Fluorescence In Situ Hybridization ( FISH )

Fluorescence in situ hybridization merupakan pendekatan metodologi yang terbaru dalam pemeriksaan perubahan genetic pada sel tubuh.FISH mampu menditeksi amplifikasi gen baik pada metaphase kromosom maupun pada interfase nuclei.

FISH dapat menentukan level amplifikasi dan pola amplifikasi(clustered signals dan multiple scattered signals) yang berhubungan sesuai dengan penguatan yang terjadi pada homogeneous staining regions dan double-minute chromosome.^1,2,3

FISH dapat menditeksi amplifikasi gen tidak hanya pada isolated nuclei dan imprinted cells akan tetapi dapat juga pada jaringan yang difiksasi formalin dan diblok paraffin serta merupakan perangkat yang mantap untuk menditeksi amplifikasi gen pada specimen tumor padat.²

FISH dapat digunakan untuk menditeksi mikrodelesi,translokasi komplek dan perubahan telomere yang tidak dapat diditeksi oleh karyotyping rutin.^1,2,3

FISH dapat juga diaplikasikan untuk mengidentifikasi  pasien-pasien kanker payudara yang terpilih untuk diterapi dengan humanized monoclonal antibody terhadap protein c-erbB2 (trastuzumab).²

Oleh karena banyaknya aplikasi dari FISH,maka ada baiknya kita mengetahui secara mendasar tentang prinsip kerja dari FISH.

Tinjauan Pustaka

Pengertian

FISH adalah suatu teknik pemeriksaan sitogenetik yang merupakan suatu tipe hibridisasi yang menggunakan complementary DNA atau RNA strand (probe) yang dilabel dengan bahan fluorescence untuk melokalisir rantai DNA|atau RNA yang spesifik yang penampilannya dapat dilihat di bawah mikroskop fluorescence.^1,2,3,4

           

Prinsip Kerja

FISH menggunakan fluorescence probes yang akan mengikat bagian dari kromosom (rantai DNA/RNA yang spesifik) yang menunjukkan derajat rantai yang mirip.Ikatan ini kemudian dilihat di bawah mikroskop fluorescence.^1,2,3,4

Teknik Pemeriksaan

Pada pemeriksaan FISH,kita menggunakan probes untuk menditeksi suatu gen yang spesifik yang terdapat di nuclei yang terisolasi.Probes yang digunakan minimal 10 Kb atau lebih dari itu.Probes yang kita jumpai di pasaran memiliki ukuran  berkisar antara 30-100 Kb.Probes tersebut terdiri dari 300 bases rantai DNA yang unik.Probes yang lebih pendek menyebabkan proses hibridisasi yang tidak spesifik.

Beberapa probes tersebut dilabel dengan biotin atau digoxigenin(indirectly labeled probes) dan haptens ini kemudian diditeksi dengan streptoavidin atau anti-digoxigenin antibody yang dilabel dengan bahan fluroscence seperti FITC (Fluoroscence Isothiocyanate) atau rhodamine.FISH probes dari Vysis secara langsung dilabel dengan bahan fluorescence seperti Spectrum Orange, Spectrum Green, SpectrumAqua, dll. FISH probes tersebut dapat diproduksi dengan PCR jika kita menginginkan rantai DNA yang lebih panjang dari 10 Kb.FISH probes yang dihasilkan bersifat spesifik,seperti untuk K-sam terlokalisir pada 10q26 sebagai amplifikasi gen pada kanker lambung.²

FISH pada suspensi nuclear yang terisolasi

Jaringan tumor yang telah diangkat sesegera mungkin dipotong sehalus-halusnya dan kemudian diproses.Jaringan yang telah disiapkan ,diinkubasi selama 60 menit pada suhu 37⁰C di dalam Eagle^’s minimal essential medium yang mengandung 5% serum anak sapi dan 0.1%colagenase(protocol 1) ,selanjutnya di-vortex.Ataupun jaringan tersebut dapat diinkubasi dalam KCl hipotonik(75 mmol/L) selama 15 menit pada suhu 37⁰C(protocol2).

