RCA (cDNA)
Revised upon 20240724_RCA_tissue_protocol.docx.
a. Add reverse transcription step for cDNA generation.
Universal
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Rnase Free H₂O
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DEPC-treated ddH₂O (for buffer and solution preparation)
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BSA (20 µg/µL) (universal stabilizer in enzyme solutions)
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KCl (1M) (used for ionic strength in buffers)
Fixation
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0.1% Glutaraldehyde (GA) in 4% Paraformaldehyde (PFA)
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4% Formaldehyde (for postfixation)
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BS-PEG9 (used in RCA fixation)
Washing and Dehydration
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PBS-Tween 0.05% (for washing)
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80% Ethanol (for washing and dehydration)
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100% Ethanol (for dehydration)
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20X SSC Buffer (3 M NaCl, 300 mM trisodium citrate)
Permeabilization
- 0.04% Pepsin in 0.1M HCl (for tissue permeabilization)
Reverse Transcription
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RT-reverse transcriptase buffer (10x)
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RNase Inhibitor (40 U/µL)
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dNTPs mix (10 mM) (for reverse transcription reaction)
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LNA primer / Random decamers (100 µM) (as primers for reverse transcription)
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TranscriptMe reverse transcriptase (5 U/µL or 20 U/µL)
Probe Hybridization
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tRNA (Ambion AM7119) (10 µg/µL) (used during blocking to prevent nonspecific probe binding)
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Probes oligo (10 µM)
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Formamide deionized (100%) (for hybridization buffer)
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Ampligase Buffer (10x)
Ligation
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SplintR (NEB) (25 U/µL)
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SplintR Buffer (10x)
RCA (Rolling Circle Amplification)
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Phi29 DNA Polymerase (10 U/µL)
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Phi29 Buffer (10x)
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Glycerol (100%) (used in RCA buffer)
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NH₂-dUTP (2 mM) (nucleotide for amplification)
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RCA Primer 1+2 (10 µM for both) (primers for rolling circle amplification)
All concentrated buffers are provided together with enzymes and stored according to vendor specification.
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Phosphate Buffered Saline (1x PBS): 137 mM NaCl, 10 mM sodium phosphate, 2.7 mM KCl, and DEPC-ddH₂O, pH 7.4.
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Washing buffer (PBS-T): 0.05% Tween 20 in 1x PBS.
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Saline-sodium citrate buffer (20x SSC): 3 M NaCl and 300 mM trisodium citrate in DEPC-ddH₂O, pH 7.
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RT-mix (100 μL per chamber): 10 μL 10x RT-reverse transcriptase buffer, 2 μL 40 U/µL RNase Inhibitor, 1 μL 20 µg/µL BSA, 5 μL 10 mM dNTPs mix, 5 μL 100 µM LNA primer or Random decamers, TranscriptMe reverse transcriptase, Fill up to 100 μL with DEPC-ddH₂O.
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Hybridization mix (100 μL per chamber): 5 μL 20x SSC, 10 μL 100% formamide, 2 μL tRNA (Ambion AM7119), Fill up to 100 μL with DEPC-ddH₂O.
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Ligation mix (100 μL per chamber): 10 μL 10x SplintR Buffer, 2 μL 25 U/μL SplintR (NEB), 5 μL 10 U/µL RNase H, 1 μL 20 µg/µL BSA, 5 μL 1M KCl, 20 μL 100% formamide, Fill up to 100 μL with DEPC-ddH₂O.
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RCA mix (100 μL per chamber): 10 μL 10x Phi29 DNA polymerase buffer, 5 μL 50% glycerol, 2 μL 10 µg/µL BSA, 2.5 μL 2 mM NH₂-dUTP, 3 μL + 3 μL RCA Primer 1+2 (10 µM each), Fill up to 100 μL with DEPC-ddH₂O.
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- fixation of the slides (~60min)
i. Incubate the slides in 200ul 0.1% GA in 4% PFA for 1**5-30 min**
Freshly prepare: 1µL 50% GA + 500 µL PFA
ii. Wash the slides in 200ul 1 x 2 min PBS-Tween 0.05%
iii. Wash the slides in 200ul 80% EtOH
iv. Assemble the PDMS chamber to glass slide
v. Wash the slides in 50 ul 1 x 2 min PBS-T
- Permeabilization of the tissue (~10min)
i. Incubate the slides in 0.04% pepsin in 0.1M HCl at 37°C for 2 min
ii. Wash the slides for 1 x 2 min with PBS-Tween 0.05%
- Dehydration (~20min)
i. Incubate the slides in 80% ETOH for 10 min
ii. Incubate the slides in 100% ETOH for 2 min
iii. wash the slides for 3 x 2 min PBS-Tween 0.05% for rehydration.
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- Reverse transcription (~2h)
| Reagents | Stock | Final | 1 x slide | x slides |
|---|---|---|---|---|
| Rnase Free H₂O | - | - | ||
| RT-reverse transcriptase buf. | 10x | 1x | 10 μL | |
| RNase Inhibitor | 40 U/μL | 0.8 U/μL | 2 μL | |
| BSA | 20 μg/μL | 0.2 μg/μL | 1 μL | |
| dNTPs | 10 mM | 0.5 mM | 5 μL | |
| Hexamer random primer | 100 μM | 5 μM | 5 μL | |
| Reverse transcriptase | 5 U/μL | 0.5 U/μL | 10μL | |
| Total |
i. Apply the RT mix(above) to the chamber, mix by gentle pipetting (3-5 times), and incubate the slides at 42°C for 2h.
