FuseFISH: Robust Detection of Transcribed Gene Fusions in Single Cells
Stefan Semrau, Nicola Crosetto, Magda Bienko, Marina Boni, Paolo Bernasconi, Roberto
Chiarle, Alexander van Oudenaarden
STEP-BY-STEP PROTOCOLS FOR PURIFIED RNA, CELLS, AND TISSUES
REAGENTS NEEDED
- RNase-free proteinase K (Ambion, catalogue no. AM2548)
- Molecular biology grade ethanol and methanol
- D-Limonene clearing agent (VWR, catalogue no. 700002-376)
- Acetic acid (Sigma, catalogue no. 338826)
- Sodium citrate (Sigma, catalogue no. 71497)
- Sodium borohydride (Sigma, catalogue no. 480886)
- Ribonucleoside Vanadyl Complex (RVC) (NEB, catalogue no. S1402S)
- 10× PBS pH 7.4 (Ambion, catalogue no. AM9625)
- 20× SSC (Ambion, catalogue no. AM9765)
- Deionized formamide (Ambion, catalogue no. AM9342)
- Nuclease-free water (Ambion, catalogue no. AM9932)
- Pepsin (Sigma, catalogue no. P6887)
- Bovine Serum Albumin (BSA) (Ambion, catalogue no. AM2616)
- Dextran sulphate (Sigma, catalogue no. D8906)
- E. coli tRNA (Sigma, catalogue no. R4251)
- Trolox (Sigma, catalogue no. 238813)
- Glucose Oxidase (Sigma, catalogue no. G2133-10KU)
- Catalase (Sigma, catalogue no. 3155)
- 4',6-diamidino-2-phenylindole (DAPI) (Sigma, catalogue no. D9542)
- Fixogum Rubber Cement (MP Biomedicals, catalogue no. 11FIXO0050)
MATERIALS NEEDED
- SecureSeal hybridization chambers (EMS, catalogue no. 70333-10)
- Thermomixer R with 4-slide exchangeable block (Eppendorf)
- Incubator or hybridization oven
1
WORKING SOLUTIONS
The following solutions can be prepared in advance and stored at the indicated temperature up to
one year.
- RNA wash buffer (RW25), store in darkness at 25 °C:
REAGENT
Formamide
20× SSC
FINAL CONCENTRATION
25%
2x
- RNA hybridization buffer (RH25), store in 500 l aliquots at –20 °C:
REAGENT
Formamide
20× SSC
Dextran Sulphate
BSA
E. coli tRNA
RVC
FINAL CONCENTRATION
25%
2x
10%
0.02%
1 mg/ml
2 mM
- Equilibration buffer (EQ), store at 25 °C:
REAGENT
20× SSC
Tris-HCl 1M pH 7.5
Glucose
FINAL CONCENTRATION
2x
10 mM
0.4%
- Imaging buffer (IB) is prepared by adding the following enzymes to a small volume of
equilibration buffer just prior to imaging:
REAGENT
Trolox
Glucose Oxidase
Catalase
FINAL CONCENTRATION
2 mM
37 ng/l
≥10 mg/ml
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PURIFIED RNA SPOTTING AND PRE-HYBRIDIZATION
- Purify the RNA of interest using a commercial kit (we have been using Qiagen’s RNeasy kit,
eluting RNA in nuclease-free water)
- Dilute RNA at 500 ng/l in nuclease-free water
- Prepare a mix 1/1 v/v of diluted RNA and proteinase K 20 mg/ml
- In the center of a microscope slide, gently release 0.5 l of RNA-proteinase K mix
- On the opposite side of the slide, mark the RNA-protein spot
- Incubate the slide in a thermomixer for 20 min at 80 °C
- On the slide, mount a hybridization chamber so that the RNA-protein spot is located
approximately at its center
- Fill the chamber with 100 l of a freshly prepared mix of methanol/acetic acid 3/1 v/v
- Incubate for 10 min at 25 °C
- Empty the chamber
- Wash the chamber with 100 l of 2× SSC/RVC 1/20 v/v
- Empty the chamber and repeat once the wash
- Fill the chamber with 100 l of RW25 buffer
- Incubate for 10 min at 25 °C
- Proceed to hybridization
3
CELL SPOTTING AND PRE-HYBRIDIZATION
For cultured cells: trypsinize adherent cells or pellet suspension cells, and wash them once with
1× PBS at 25 °C
For peripheral blood or bone marrow cells: purify the white blood cell fraction using a commercial
kit, then wash the cells once with 1× PBS at 25 °C
- Pre-fix the cells by slowly adding a freshly prepared mix of methanol/acetic acid 3/1 v/v to an
equivalent volume of cell suspension in 1× PBS
- Pellet the cells by centrifugation for 5 min at 200 g at 25 °C
- Aspirate most of the supernatant, and then tap the tube to dislodge the pellet
- Slowly resuspend the cells in methanol/acetic acid
- Pellet the cells by centrifugation for 5 min at 200 g at 25 °C
- Repeat the fixation twice
- Resuspend the cells in a volume of methanol/acetic acid so that the cell concentration is
approximately 10,000/l
NOTE: at this point, cells can be kept in methanol/acetic acid at 4 °C up to six months or at –80
°C indefinitely
- In the center of a microscope slide, manually release 4-5 l of fixed cell suspension, and let the
fixative evaporate. Alternatively, use a Cytospin instrument to spot 4-5×104 cells on a microscope
slide.
