5. Molecularbiology and In-situ-Hybridization - quartett and In-situ... · Molecularbiology and In-situ-Hybridization ... slide and coverslip. ... •Focus plane cannot be adjusted

227

5. Molecularbiology and In-situ-Hybridization

5.1. Fluorescence-In-situ-Hybridization (FISH)FISH is a molecularbiological method to detect chromosomal abberrations, such as deletions, translocations, inversions, insertions, amplifications and aneuploidies. Therefore, it is a major tool in cytogenetic, tumor diagnosis and human chromosomal research.

Artificially produced, fluorescence labeled DNA probes bind by complementary base pairing to specific target DNA sequences in situ. This is called hybridization. ProTaqs® jellyFISH probes are used to visualize genomic DNA in formalin-fixed paraffin-embedded sections, in living cells, in metaphase chromosome spreads and interphase nuclei by fluorescence microscopy.

▶ A: Fluorescence labeling of DNA probe ▶ B: Target DNA sequence ▶ C: Denaturation of DNA double strands

(probe DNA and target DNA) ▶ D: In-situ-Hybidization ▶ E: Fluorescence signal in sample seen in

fluorescence microscopy

A

C

B

D E

E

Scheme of FISH:

▶ Most proximal marker covered by DNA probe

▶ Size and color of fluorescence signal of DNA probe

▶ Target gene detected by DNA probe

▶ Most distal marker covered by DNA probe

▶ Chromosom - Centromere

▶ Chromosom - Telomere

▶ Chromosom - Long arm

▶ Chromosom - Short arm

▶ Chromosom - Telomere

Scheme of probe map:

228

5.1.1. ProTaqs® jellyFISH ProbesThe detection of genetic abberrations with ProTaqs® jellyFISH technology is a powerful aid to guide more confident diagnoses and treatment decisions. quartett‘s expanding product portfolio includes a variety of direct labeled DNA probes for oncology to detect solid tumors and hematologic disorders:

▶ Bi-Color probes (Orange & green signal) ▶ Tri-Color probes (Orange, green and blue signal)

The following fluorophores are used:

Cat.-No. Product Name Color Signals Spezification Indication Probe Map Application Quantity

FISH011-37FISH011-38

ProTaqs® jellyFISH ALK Probe

Bi-Color Break-apart probe, orange labeled probe hybridizes to the proximal region within ALK in 2p23, green labeled probe hybridizes distal to ALK.

Lung carcinoma (NSCLC), B cell lymphoma, Neuroblastoma, Myofibroblastic tumors

FISH 80 µl160 µl


ProTaqs® jellyFISH ROS1 Probe

Bi-Color Break apart probe, orange labeled probe hybridizes proximal to the ROS1 gene at 6q22, green labeled probe hybridizes to the distal region of ROS1; also suitable to analyze the FIG-ROS1 fusion caused by an interstitial 240 kb deletion.

Lung carcinoma (NSCLC)

FISH 80 µl160 µl


ProTaqs® jellyFISH RET Probe

Bi-Color Break apart probe, orange labeled part hybridizes proximal to the RET gene at 10q11, green labeled probe hybridizes to the distal region of RET.

Lung carcinoma (NSCLC), Thyroid carcinoma

FISH 80 µl160 µl

Fluorophor Absorption max. [nm] Emission max. [nm]

Blue 426 480Green 505 530

Orange 552 576

Troubleshooting

Problem Potential Cause(s) Recommended Solution

No FISH signals can be detected in the microscope.

• Reflected light shutter closed / stop slider in light path.

• Fluorescent lamp is off. • Wrong fluorescence filter in light path. • Objective out of position. • Phototube in camera position.

• Open shutter / move stop slider out of the light path.

• Switch on fluorescent lamp. • Move correct filter into light path. • Swing objective into light path. • Direct light path to eyepieces.

Hybridization signals become weak after a while.

• Immersion oil soaked in-between slide and coverslip.

• Replace coverslip and DAPI/antifade. Use 24 x 32 mm² coverslip even if only a small region is hybridized.

Diffuse signals. • Preparation not adequately illuminated.

• Focus plane cannot be adjusted properly.

• Antifade layer is too thick for focusing.

• Check optical pathway of microscope. Adjust the UV light properly. Check the lifetime of the UV lamp.

• Use enough immersion oil. Do not mix different immersion oils. Use immersion oil suitable for fluorescence.

• Do not use too much DAPI/antifade. 10 µl per slide (24 mm x 32 mm coverslip) are sufficient.

Weak signals. • Chromosome slide preparation too old. Denaturation of chromosomes not adequate.

• Slides should not be older than two weeks. • Aging, baking or further fixation may inhibit the

hybridization and is not recommended.

High diffuse background in green color channel.