The optimal incubation time needs to be determined experimentally. Typically, 1h is sufficient when using LNA-modified target-specific primers, while RT with random decamers is conducted overnight.
ii. Wash the slides for 1 x 2 min with 1X PBS-T.
- Postfixation
i. Incubate the slides in 4% PFA for 30 min.
ii. Wash the slides for 2 x 1 min with 1X PBS-T.
At this point, the protocol can be halted, and samples can be stored for a few days at 4°C in 1X PBS.
- Blocking (30min)
| Reagents | stock | final | 1 x slide | x slides |
|---|---|---|---|---|
| Rnase Free H₂O | ||||
| Ampligase Buffer | 10x | 1x | 10μl | |
| KCl | 1M | 0.05M | 5μl | |
| Formamide deionized | 100% | 20% | 20μl | |
| Probes oligo | 10μM | 0.05μM | 1μl | |
| BSA | 10μg/μl | 0.2μg/μl | 2μl | |
| RiboLock (Thermo) | 40U/μl | 1U/μl | 2.5μl | |
| tRNA (Ambion AM7119) | 10μg/μl | 0.2μg/μl | 2μl | |
| Total | 100μl |
i. Add the blocking solution into the chamber, mix by gentle pipetting (3-5 times) and incubate the slides at room temperature (RT) for 30 min.
At this point, the protocol can be halted, and samples can be stored for a few days at 4°C in 1X PBS.
- Hybridization of Padlock probes (3h)
| Reagents | stock | final | 1 x slide | x slides |
|---|---|---|---|---|
| Rnase Free H₂O | ||||
| Ampligase Buffer | 10x | 1x | 10μl | |
| KCl | 1M | 0.05M | 5μl | |
| Formamide deionized | 100% | 20% | 20μl | |
| Probe | 0.2μM | |||
| BSA | 10μg/μl | 0.2μg/μl | 2μl | |
| RiboLock (Thermo) | 40U/μl | 1U/μl | 2.5μl | |
| tRNA (Ambion AM7119) | 10μg/μl | 0.2μg/μl | 2μl | |
| Total | 100μl |
i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal.
ii. Incubate the slides at 55°C for 18 min for denaturation and at 45°C for 120 min for hybridization of the probes onto the target mRNA.
iii. Wash the slides for 3 x 10 min with washing buffer (10% formamide in 2X SSC) to remove unhybridized probes. 100µL 20X SSC + 100µL formamide + 800µL H₂O.
iv. Wash the slides for 3 x 1 min with PBS-Tween 0.05% to remove the remaining formamide, as it can deactivate the enzyme in the following step.
At this point, the protocol can be halted, and samples can be stored for a few days at 4°C in 1X PBS.
- Ligation of Padlock probes (~2h)
The ligation of the hybridized padlock probes is mediated by T4 DNA Ligase
| Reagents | stock | final | 1 x slide | x slide |
|---|---|---|---|---|
| RNase Free H₂O | ||||
| T4 Buffer | 10X | 1X | 10 ul | |
| BSA | 10 ug/ul | 0.2 ug/ul | 2 ul | |
| T4 DNA ligase (ThermoFisher, EL0011) | 25 U/ul | 2.5 U/ul | 10 ul | |
| Total | 100 ul |
i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal
ii. Incubate the slides at 37°C for 2 hours
iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%.
At this point, the protocol can be halted, and samples can be stored for a few days at 4°C in 1X PBS.
- Rolling circle amplification (RCA, ~3h-17h)
| Reagents | stock | final | 1 x slide | x slide |
|---|---|---|---|---|
| RNase Free H₂O | 64.5 ul | |||
| Phi29 Thermo | 10 U/ul | 0.25 U/ul | 2.5ul | |
| Phi29 Buffer | 10X | 1X | 10 ul | |
| Glycerol | 100% | 0.2 ug/ul | 10 ul | |
| dNTPs | 10 mM | 0.25 mM | 2.5 ul | |
| NH₂-dUTP | 2 mM | 0.05 mM | 2.5 ul | |
| BSA | 10 ug/ul | 0.2 ug/ul | 2 ul | |
| RCA Primer 1+2 | 10uM for both | 0.3uM for both | 3 + 3 ul | |
| Total | 100 ul |
i. Add the solution into the chamber, mix by gentle pipetting (3-5 times) and seal
ii. Incubate the slides at 30°C for 12-16 hours (For 2h, need to incubate at 37C)
iii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%.
iv. Add 0.2% PEG9 in PBS-T mix by gentle pipetting (3-5 times) and incubate for 15-30 min at RT to fix the RCA products on the tissue.
Freshly prepare: 2µL PEG9 BS + 98µL PBS-T
v. Wash the slides for 3 x 1 min with PBS-Tween 0.05%.
vi. Wash the slides for 3 x 2-10 min with 65% formamide at 30°C on a PCR machine block or an Eppendorf Thermostat. 650 µL Formamide + 350 µL H₂O
vii. Wash the slides for 2 x 1 min with PBS-Tween 0.05%
At this point, the protocol can be halted, and samples can be stored for a few days at 4°C in 1X PBS.