NOTE: at this point, spotted dried cells can be stored at room temperature for several months
before hybridization (we have tested samples 6 months after spotting, obtaining good results)
- On the slide, mount a hybridization chamber so that the cell spot is located approximately at its
center
- Fill the chamber with 100 l of Triton X-100 0.5% in 1× PBS
- Incubate for 10 min at 25 °C
- Empty the chamber
- Wash the chamber with 100 l of 2× SSC/RVC 1/20 v/v
- Empty the chamber and repeat once the wash
- Fill the chamber with 100 l of RW25 buffer
- Incubate for 10 min at 25 °C
- Proceed to hybridization
4
RNA RETRIEVAL IN TISSUE SECTIONS AND PRE-HYBRIDIZATION
- Using a clean blade and wearing protective gloves, cut 5 mm sections from a Formalin Fixed
Paraffin Embedded (FFPE) tissue, and mount each section onto a microscope slide
NOTE: at this point, tissue sections can be stored at room temperature for several months before
hybridization (we have tested samples year after sectioning, obtaining good results)
- In plastic staining jar, place up to 8 slides
NOTE: all steps below are performed in plastic staining jars until tissue sections are mounted with
hybridization chambers
- Bake tissue sections for 16 h at 56 °C in a hybridization oven
- Immerse the slides in D-Limonene for 10 min at 25 °C
- Repeat twice, each time immersing the slides in fresh limonene
- Transfer the slides in ethanol 100% for 5 min at 25 °C
- Repeat once, immersing the slides in fresh ethanol
- Transfer the slides in a freshly prepared mix of methanol/acetic acid 3/1 v/v for 5 min at 25 °C
- Transfer the slides in ethanol 100% for 5 min at 25 °C
- Transfer the slides in ethanol 85% for 3 min at 25 °C
- Transfer the slides in ethanol 70% for 3 min at 25 °C
- Transfer the slides in nuclease-free water for 3 min at 25 °C
- Transfer the slides in sodium citrate 0.01 M pH 6/RVC 1/20 v/v pre-warmed at 80 °C
- Incubate the slides for 45 min @ 80 °C
- Transfer the slides in nuclease-free water, and incubate for 3 min at 25 °C
- Transfer the slides in ethanol 70% for 3 min at 25 °C
- Transfer the slides in ethanol 85% for 3 min at 25 °C
- Transfer the slides in ethanol 100% for 3 min at 25 °C
- Dry the slides on a piece of Parafilm, then mount each tissue section with a hybridization
chamber
- Fill each chamber with 100 l of ethanol 100%, and incubate for 3 min at 25 °C
- Empty the chambers
- Fill each chamber with 100 l of ethanol 85%, and incubate for 3 min at 25 °C
- Empty the chambers
- Fill each chamber with 100 l of ethanol 70%, and incubate for 3 min at 25 °C
- Empty the chambers
- Fill each chamber with 100 l of nuclease-free water, and incubate for 3 min at 25 °C
- Empty the chambers
- Fill each chamber with 100 l of pepsin 0.025% in HCl 0.01 M, and incubate for 15 min at 37 °C
- Empty the chambers
- Fill each chamber with 100 l of nuclease-free water, and incubate for 3 min at 25 °C
5
- Empty the chambers
- Fill each chamber with 100 l of NaBH4 1% in 1× PBS, and incubate for 15 min at 25 °C
NOTE: since the borohydride solution is effervescent, replenish the chambers with fresh solution
multiple times during the course of incubation
- Empty the chambers
- Wash the chambers with 100 l of nuclease-free water
- Repeat the washes with water 4-5 times
- Empty the chambers
- Fill the chambers with 100 l of RW25 buffer
- Incubate for 10 min at 25 °C
- Proceed to hybridization
6
HYBRIDIZATION AND IMAGING
- Empty the hybridization chamber
- Slowly fill in the chamber with 100 l of RH25 buffer containing 40 ng of the desired probe (if
more than one probe is needed, use 40 ng of each probe), and then seal the injection ports
- Incubate the sample for 16 h at 37 °C
- Using RNase-free tweezers remove the seals, and then slowly empty the chamber
- Wash the chamber with 100 l of RW25 buffer
- Repeat the wash twice
- Fill in the chamber with 100 l of RW25 buffer
- Incubate the sample for 1 h at 37 °C
- Empty the chamber
- Fill in the chamber with 100 l of RW25 buffer supplemented with DAPI 20 ng/ml
- Incubate the sample for 30 min at 37 °C
- Empty the chamber
- Fill in the chamber with 100 l of EQ buffer
- Incubate the sample for 1-2 min at 25 °C
- Empty the chamber
- Peel the hybridization chamber off the slide, and remove all glue residues with RNase-free
tweezers
- Overlay the cells or tissue with 10 l of IB buffer, and the cover them with a coverglass
- Seal the coverglass with Fixogum, and then proceed to imaging
7