Setelah itu difiltrasi melalui nylon mesh(50um) dan filtratnya dicuci  sebanyak dua kali dengan phosphate-buffered saline yang dicampur dengan Carnoy^,s solution dan kemudian disimpan pada suhu -20⁰C sampai kita gunakan.

Bila dibandingkan kedua protocol diatas,maka tidak terdapat perbedaan yang berarti,walaupun pada protocol 2 diperlukan waktu yang lebih singkat.

Langkah selanjutnya suspensi nuclear tadi dihapuskan pada silanized glass slide.²

FISH pada jaringan yang difiksasi formalin dan diblok paraffin

Parameter fiksasi seperti keterlambatan fiksasi,waktu fiksasi,pH ,konsentrasi formalin,ukuran blok dan suhu pada saat proses blok paraffin sangat mempengaruhi hasil FISH.

Tingkat keberhasilan dalam pemeriksaan FISH tergantung pada pemisahan protein untuk memperoleh DNA untuk hibridisasi.Metode digesti yang sering digunakan adalah dengan sodium bisulfate/proteinase K.

Sayatan jaringan yang telah diparaffinisasi dan direhidrasi diletakkan di silanized slides dan kemudian diinkubasi dalam 20% sodium bisulfate/2xstandard saline citrate(2xSSC) pada suhu 43⁰C selama 20 menit.Kemudian setelah dicuci dengan 2xSSC,pada slide diberikan proteinase K(25ng/ml) dan diinkubasi pada suhu 37⁰C selama 30 menit.Selanjutnya dicuci lagi dengan 2xSSC dan didehidrasi dengan etanol,dikeringkan dan didenaturasi dengan probe.²

Amplifikasi onkogen pada sel-sel tumor

Pada sel-sel mammalia,amplifikasi DNA yang tinggi dapat dijumpai pada dua struktur yaitu homogenously staining region (HSRs) dan double minute chromosome(DMs).HSRs terlokalisasi di dalam suatu kromosom dalam bentuk meluas sepanjang chromosome region,sedangkan DMs merupakan struktur bebas yang mengelilingi sentromer.

Amplifikasi gen pada  HSRs berupa clustered signals dan pada DMs berupa multiple scattered signals.

Pada pemeriksaan FISH dapat digunakan single colour ataupun dual-colour.Pada single colour FISH ,dikatakan amplifikasi gen bila terlihat lebih dari 4 signal per nucleus.

Sedangkan pada dual-colour FISH,digunakan gene specific probe dan centromere specific probe yang dilabel secara berbeda.Kemudian dihitung ratio onkogen signal terhadap sentromer signal.Apabila rationya lebih besar dari 3 atau 4,maka dapat dikatakan amplifikasi pada beberapa studi.²

 DAFTAR PUSTAKA

1.Kumar,Abbas,Fausto.Pathologic Basis of Disease.Seventh edition.Elsevier     

   Saunders.Philadelphia.2005

2.Oncogene Amplification Detection by Fluorescence In Situ Hybridization,

    available at :http://npg.nature.com/modpathol/keyword_index/kiiy.html

3.Fluorescence In Situ Hybridization,available at:http://en.wikipedia.org/wiki/

   Fluorescence_in_situ_hybridization

4.In Situ Hybridization,available at:http://en.wikipedia.org/wiki/In_situ_hybridization

5.Fluorescence In Situ Hybridization,available at:http://members.aol.com/chrom info

   /fishinfo.htm

6.Protocol for Fluorescence In Situ Hybridization,available at:http://www.hku.hk/

   Oncology/lcg/ProtocolforFISH.htm

 LAMPIRAN⁶

Protocol for Fluorescence in situ Hybridization (FISH)

 
FISH is a very widely used technique on not only cytogenetic studies, but also other biological fields. It include metaphase & interphase FISH. This protocol will be divided into three parts: Probe labeling, Hybridization, and Washing.

v    v                 Probe labeling:

    Several methods are used to do probe labeling: Nick translation, Random Priming, and PCR.

