slide at RT for ~20 min. (see Note 16)
[Decorator probe]
Note 16: In a multiplexed reaction (when more than one decorator probe is used), we recommend incubation at 37°C for 30 min (to minimize nonspecific binding of the oligonucleotides). In such case, cover the chamber inlets to prevent mix evaporation.
?. [SNP Detection Protocol: Decorator Probe Hybridization and Nuclei Counterstaining]
Remove reagents and wash the chamber twice with PBS-T.
?. [SNP Detection Protocol: Decorator Probe Hybridization and Nuclei Counterstaining]
Mark the position of the chamber on the backside of the slide with a diamond pen and remove the Secure-Seal chamber. (see Note 17)
Note 17: A double edge razor can be used to facilitate complete removal of the Secure-Seal chamber.
?. [SNP Detection Protocol: Decorator Probe Hybridization and Nuclei Counterstaining]
Dehydrate the slides through an ethanol series (70, 85, and 99.5% ethanol, each for 3 min).Allow slide to air dry.
[70% EtOH]
[85% EtOH]
[99.5% EtOH]
?. [SNP Detection Protocol: Decorator Probe Hybridization and Nuclei Counterstaining]
Apply Slow-Fade medium to the speciment and gently, mount the specimen. (see Note 5 and 18)
Note 5: Slow-Fade® Gold Antifade Mountant works best in our experience to prevent photobleaching.Note 18: We typically apply 5-7 μL of mounting medium for single, 50 μL Secure-Seal chamber. Remove excess of the medium by gently pressing the slide and coverslip against a paper towel (excess of medium will be absorbed by the paper towel).
?. [Specimen Pretreatment: Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue]
Sectioned tissues are mounted on microscope slides (see Note 12) and can be stored at -80°C until use.
Note 12: Any slides that enhance adhesion of tissue sections can be used. (SuperFrost Plus® from Menzel-Glaser work very well in our hands). Sections should be as thin as possible (preferably few cell layer). Thinner sections are more prone to break and fold during cutting. We commonly use 10 μm thick sections.
?. [Specimen Pretreatment: Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue]
Frozen samples are thawed at RT.
?. [Specimen Pretreatment: Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue]
Dewax samples by submerging slides in a solvent series in separate Coplin jars: 15 min xylene 10 min xylene 2X 2 min 100% ethanol 2X 2 min 95% ethanol 2X 2 min 70% ethanol 5 min DEPC-H2O 5 min PBS
[Xylene (1)]
[Xylene (2)]
[100% EtOH (1)]
[100% EtOH (2)]
[95% EtOH (1)]
[95% EtOH (2)]
[70% EtOH (1)]
[70% EtOH (2)]
[H2O]
[PBS]
Safety precautions: Xylene
?. [Specimen Pretreatment: Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue]
Permeabilize the tissue by incubating with pre-warmed pepsin in 0.1 M HCl at 37°C for 5 min (see Notes 4 and 13).
[HCl]
Safety precautions: Hydrochloric acid
Note 4: Lyophilized pepsin batches may vary in activity, even from the same supplier. Every new batch of pepsin should be tested on established model. We typically detect housekeeping gene (ACTB or GAPDH) in the same tissue type to evaluate detection reproducibility.Note 13: The fixation time needs to compromise optimal fixative diffusion and minimize loss of tissue content. We recommend pragmatic evaluation of fixation parameters (consecutive sections should be used for each condition). Fixation time may vary for different tissues and different specimen thicknesses. Housekeeping gene is a good candidate for such optimization studies. Use conditions showing maximal signal amount.
?. [Specimen Pretreatment: Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue]
Wash with PBS for 5 min.
[PBS Wash]
?. [Specimen Pretreatment: Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue]
Postfix the tissue in 3.7% formaldehyde for 10 min at RT.
[Postfix]
?. [Specimen Pretreatment: Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue]
Wash twice with PBS for 5 min.
[PBS Wash (1)]
[PBS Wash (2)]
?. [Specimen Pretreatment: Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue]
Dehydrate the tissue section in the ethanol series (70, 85, and 99.5% ethanol, each step for 3 min).
