FRET-FISH probe preparation
DAY 1
PCR (Polymerase Chain Reaction)
1. Mix the following reagents in a PCR tube:
● Forward primer (12.5 μM) 1.6-0.4 μL (depending on the qPCR profile)
● Reverse primer (12.5 μM) 1.6-0.4 μL (depending on the qPCR profile)
● Oligo-pool (10 nM) 2.0-0.4 μL (depending on the qPCR profile)
● SYBR master mix (2x) 12.5 μL
● Nuclease-free water 11.3-4.5 μL (to make up 25 μL)
2. Transfer the PCR product into a 1.5 mL DNA LoBind tube
3. Pipette the bead solution gently to achieve homogeneous suspension before use. Add bead suspension with 1.8x the volume of the PCR product
4. Mix well and incubate 5 min at room temperature (rt)
5. Place the reaction tube onto a DynaMag magnetic rack and wait for 5 min at rt until the beads clearly separate from the solution
6. Slowly aspirate the cleared solution and discard it. This step must be performed while the reaction tube is still on the magnetic rack. Do not disturb the separated magnetic beads
7. Add 100 μL of 70% ethanol to the tube and incubate for 30 sec at rt
8. Aspirate the ethanol and discard it while the tube is still on the magnetic rack. Do not disturb the separated magnetic beads. Be sure to remove all of the ethanol
9. Open the lid of the tube and let the beads air-dry for 10 min. The beads should air-dry until the last visible traces of ethanol evaporate
Note: over-drying the sample may result in a lower recovery
10. Add 35 μL of RNase-free water to the tube, pipette-mix at least 10 times, then incubate for 2 min at rt
11. Place the reaction tube on the magnetic rack for 1 min until the beads separate from the solution
12. Transfer the eluate to a new tube
13. Measure dsDNA concentration using Qubit
Note: to measure dsDNA concentration, follow the instructions of the Qubit dsDNA HS Assay Kit
IVT (In-Vitro Transcription)
1. Prepare and mix the following solution, incubate the solution at 37°C in a PCR cycler for 12-16h
● Template dsDNA 16.5 μL
● TNP buffer (20 mM)* 10 μL
● RNaseOUT (40 U/μL) 1.5 μL
● T7 RNA polymerase mix* 2 μL
*Components included in HiScribe T7 Quick high yield RNA synthesis kit
DAY 2
Removal of DNA in the IVT product
1. Add 20 μL nuclease-free water and 2 μL of DNase I to the IVT solution
2. Incubate at 37°C for 15 min
RNA purification using RNAClean XP beads
Note: In this step, it is very important to avoid RNase contamination. Make sure that all the reagents and tools are RNase-free. Proceed as quickly as possible.
1. Pre-warm the beads for at least 30 min at rt.
2. Transfer the IVT product into a 1.5 ml DNA LoBind tube
3. Pipette the bead solution gently to achieve homogeneous suspension before use. Add the bead suspension with 1.8x the volume of the PCR product
4. Mix well and incubate 3-5 min at rt
5. Place the reaction tube onto a DynaMag magnetic rack and wait for 5 min at rt until the beads clearly separate from the solution
6. Slowly aspirate the cleared solution and discard it. This step must be performed while the reaction tube is still on the magnetic rack. Do not disturb the separated magnetic beads
7. Add 150 μL of 70% ethanol to the tube and incubate for 30 sec at rt
8. Aspirate the ethanol and discard it while the tube is still on the magnetic rack. Do not disturb the separated magnetic beads. Be sure to remove all of the ethanol
9. Repeat the steps 7 and 8 twice
10. Open the lid of the tube, let the beads air-dry for 10 min. The beads should air-dry until the last visible traces of ethanol evaporate
Note: over-drying the sample may result in a lower recovery
11. Add 25 μL of RNase-free water to the tube, pipette-mix at least 10 times, then incubate for 2 min at rt
12. Place the reaction tube on the magnetic rack for 1 min until the beads separate from the solution
13. Transfer the eluate into a new tube
14. Measure RNA concentration using Qubit
Note: to measure RNA concentration, follow the instructions of the Qubit RNA BR Assay Kit
RT (Reverse Transcription)
1. Prepare and mix the following reagents:
● dNTP mix (10 mM) 4.5 μL
● forward primer (100 μM) 6 μL
● RT buffer (5x) 6 μL
● Maxima H reverse transcriptase (200U/μL) 1.5 μL
● RNaseOUT (40U/μL) 1.5 μL
● Template RNA 10.5 μL
Note: Add the template RNA as last
2. Incubate the solution at 50°C for 1 h
3. Terminate the reaction at 85°C for 5 min
Removal of the RNA in RT products
1. Add 20 μL of 0.5M EDTA and 20 μL of 1 N NaOH to the RT product
2. Incubate at 95°C in a PCR cycler for 15 min
3. Put the reactions on ice to cool down before proceeding
Purification of the RT product
1. Add the following reagents to the RT product prepared above:
● Oligo binding buffer 120 μL
● 100% EtOH 480 μL
2. Mix well and transfer the entire volume into a Zymo Spin IC column
3. Centrifuge at 10,000g for 30 sec and discard the flow-through
4. Wash the column with 750 μL of DNA wash buffer
5. Centrifuge at 10,000g for 30 sec and discard the flow-through
6. Elute the product using 6-20 μL of nuclease-free water
7. Centrifuge at 10,000g for 1 min
Note: In this step, you may also divide the 40 μL elution into 10 μL or 20 μL per time. This can improve the yield of the final probe. The ssDNA obtained at this step represents a ready-to-use DNA FISH probe
8. Measure ssDNA concentration with Qubit and store the probe at -20°C
Note: to measure ssDNA concentration, follow the instructions of the Qubit ssDNA Assay Kit
FRET-FISH sample preparation and hybridization
Fixation
1. Submerge the coverslips in 70% ethanol for at least 5 min
2. Take out the coverslips, put them in 6 well plate and let them airdry under the cell hood until the last traces of ethanol are gone
3. Grow adherent cells on the coverslips until desired confluency (around 90%)
4. Heat up 1 x PBS (w/ CaCl2 and MgCl2) to 37°C
5. Prepare the following buffers:
a. 1x PBS/4% PFA
b. 1x PBS/125 mM Glycine
c. 1x PBS
Note: Prepare 1x PBS/4% PFA right before the fixation
6. Rinse the coverslips twice with 1xPBS w/ CaCl2 and MgCl2 at 37°C
7. Wash the coverslips with 1x PBS/4% PFA for 10 min at rt
Note: Use an excess of 1x PBS/4% PFA to make sure PFA will also act on the sides of the well
8. Wash the coverslips twice, 5 min each, with 1x PBS/125 mM glycine at rt
9. Wash the coverslips twice, 5 min each, with 1x PBS at rt
10. Store the coverslips in 1x PBS at 4°C for maximum two weeks or in 1x PBS/0.05% NaN3for a longer storage, otherwise proceed with permeabilization
Permeabilization
1. Prepare the following buffers:
a. 1x PBS/0.5% Triton X-100
b. 1x PBS/0.05% Triton X-100
c. 1x PBS/0.1 N HCl
d. 2x SSC/50% FA/50 mM sodium phosphate buffer
Note: 2x SSC/50% FA/50 mM sodium phosphate buffer can be stored at 4°C in darkness but it has to be brought up to rt in darkness before proceeding to the next steps
2. If samples are stored at 4°C, exchange for fresh 1x PBS at rt
3. Wash with 1x PBS/0.5% Triton X-100 for 20 min at rt
4. Rinse twice with 1x PBS/0.05% Triton X-100 at rt
5. Wash twice, 5 min each, with 1x PBS/0.05% Triton X-100 at rt, while shaking gently
6. Rinse with 0.1 N HCl at rt
7. Wash with 0.1 N HCl at rt for 5 min
Note: Cells should be in contact with HCl for exactly 5 min. The timer should be set from the start of the rinsing process
8. Repeat steps 4 and 5
9. Wash the coverslips with 2x SSC at rt for 5 min
10. Rinse the samples with 2x SSC/50%FA/sodium phosphate buffer at rt
11. Exchange for fresh 2x SSC/50%FA/sodium phosphate buffer and store at rt overnight
Probe hybridization
DAY 1 – Hybridization of primary oligos
1. Prepare the following solutions:
a. Pre-hybridization buffer: 2x SSC/50% formamide/5x Denhardt’s solution/50 mM sodium phosphate buffer/1 mM EDTA/100 μg/mL ssDNA/100 μg/mL Cot-1 DNA, pH 7.5-8.0
b. Hybridization buffer: 2.2x SSC/55% formamide/5.5x Denhardt’s solution/55 mM sodium phosphate buffer/1.1 mM EDTA/100 μg /mL ssDNA/100 μg/mL Cot-1 DNA/11% dextran sulfate, pH 7.5-8.0
Note: Pre-hybridization buffer and hybridization buffer can be stored at –20°C, but must be brought up to rt in darkness before proceeding to the next steps
2. Prepare a humidity chamber by placing wet paper towels around a square petri dish
3. Place a squared piece of Parafilm on the bottom of the humidity chamber
4. Pipette a drop of 200 μL of pre-hybridization buffer per coverslip onto the piece of Parafilm
5. Exchange the 2xSSC/50% formamide/50 mM sodium phosphate solution in the plate containing the coverslips with 2x SSC
6. Gently lift each coverslip using tweezers and gently blot the edge of the glass and side without the cells on a piece of paper towel
Note: make this step as quickly as possible to prevent drying out of the cells
7. Place the coverslips onto the drops of pre-hybridization buffer pre-dispensed on Parafilm, with the cells facing down and touching the solution
8. Seal the humidity chamber with Parafilm
9. Incubate for 1 h at 37°C in darkness
10. Towards the end of the incubation time, prepare the hybridization mix by mixing the primary probes with hybridization buffer at a ratio of 1:9 vol./vol. so that the final concentration of each single oligo is ~0.05 nM
11. Mix well by vortexing and centrifuge at 20,000–30,000 x g for 1 min
Note: In case of debris formation, the supernatant can be transferred to a separate tube and used for hybridization