Probes Labeling for FISH by Nick Translation

Materials:

Nick Translation Kit (Gibco, Cat#: 18247-015)

Components:

10X dNTP Mix

0.2 mM         each dCTP, dGTP, dTTP

0.1 mM         dATP

0.1 mM         biotin-14-dATP

500 mM       Tris-HCl (pH 7.8)

50  mM         MgCl2

100 mM       beta-mercaptoethanol

100 ug/ml    nuclease-free BSA

10X Enzyme Mix

0.5 U/μl        DNA Polymerase I

0.007 U/ul    DNase I

50 mM          Tris-HCl (pH 7.5)

5 mM            magnesium chloride

0.1 mM         phenylmethylsulfonyl fluoride

50% (v/v)     glycerol

100 ug/ml    nuclease-free BSA

Stop Buffer 0.5 M EDTA (pH 8.0)

Distilled H2O

 Procedures:

1. 1.  Place a 0.6mL microcentrifuge tube on ice and allow the tube to cool.

2. 2.  Pipet the following components to the tube:

                                                                                   

Volume	Reagent
5 mL	10X dNTP mix (minus dATP)
5 mL	10X dATP + Biotin-dATP
x mL	1mg DNA (YAC, BAC)
35– x mL	dH2O
45 mL	Total volume

           

                          

3. 3.  Mix the tube briefly. Add 5uL Pol 10X Enzyme Mix.  Mix thoroughly but gently.  Centrifuge briefly in a centrifuge to bring liquid to the bottom of the tube.

4. 4.  Incubate at 15°C for 1-2 hours in a PCR machine.

5. 5.  At 1 hour, stop the reaction by placing the tubes in -20°C.

6. 6.  Check the size of the labeled probes (2mL) by gel electrophoresis in 0.7~2% agarose gel, looking for the peak size between 50 – 500 bp (or 100-300 bp) DNA fragments.

7. 7.  If the size range is larger than this, add a further 5mL enzyme mix, place at 15ºC for a further 30-60 min, and run another on a gel to test the size.

8. 8.  Stop the reaction by either adding the Stop Buffer or heating in a 75°C water bath for 10 minutes (or incubate at 75°C in a PCR machine).

9. 9.  Chill on ice.

Precipitating the biotin-labeled probe

10.      10. Combine the following in a 1.5mL microcentrifuge tube:

48mL                       Biotin-labeled DNA

1mL             glycogen or 50mg salmon sperm DNA

11.      11. Then, add 51mL dH₂O to make up to 100mL.

12.      12. Add 0.1 volume 3M Sodium Acetate solution (pH 5.6) into the DNA sample; e.g., 100mL DNA solution + 10mL 3M Sodium Acetate solution.

13.      13. Add 2.5X volume (250mL) cold absolute ethanol to the mixture, mix well.

14.      14. Incubate the mixture at –20°C for at least 30-60 minutes.

15.      15. Centrifuge the mixture for 20 minutes at highest speed at 4°C.

16.      16. Discard the supernatant, then vacuum dry the DNA for about 10 minutes.

17.      17. Store the DNA probe in dry form at –20°C until use.

18.      18. Add x mL of dH₂O (usually 10-20 mL) to the precipitated DNA (according to the size of the pellet). To give a final concentration of 50ng/mL.  Allow the DNA to dissolve at RT for 1-2 h or at 4°C overnight with occasional mixing.  Purified, labeled probes are stable for several years stored at –20°C.