• pH value of washing solutions to low. DAPI intensity is too high resulting in crosstalk to FITC filter.

• Ensure that pH value is between 7.0 and 7.5 of solutions. FITC fluorophores are sensitive to pH below 7.

• Reduce DAPI/antifade concentration. If the recommended measures do not solve the problem, or your problem is not listed, please contact MetaSystems.

Customer Support

Please contact MetaSystems GmbH in Germany or MetaSystems Group, Inc. in the USA by telephone or e-mail (contact details, see below). MetaSystems disclaims any proprietary interest in the marks and names of others.

M MetaSystems GmbH Representative USA MetaSystems Group, Inc.

Robert-Bosch-Str. 6 70 Bridge Street, Suite 100

D-68804 Altlussheim 02458 Newton, MA

Germany United States

Tel.: +49 (0)6205 39610 Tel.: (888)489-9959 (toll free)

Fax: +49 (0)6205 32270 Fax: +1 617 924-9954

email: [email protected] email: [email protected]

URL: www.metasystems.de URL: www.metasystems.org

Revision: Rev H 130814

Symbols Used

Symbol Symbol Description

ASR Analyte -specific reagent

Analytical and performance characteristics are not established.

h Reference no

s Maximum storage temperature

M Manufacturer g Lot number

X No of tests

Y All warnings are marked by warning triangle with exclamation mark. Depending on their character they are supplemented with the words ATTENTION or CAUTION

H Expiry date

The XT ROS1-GOPC probe is designed as a break apart probe. Its green labeled part hybridizes proximal to the ROS1 gene at 6q22, the orange labeled probe hybridizes to the distal region of ROS1. This probe is also suited to analyze the FIG-ROS1 fusion, caused by an interstitial deletion of 240 kb in size. This probe is intended for Tissue-FISH applications and has been optimized for combined use with MetaSystems‘ pretreatment kit. Analytical and performance characteristics are not established. Probe Diagram:

V 140307 Chromosome 6

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XT ROS1-GOPC BA orange/green D-6029-100-OG 100µl jel

ly FISHby ProTaqs®

229



ProTaqs® jellyFISH EGFR Probe

Bi-Color Detects amplifications in the short arm of chromosome 7, orange labeled probe hybridizes to the EGFR locus at 7p11, green labeled probe hybridizes to the 7cen region.

Carcinoma in head and neck, brain, bladder, stomach, breast, lung, endometri-um, cervix, ovar, esophagus

FISH 80 µl160 µl


ProTaqs® jellyFISH ERG-TMPRSS2 Probe

Tri-Color Detects deletions by building a fusion product between ERG and TMPRSS2, detects translocations involving the ERG locus in 21q22, green labeled probe detects a region proximal to the ERG locus, orange labeled probe hybridizes specifically to the distal region adjacent to the ERG gene, blue labeled probe detects a region distal to the TMPRSS2 region which can be fused to ERG by a deletion of sequences between ERG and TMPRSS2.

Prostata carcinoma

FISH 80 µl160 µl


ProTaqs® jellyFISH PTEN/GRID1 Probe

Tri-Color Locus-specific probe detects deletions in 10q23, PTEN specific probe is labeled in orange, green labeled probe functions as a reference hybridizing to the GRID1 locus, centromere of chromosome 10 is detected by a blue labeled probe.

Glioma, carcinoma of prostate and breast

FISH 80 µl160 µl


ProTaqs® jellyFISH MYC Probe

Bi-Color Detects amplifications in the long arm of chromosome 8, orange labeled probe hybridizes to the MYC locus at 8q24, green labeled probe hybridizes to the 8cen region and functions as a control probe.

Carcinoma of breast, lung, esophagus, bladder, cervix; leukemia (CLL)

FISH 80 µl160 µl


ProTaqs® jellyFISH Her2/Neu Probe

Bi-Color Detects amplifications in the long arm of chromosome 17, orange labeled probe hybridizes to the Her2/Neu (ERBB2) locus at 17q12, green labeled probe hybridizes to the 17cen region and functions as a control probe.

Carcinoma of breast, prostate, lung, colon, ovar

FISH 80 µl160 µl

Troubleshooting























Customer Support


M MetaSystems GmbH RepresentativeRepresentativeRepresentativeRepresentative USAUSAUSAUSA MetaSystems Group, Inc.