[70% EtOH]
[85% EtOH]
[99.5% EtOH]
?. [Specimen Pretreatment: Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue]
Air dry the slides and mount Secure-Seal chambers.
?. [Specimen Pretreatment: Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue]
Rehydrate the tissue with PBS-T.
?. [SNP Detection Protocol: Image Acquisition and Analysis]
Mounted slides can be imaged immediately. Conventional wide-field epifluorescence microscopes are usually sufficient to image RCA products in tissue sections and cells. (see Note 19)
Note 19: We use a Zeiss Axioplan II Epifluorescence microscope equipped with either a metal hallide lamp or LED light source (Lumencor SpectraX) and a Hamamatsu, sCMOS camera. The following filter setup provides good wavelength separation and minimal crosstalk between different channels. 38HE (Zeiss) for imaging GFP/FITC/FAM dyes; SP102v2 (Chroma) for imaging Cy3 (minimal crass talk with 38HE filter); SP103v2 (Chroma) for imaging Cy3.5/TexasRed; SP104v2 (Chroma) for imaging Cy5; 49007 (Chroma) for imaging Cy7/Alexa 7.5 dyes.
Make sure to use filters appropriate for nuclear staining and fluorophores used in the experiment.Depending on the level of detail desired, use appropriate objective (we typically use 10X, 20X and 40X high numerical aperture objectives).To ensure accurate signal quantitation during image analysis, carefully adjust exposure time for a signal channels (abundant RCPs might be hard to segment if overexposed).We typically acquire 5-10 μm thick z-stack of images to capture RCPs in all focal planes (if possible, preview the specimen to define "first" and "last" stack corresponding to RCPs present in the lowest and highest stack and capture all images within this range).
?. [SNP Detection Protocol: Image Acquisition and Analysis]
Z-Stacks with multiple focal planes are combined to a single 2D maximum intensity projection (MIP) image that can be used in image analysis. We recommend taking images from several positions for each experiment (depending on cell density) to account for cell-to-cell expression variations.
?. [SNP Detection Protocol: Image Acquisition and Analysis]
Open-source programs such as CellProfiler or ImageJ can be used for image analysis. (see Note 20)
Note 20: CellProfiler is a great, user-friendly tool to aid biologists in image processing and analysis. With respect to presented protocol, CellProfiler offers scripts for cell segmentation (definition of the nucleus and the cytoplasm), RCP segmentation, and assignment of RCPs to individual cells or fluorescence measurements. All scripts can be implemented in automated pipeline, allowing for batch image processing. An example script for cell and RCP identification is available at CellProfiler website http://www.cellprofiler.org. Briefly, gray scale TIFF images (offering highest resolution, JPEG images are processed faster and can also be used) from individual fluorescence channels are loaded into the pipeline. Cells are segmented to nuclei and cytoplasm and RCPs are identified and related to neighboring cells. Finally, number of RCPs for each cell is exported as a .csv file, which can be used for post-analysis processing.
?. [Specimen Pretreatment: Adherent Cells]
Aspirate Trypsin-EDTA and monitor cells trypsinization under a brightfield microscope.
?. [SNP Detection Protocol: Postfixation]
Remove reverse transcription reagents and wash the chamber once with PBS-T.
?. [Anticipated Results]
Every discrete, fluorescent detected signal originates from a single, successful detection of the SNPAll RCPs should be localized to the cytoplasm, however RCPs can occasionally dissociate out of cells and generate RCPs on the glass slide (such signals should be disregarded).
For anticipated results, we refer to the accompanying publiction.https://link.springer.com/protocol/10.1007%2F978-1-4939-7213-5_14
?. [SNP Detection Protocol: Postfixation]
Fix cells with 3.7% formaldehyde. (See Note 3)For cultured cells, 10 min.For tissue sections, up to 45 min.
[Cell postfixation]
[Tissue postfixation]
Note 3: We recommend using freshly prepared formaldehyde solutions in DEPC-PBS. Working solutions can be prepared form either 37% methanol-stabilized stock solution or from paraformaldehyde powder. Aliquots of 3.7% formaldehyde in DEPC-PBS in 1 mL (used during the experiment) and 15 mL (for cell fixation) can be stored at -20°C. Do not freeze and thaw.
Safety precautions: Formaldehyde