Probes Labeling for FISH by Random Priming Method

Materials:

BioPrime DNA Labeling System (Cat#: 18094-011)

Components:

2.5X Random Primers Solution:

[125 mM Tris-HCl (pH 6.8), 12.5 mM MgCl2, 25 mM 2-mercaptoethanol, 750 ug/ml oligodeoxyribonucleotide primers (random octamers)]

 

10X dNTP Mixture:

 [1 mM biotin-14-dCTP, 1 mM dCTP, 2 mM dATP, 2 mM dGTP, 2 mM dTTP in 10 mM Tris-HCl (pH 7.5), 1 mM Na2EDTA]

Klenow Fragment (Large Fragment of DNA Polymerase I):

[40 U/ul Klenow Fragment in 50 mM Potassium Phosphate (pH 7.0), 100 mM KCl,

1 mM DTT, 50% Glycerol]

Stop Buffer: [0.5 M Na2EDTA (pH 8.0)]

Distilled Water

Procedures:

1. Dissolve 100 ng DNA in 5-20 ul of dilute buffer in a microcentrifuge tube. On ice, add 20 μl 2.5X Random Primers Solution.

2. 2.  Denature by heating for 5 min in a boiling water bath; immediately cool on ice. (The amount of template per reaction has been varied from 25-500 ng with satisfactory results.)

Perform the following additions on ice:

5 ul 10X dNTP Mixture

Distilled Water to a total volume of 49 ul

3. 3.  Mix briefly.

4. 4.  Add 1 ul Klenow Fragment. Mix gently but thoroughly. Centrifuge 15-30 sec.

5. 5.  Incubate at 37°C for 60 min.

6. 6.  Add 5 ul Stop Buffer.

Probes Labeling for FISH by DOP-PCR: (see Protocol of Chromosome Microdissection)

v    v                 Hybridization:

 

1. Materials:

1.1. Slide Pretreatment

1.              (Optional: not recommended for metaphase FISH): PK stock solution: 5 mg proteinase K (Boehringer, Mannheim, Germany), 50 mL 1M Tris-HCl (pH 7.5), 20 mL 0.5M EDTA (pH 7.0), 2 mL 5M NaCl, make up to 1 mL in filtered double- distilled water; make fresh as required.

2.              20X standard saline citrate (SSC) stock solution: 3.0M NaCl, 0.3M Na-citrate; set up with double-distilled water, adjust to pH 7.0, autoclave, and store at room temperature.

3.              RNase stock solution: 10 mg/mL of RNase type A (Boehringer); set up with filtered double- distilled water; aliquot and store at -20ºC.

4.              RNase solution: per slide 200 mL 2X SSC plus 10 mL of RNase stock solution are necessary; make fresh as required.

5.              (Optional: not recommended for metaphase FISH): Pepsin stock solution 10% (w/v): dissolve 100 mg pepsin (Serva, Heidelberg, Germany) in 1 mL of filtered double-distilled water at 37ºC; aliquot and store at -20ºC.

6.              (Optional: not recommended for metaphase FISH): Pepsin buffer: Add 1 mL of 1M HCl to 99 mL of distilled water and incubate at 37ºC for about 20 min; then add 50 mL of the pepsin stock solution 10% (wlv) and leave the coplin jar at 37ºC; make fresh as required.

7.   7.    (Optional: not recommended for metaphase FISH): 1X PBS/ MgCl₂: 5% (v/v) 1M MgCl₂ in 1X PBS. (2.5mL 1M MgCl₂ in 47.5mL 1X PBS)

8.   8.    (Optional: not recommended for metaphase FISH): Formalin buffer: 3% (v/v) of acid-free formaldehyde (37%; Roth) in 1X PBS; make fresh as required.

1.2. Fluorescence In Situ Hybridization (FISH)

1.2.1. Slide Denaturation

1. 1.    Denaturation buffer(preferred): 70% (v/v) deionized formamide, 10% (v/v) filtered double-distilled water, 10% (v/v) 20X SSC, 10% (v/v) phosphate buffer; make fresh as required.

OR:  Denature solution: 70% (v/v) formamide, 2X SSC (pH7.0), 0.1mM EDTA, pH7.0

Add 175mL formamide, 25mL 20X SSC (pH7.0), 50mL 0.5M EDTA, pH7.0 and 50mL purified H₂O to make 250mL solution and mix thoroughly.  Verify that the pH is 7.0-7.5 by measuring the pH at ambient temperature.  Between use, store covered at 4°C.  Discard after 7 days.