Tel.: +49 (0)6205 39610 Tel.: (888)489-9959 (toll free)

Fax: +49 (0)6205 32270 Fax: +1 617 924-9954



RevisionRevisionRevisionRevision: Rev : Rev : Rev : Rev HHHH 130130130130814814814814

Symbols Used


ASRASRASRASR Analyte -specific reagent


hhhh Reference no

ssss Maximum storage temperature

MMMM Manufacturer gggg Lot number

XXXX No of tests

YYYY All warnings are marked by warning triangle with exclamation mark. Depending on their character they are supplemented with the words ATTENTION or CAUTION

HHHH Expiry date

The XT ERG-TMPRSS2 probe detects deletions and translocations involving the ERG locus in 21q22. The green labeled probe detects a region proximal to the ERG locus, an orange labeled probe hybridizes specifically to the distal region adjacent to the ERG gene. A blue (aqua) labeled probe detects a region distal to the TMPRSS2 region which can be fused to ERG by a deletion of sequences between ERG and TMPRSS2. This probe is intended for Tissue-FISH applications and has been optimized for combined use with MetaSystems‘ pretreatment kit. Analytical and performance characteristics are not established. Probe Diagram:

D21S1917D21S1917D21S1917D21S1917

ERGERGERGERG

400 kb

21q22

DSCR4DSCR4DSCR4DSCR4

TMPRSS2TMPRSS2TMPRSS2TMPRSS2

Gap ~2 Mb

360 kb

340 kb

D21S228D21S228D21S228D21S228

D21S1820D21S1820D21S1820D21S1820 not not not not totototo scalescalescalescale

V 130110 Chromosome 21 M

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XT ERG-TMPRSS2 BA,del orange/green/blue D-6006-100-TC 100µl

Troubleshooting























Customer Support






Tel.: +49 (0)6205 39610 Tel.: (888)489-9959 (toll free)

Fax: +49 (0)6205 32270 Fax: +1 617 924-9954




Symbols Used




hhhh Reference no



XXXX No of tests


HHHH Expiry date

The XT EGFR probe detects amplifications in the short arm of chromosome 7. The orange labeled probe hybridizes to the EGFR locus at 7p11. A green labeled probe hybridizes to the 7cen region. This probe is intended for Tissue-FISH applications and has been optimized for combined use with MetaSystems‘ pretreatment kit. Analytical and performance characteristics are not established. Probe Diagram:

D7S2276

EGFREGFREGFREGFR

7p11.2

not not not not totototo scalescalescalescale

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7p11-q11

RH122798


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230



ProTaqs® jellyFISH MDM2 Probe

Bi-Color Detects amplifications in the long arm of chromosome 12, orange labeled probe hybridizes to the MDM2 locus at 12q15, green labeled probe hybridizes to the 12cen region and functions as a control probe.

Soft tissue sarcoma, such as fibrosarcoma and chronic lymphocytic leukemia

FISH 80 µl160 µl


ProTaqs® jellyFISH MET Probe

Bi-Color Detects amplifications in the long arm of chromosome 7, orange labeled probe hybridizes to the MET locus at 7q31, green labeled probe hybridizes to the 7cen region and functions as a control probe.

Hematologic disorders, solid tumors and deriving metastases, such as carcinoma of stomach, head and neck, liver, ovar, lung, thyroid

FISH 80 µl160 µl


ProTaqs® jellyFISH MALT1 Probe

Bi-Color Break apart probe, orange labeled part hybridizes proximal to the MALT1 gene at 18q21, green labeled probe hybridizes to the distal region of MALT1.

Lymphoma FISH 80 µl160 µl


ProTaqs® jellyFISH Bcl-2 Probe

Bi-Color Break apart probe, orange labeled part hybridizes proximal to the Bcl-2 gene at 18q21, green labeled probe hybridizes to the distal region of Bcl-2.

Lymphoma FISH 80 µl160 µl


ProTaqs® jellyFISH JAK2 Probe

Bi-Color Detects amplifications in the short arm of chromosome 9, orange labeled probe hybridizes to the JAK2 locus at 9p24, green labeled probe hybridizes to a region in 9q22 and functions as a control probe.

Myeloproliferative neoplasm

FISH 80 µl160 µl

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Troubleshooting























Customer Support






Tel.: +49 (0)6205 39610 Tel.: (888)489-9959 (toll free)

Fax: +49 (0)6205 32270 Fax: +1 617 924-9954




Symbols Used




hhhh Reference no



XXXX No of tests


HHHH Expiry date

The XT MET probe detects amplifications in the long arm of chromosome 7. The orange labeled probe is designed to hybridize to the MET locus at 7q31. A green labeled probe hybridizes to the 7cen region and functions as a control probe. This probe is intended for Tissue-FISH applications and has been optimized for combined use with MetaSystems‘ pretreatment kit. Analytical and performance characteristics are not established. Probe Diagram:

395 kb

D7S522

TESTESTESTES

D7S486

7q31

METMETMETMET

7p11-q11

D7S2460


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XT MET amp orange/green D-6013-100-OG 100µl

Troubleshooting























Customer Support






Tel.: +49 (0)6205 39610 Tel.: (888)489-9959 (toll free)