12. Pipette 20 μL of hybridization mix per coverslip onto a microscope slide
13. Transfer each coverslip onto the drop of hybridization mix using tweezers
14. Remove the excess of hybridization mix from underneath the coverslip by absorbing it with a filter paper
Note: Remove as much hybridization mix as possible from the sides of the microscope slide since excessive solution might prevent Fixogum from sticking to the glass
15. Seal the space between the coverslip and the microscope slide with Fixogum
16. Wait for the Fixogum to completely dry
17. In the meantime, set up a heating block to 75°C
18. Spray a bit of water onto the heating block and wait for it to reach the required temperature (75°C)
19. When Fixogum is completely solidified, place the microscope slides onto the drops of water on the heat block
Note: Make sure that the water does not overflow on the microscope slides and does not contact fixogum as this risks leaking in and diluting the hybridization mix
20. Incubate on the heating block for 2 min and 30 sec
Note: The exact denaturation timing needs to be determined empirically whenever a new cell line is used
21. Assemble another humidity chamber
22. Place the microscope slide in the new humidity chamber and seal it with Parafilm
23. Incubate the slides at 37°C overnight
DAY 2 – Hybridization of secondary oligos
1. Prepare the following solutions:
a. RNA hybridization buffer: 2x SSC/25% formamide/10% Dextran sulfate/1 mg/mL E.colitRNA/0.02% BSA/2 mM vanadyl-ribonucleoside complex
b. RNA wash buffer: 2s SSC/25% formamide
c. 2x SSC/0.2% Tween20
d. 0.2x SSC/0.2% Tween20
Note: RNA wash buffer can be stored at 4°C and RNA hybridization buffer can be stored at –20°C. Bring them to rt in darkness before proceeding to the following steps
2. Preheat the 0.2x SSC/0.2% Tween20 solution to 65°C in a water bath
3. Fill a 6-well plate and a Petri dish with 2x SSC/0.2% Tween20
4. Take the microscope slides out of the overnight incubation solution at 37°C
5. Place the slides in the Petri dish filled with 2x SSC/0.2% Tween20 and gently peel of the Fixogum from the glass, making sure that the coverslip is not dislocated
6. Gently slide each coverslip to the side of the corresponding microscope slide, lift it up with tweezers and transfer it into a well in a 6-well plate, so the side with cells is facing up
7. Quickly wash each coverslip with fresh 2x SSC/0.2% Tween
8. Wash the coverslips twice, 7 min each, with 0.2x SSC/0.2% Tween20 at 65°C by allowing the plate to float in a water bath
9. In the meantime, prepare the secondary hybridization mix by mixing the desired fluorescently labelled secondary oligo solutions with RNA hybridization buffer at a ratio of 1:99 vol./vol., so the final concentration of each fluorescent oligo is ~20 nM
10. Mix by vortexing and centrifuge at 20,000–30,000 x g for 1 min
11. Rinse each coverslip twice with 2x SSC
12. Rinse each coverslip once with RNA wash buffer
13. Exchange the RNA wash buffer in each well to fresh RNA wash buffer
14. Prepare a humidity chamber by placing wet paper towels on the sides of a square Petri dish
15. Place a piece of Parafilm on the bottom of the humidity chamber and pipette one drop of 200 μL of secondary hybridization mix for each coverslip
16. Place each coverslip on a drop of secondary hybridization mix, with cells facing down
17. Seal the humidity chamber and incubate at 30°C overnight in darkness
DAY 3 – Mounting
1. Prepare the following solutions:
a. RNA wash buffer: 2x SSC/25% formamide (keep in darkness)
b. RNA wash buffer + Hoechst: 2x SSC/25% formamide/1 ng/μL Hoechst (keep in darkness)
c. Anti-bleach buffer (GLOX): 2x SSC/10 mM Tris-HCl (pH 7.5)/0.4% glucose/10 mM TROLOX/37 ng/μL Glucose Oxidase/32 mM catalase
Note: RNA wash buffer can be stored at 4°C but needs to be brought up to rt in darkness before each new experiment. The RNA wash buffer + Hoechst and GLOX solutions should be made fresh each time and used on the same day
2. Transfer the coverslips from hybridization chamber to a 6-well plate pre-filled with RNA wash buffer
3. Exchange the RNA wash buffer to fresh one and incubate at 30°C for 1 h in darkness
4. Exchange the solution for RNA wash buffer + Hoechst and incubate at 30°C for 30 min in darkness
5. Wash the coverslips twice with 2x SSC
6. Mount the coverslips in fresh GLOX solution and seal the edges with Fixogum
7. Image the coverslips on the same day
Note: Coverslips can be stored in GLOX solution for a few days at 4°C. Before imaging we recommend mounting them in fresh GLOX solution.