2. Deionized formamide: Add 5 g of ion exchanger Amberlite MB1 (Serva) to 100 mL of formamide (Merck, Darmstadt, Mannheim, Germany) stir for 2 h (room temperature) and filter twice through Whatmann no. 1 filter paper. Aliquot and store at -20ºC.

3. Phosphate buffer: prepare 0.5M Na₂HPO₄ and 0.5M NaH₂PO₄, mix these two solutions (1: 1) to get pH 7.0, then aliquot and store at -20ºC.

1.2.2. Probe Denaturation

1.   Hybridization buffer: Dissolve 2 g dextran sulfate in 10 mL 50% deionized formamide/2X SSC/50 mM phosphate buffer for 3 h at 70ºC. Aliquot and store at -20ºC.

OR:     Hybridization solution: MM2.1:           5.5mL formamide

1g Dextran sulfate

0.5mL 20X SSC

Heat to 70°C for several hours to dissolve the dextran sulfate, then cool and adjust to pH 7.0 and add water to volume of 7mL.

1.2.3. Posthybridization and Detection Washing

 

1.   1.    Washing solution I: 50% (v/v) formamide (Merck), 10% (v/v) 20X SSC, 40% (v/v) distilled water; make fresh as required.

2.   2.    Washing solution 2 (WS-2)(4X SSC/0.05% Tween 20).

3.   3.    Washing solution 3 (WS-3)(4X SSC).

4.   4.    Blocking solution: 1-3% (w/v) BSA in 4X SSC, 0.05% (v/v) Triton X-100 (make up fresh).

5.   5.    PN buffer: 0.1M NaH₂PO₄/0.1M Na₂HPO₄ M, pH 8.0; 0.1% NP-40.

6.   6.    PNM buffer:

Add 5% (w/v) non fat dry milk to PN buffer plus 0.02% (w/v) Na-azide, incubate at 37°C overnight.  It will look terrible.  Centrifuge the solution for 5 minutes at 1000g. Transfer the supernatant to a clean tube and store at 4°C.

7.   7.    FITC-Avidin (2mg/2mL): Add 398mL PNM buffer to make up 5mg/mL.

OR:  Solution 1: FITC-avidin (CAMON Vector Laboratories)/4X SSC/0.2 %Tween/5% BSA (1: 300 both Sigma, St. Louis, MO); make fresh as required.

8.   8.    Anti-Avidin (2mg/2mL): Add 398mL PNM buffer to make up 5mg/mL.

 OR: Solution ll: Biotinylated antiavidin (CAMON Vector Laboratories)/Anti-digoxigenin- rhodan-dne (Boehringer Mannheim, Germany)/4X SSC/0.2%Tween/5% BSA (1: 20: 100); make fresh as required.

9.   9.    Antifade solution

100mg p-phenylenediamine dihydrochloride in 10mL PBS.  Adjust to pH 8.0 with 0.5M carbonate-bicarbonate buffer (0.42g NaHCO₃ in 10mL dH₂O, adjust pH to 9.0 with NaOH).  Add to 90mL with glycerol.  Filter with 0.22m membrane to remove undissolved particals.  If necessary, add 0.5-1mg/mL DAPI.

OR:  DAPI-solution: Dissolve 5 mL of DAPI (4,6-diamidino-2-phenylindol.2HCl stock- solution; Serva) in 100 mL 4X SSC/0.2% Tween; make fresh as required.

OR:  70% (v/v) deionized formamide, 10% (v/v) filtered double-distilled water, 10% (v/v) 20X SSC, 10% (v/v) phosphate buffer, make fresh as required.

2. Procedures for Fluorescence In Situ Hybridization (FISH):

Pre-treatment of slides

1. 1.  (Optional) If the slide is not dry enough, dehydrate the slide by immersing the slide into 100% ethanol for 1 min.  Air dry.

2. 2.  Pretreat slide with 200mL diluted RNase solution (0.1mg/mL) for about 30 to 60 minutes at 37°C.

3. 3.  Wash slide with 2X SSC for 5 minutes (with agitation).