Fax: +49 (0)6205 32270 Fax: +1 617 924-9954




Symbols Used




hhhh Reference no



XXXX No of tests


HHHH Expiry date

The XT MALT1 probe is designed as a break apart probe. Its orange labeled part hybridizes proximal to the MALT1 gene at 18q21, the green labeled probe hybridizes to the distal region of MALT1. This probe is intended for Tissue-FISH applications and has been optimized for combined use with MetaSystems‘ pretreatment kit. Analytical and performance characteristics are not established. Probe Diagram:

18q21

ALPK2

D18S887

not not not not totototo scalescalescalescale

NEDD4L

290 kb

D18S881

MALT1MALT1MALT1MALT1

330 kb

D18S529

ZNF532


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Troubleshooting























Customer Support






Tel.: +49 (0)6205 39610 Tel.: (888)489-9959 (toll free)

Fax: +49 (0)6205 32270 Fax: +1 617 924-9954




Symbols Used




hhhh Reference no



XXXX No of tests


HHHH Expiry date

The XT JAK2 probe detects amplifications in the short arm of chromosome 9. The orange labeled probe is designed to hybridize to the JAK2 locus at 9p24. A green labeled probe hybridizes to the 9cen region and functions as a control probe. This probe is intended for Tissue-FISH applications and has been optimized for combined use with MetaSystems‘ pretreatment kit. Analytical and performance characteristics are not established. Probe Diagram:


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231

Cat-No. Product Name Synonym Delivered/Stock Solution

Working Solution Format pH Application

400900492 Post Hybridization Wash Solution

6 x 20 ml (20x) 6 x 400 ml Concentrate ISH

400900192 SSC Buffer Concentrate Saline Sodium Citrate Buffer 6 x 20 ml (20x) 6 x 400 ml Concentrate 7.0 Southern Blot, ISH, DNA Microarray,

Northern Blot

Description: SSC buffer is used as a hybridization buffer.

402001592 TdT Buffer (TUNEL) Terminal Deoxynucleotidyl Transferase Buffer

6 x 20 ml (5x) 6 x 100 ml Concentrate 7.2 Molecularbiology

402001692 TB Buffer (TUNEL) Termination Buffer 6 x 20 ml (5x) 6 x 100 ml Concentrate Molecularbiology

Description: TUNEL technology (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) is used for detection and quantification of apoptosis at single cell level, based on labeling of DNA strand breaks.

400900292 TES Washbuffer Concentrate

N-[TRIS(hydroxymethyl)methyl]-2-aminoethanesulfonic Acid

6 x 20 ml (25x) 6 x 500 ml Concentrate 7.5 Molecularbiology

Description: It is one of the Good's buffers and can be used to make buffer solutions in the pH range 6.8 – 8.2.

5.2. Wash Solutions and Buffer

5.1.2. Preatreatment and FISH on Paraffin TissuesProTaqs® jellyFISH Pretreatment Kit is recommended for pretreatment of FFPE tissue sections. The kit is optimized for ProTaqs® jellyFISH probes. The kit and additional reagents are performed to increase the permeabilization of the cell membranes to facilitate penetration of the ProTaqs® jellyFISH probes.

Cat.-No. Product Name Format Application QuantityFISH001 ProTaqs® jellyFISH Pretreatment Kit Concentrate FISH Pretreatment of FFPE tissues 20 - 30

Tests

ProTaqs® jellyFISH Pretreatment Kit contains Pretreatment Buffer, Protease and Protease Buffer. Please find below all additionally required reagents.

Cat.-No. Product Name Synonym Format pH Application Quantity400801301 ProTaqs® Alcohol 100 % Ethanol Ready-to-use FISH: Deparaffinization 1 l

400801201 ProTaqs® Xylene 100 % Ready-to-use FISH: Deparaffinization 1 l

400301105 ProTaqs® Clear Xylene Alternative Ready-to-use FISH: Deparaffinization 5 l

400900501 SSC Buffer (2x) Saline Sodium Citrate Buffer Ready-to-use 7.0 FISH: Pretreatment, Post-Hybridization 1 l

400900601 SSC Buffer (0.4x) Saline Sodium Citrate Buffer Ready-to-use 7.0 FISH: Post-Hybridization 1 l

400900701 SSC Buffer (2x) w. 0.05 % Tween20

Saline Sodium Citrate Buffer Ready-to-use 7.0 FISH: Post-Hybridization 1 l

401603392 ProTaqs® Mount Fluor Anti Fading

Ready-to-use FISH: Counterstain 15 ml

Description: ProTaqs® Mount Fluor Anti Fading contains 500 ng/ml DAPI for counterstain step.

5. Molecularbiology and In-situ-Hybridization - quartett and In-situ... · Molecularbiology and In-situ-Hybridization ... slide and coverslip. ... •Focus plane cannot be adjusted

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