4. 4.  Pepsin treatment: (Optional, if the chromosome targets are bone marrow smear, bone marrow progenitors from methyl cellulose-grown colony assays, tumour preparations)

i.   i.         Add 200mL diluted (with 0.01M HCl) pepsin to the slide

ii.  ii.       Incubate slides at 37°C for 5-10 minutes.

iii. iii.      Wash 2X with 1X PBS for 5 min at RT with shaking.

Note: over-digestion can also cause problems (loss of cells from the slide), so only use when absolutely necessary.

5. 5.  Place slides in PBS/50mM MgCl₂ for 5 min.

6. 6.  (Optional) Fix in PBS/50mM MgCl₂/1% formaldehyde for 10 min.

7. 7.  Wash in PBS for 5 min (with agitation).

8. 8.  Dehydrate the slide with 70%, 90%, and 100% ethanol for 1-2 min each and allow to air dry.  Slides can be stored desiccated at 4°C for up to one month before use.

Pre-hybridization of DNA probes

9. 9.  During incubation step 1, prepare hybridization mixture as follows:

i.    i.      Add 2mL (100ng) diluted Biotin-labeled DNA + 1mL (2.5mg) Cot-1 DNA + 7mL MM2.1(warmed to RT).

ii.   ii.     Denature the hybridization mix at 75°C for 5-7 minutes.

iii.  iii.   Place the probe on ice for 3-5 mins. (very IMPORTANT!)

iv.  iv.   Transfer the denatured probe to 37°C for 15 min – 2 h for prehybridization.

 Denature metaphase chromosome slides

10.     10.        Incubate slides in denaturing solution (in water-bath) for 2 minutes at 75°C.

11.     11.        Wash slides in cold 2X SSC, followed by two changes of 2X SSC.

12.     12.        Dehydrate the slide through a cold alcohol series (70%, 90%, and 100% ethanol for 1 min each)..

13.     13.        Air dry the slides and place on a hot plate at ~42°C.

14.     14.        Apply 10mL of denatured probe mix to the slide.

15.     15.        Immediately apply a coverslip and seal with rubber cement.  Keep the slide in a moist chamber at 37°C overnight – four days for hybridization.

 

Washing slides

16.     16.        Place the wash tanks containing WS-1 in a 45°C water bath for at least 30 minutes prior to use.

17.     17.        Remove rubber cement by using forceps.  Coverslips can then be removed either by soaking in 2X SSC or gently tipping them off into the glass disposal bin (never pull them off!).

18.     18.        Wash the slides 3 times with WS-1 at 45°C for 5 –10 min each (usu. 5min).

19.     19.        Wash the slides 3 times with WS-2 at RT for 2 min each.

20.     20.        (Optional)        a) Blocking treatment with 1-3% BSA in 4X SSC for 20 min at RT.

21.     21.        b) Wash the slides 3 times with WS-2 at RT for 2 min each.

22.     22.        Wash the slides with WS-3 for 2 min at RT.

Note: make sure the slides are not dried in any point during the detection and washing steps.

Signal enhancement

23.     23.        Add 40uL Avidin-FITC (5ug/mL Avidin in PNM buffer) onto slides and cover with coverslip.  Keep the slides in a moist chamber in dark for 20 min at RT.

24.     24.        Wash the slide as steps 19, 21 (do not perform step 18 & 20). Then, proceed from 24 to 26 steps or directly jump to steps 27-29.

25.     25.        (Optional): Add 40uL anti-Avidin (5ug/mL anti-Avidin in PNM buffer) onto slides and cover with coverslip.  Keep the slides in a moist chamber in dark for 20 min at RT.

26.     26.        Wash the slides as steps 23.

27.     27.        Repeat steps 22-23 for one additional Avidin-FITC treatment.

Visualizing the hybridization

28.     28.        Dehydrate the slides with 70%, 90%, and 100% ethanol for 1-2 min each and allow to air dry. 

29.     29.        Apply 40uL of DAPI II counterstain and a coverslip to hybridization location.

30.     30.        Store in dark if not use, otherwise, examine the slide at once under fluorescence microscope.