5. Molecularbiology and In-situ-Hybridization

Transcription

1 5. olecularbiology and In-itu-ybridization 5.1. Fluorecence-In-itu-ybridization (FIS) FIS i a molecularbiological method to detect chromoomal abberration, uch a deletion, tranlocation, inverion, inertion, amplification and aneuploidie. Therefore, it i a major tool in cytogenetic, tumor diagnoi and human chromoomal reearch. Artificially produced, fluorecence labeled DNA probe bind by complementary bae pairing to pecific target DNA equence in itu. Thi i called hybridization. probe are ued to viualize genomic DNA in formalin-fixed paraffin-embedded ection, in living cell, in metaphae chromoome pread and interphae nuclei by fluorecence microcopy. Scheme of FIS: A C D E B E A: Fluorecence labeling of DNA probe B: Target DNA equence C: Denaturation of DNA double trand (probe DNA and target DNA) D: In-itu-ybidization E: Fluorecence ignal in ample een in fluorecence microcopy Scheme of probe map: ot proximal marker covered by DNA probe Chromoom - Telomere Size and color of fluorecence ignal of DNA probe Chromoom - Short arm Target gene detected by DNA probe Chromoom - Centromere ot dital marker covered by DNA probe Chromoom - Long arm Chromoom - Telomere 227

2 Probe The detection of genetic abberration with technology i a powerful aid to guide more confident diagnoe and treatment deciion. quartett expanding product portfolio include a variety of direct labeled DNA probe for oncology to detect olid tumor and hematologic diorder: probe (Orange & green ignal) Tri- probe (Orange, green and blue ignal) FIS by ProTaq jelly The following fluorophore are ued: Fluorophor Aborption max. [nm] Emiion max. [nm] Blue Green Orange Cat.-No. Spezification Indication Probe ap Application Quantity FIS FIS ALK Probe Break-apart probe, orange labeled probe hybridize to the proximal region within ALK in 2p23, green labeled probe hybridize dital to ALK. (NSCLC), B cell lymphoma, Neuroblatoma, yofibroblatic tumor FIS FIS ROS1 Probe Break apart probe, orange labeled probe hybridize proximal to the ROS1 gene at 6q22, green labeled probe hybridize to the dital region of ROS1; alo uitable to analyze the FIG-ROS1 fuion caued by an intertitial 240 kb deletion. (NSCLC) FIS FIS RET Probe Break apart probe, orange labeled part hybridize proximal to the RET gene at 10q11, to the dital region of RET. (NSCLC), Thyroid carcinoma 228

3 Cat.-No. FIS FIS Cutomer Support Pleae contact etasytem Gmb in or etasytem Group, Inc. in the USA by telephone or (contact detail, ee below). etasytem diclaim any proprietary interet in the mark and name of other. etasytem Gmb Repreentative USA etasytem Group, Inc. Robert-Boch-Str Bridge Street, Suite 100 D Altluheim Newton, A EGFR Probe Spezification Indication Probe ap Application Quantity Fax: +49 (0) Fax: probe@metaytem.de URL: Reviion: : Rev Symbol Ued Symbol Symbol Decription Analyte -pecific reagent Analytical and performance characteritic are not etablihed. Detect amplification upport@metaytem.org in the hort arm of chromoome URL: 7, to the EGFR locu at 7p11, to the 7cen region. h Reference no aximum torage temperature The XT EGFR probe detect amplification in the hort arm of chromoome 7. The to the EGFR locu at 7p11. A to the 7cen region. Thi probe i intended for Tiue-FIS application and ha been optimized for combined ue with etasytem pretreatment kit. Analytical and performance characteritic are not etablihed. Carcinoma in head and neck, brain, bladder, tomach, breat, lung, endometrium, cervix, ovar, eophagu Product R Label Order 7p11.2 No. Pack Size XT ERG-TPRSS2 BA,del EGFR orange/green/blue D TC 100µl D7S kb 7p11-q11 anufacturer g Lot number X No of tet FIS FIS ERG-TPRSS2 Probe Tri- Detect deletion by building a fuion product between ERG and TPRSS2, detect tranlocation involving the ERG locu in 21q22, green labeled probe detect a region proximal to the ERG locu, pecifically to the dital region adjacent to the ERG gene, blue labeled probe detect a region dital to the TPRSS2 region which can be fued to ERG by a deletion of equence between ERG and TPRSS2. Protata V Chromoome 7 D21S1917 carcinoma DSCR4 ERG D21S228 Gap ~2 b TPRSS2 D21S kb 360 kb 340 kb 21q22 FIS FIS PTEN/GRID1 Probe Tri- Locu-pecific probe detect deletion in 10q23, PTEN pecific probe i labeled in orange, green labeled probe function a a reference hybridizing to the GRID1 locu, centromere of chromoome 10 i detected by a blue labeled probe. Glioma, carcinoma of protate and breat FIS FIS C Probe Detect amplification in the long arm of chromoome 8, to the C locu at 8q24, to the 8cen region and function a a control probe. Carcinoma of breat, lung, eophagu, bladder, cervix; leukemia (CLL) FIS FIS er2/neu Probe Detect amplification in the long arm of chromoome 17, to the er2/neu (ERBB2) locu at 17q12, green labeled probe hybridize to the 17cen region and function a a control probe. Carcinoma of breat, protate, lung, colon, ovar 229

4 Cat.-No. FIS FIS Troublehooting Problem Potential Caue() Recommended Solution No FIS ignal can be detected in the microcope. Reflected light hutter cloed / top lider in light path. ProTaq ybridization ignal jellyfis become weak after a while. D2 Probe Diffue ignal. Weak ignal. igh diffue background in green color channel. Open hutter / move top lider out of the light path. Switch on fluorecent lamp. Fluorecent lamp i off. Wrong fluorecence filter in light path. Objective out of poition. Swing objective into light path. Phototube in camera poition. Direct light path to eyepiece. Immerion oil oaked in-between lide and coverlip. Preparation not adequately illuminated. Focu plane cannot be adjuted properly. Spezification ove correct filter into light path. Indication Probe ap Application Quantity Detect Replace amplification coverlip and DAPI/antifade. in Ue the 24 x 32 mm² coverlip even if only a mall region i long arm of chromoome 12, hybridized. orange Check labeled optical pathway probe of microcope. hybridize Adjut to the D2 the UV light properly. locu Check the at lifetime 12q15, of the UV lamp. green labeled Ue enough immerion probe oil. Do not hybridize mix different to the 12cen immerion oil. region Ue immerion and oil uitable for fluorecence. function a a control probe. Antifade layer i too thick for focuing. Do not ue too much DAPI/antifade. 10 µl per lide (24 mm x 32 mm coverlip) are ufficient. Chromoome lide preparation too Slide hould not be older than two week. old. Aging, baking or further fixation may inhibit the Denaturation of chromoome not hybridization and i not recommended. adequate. p value of wahing olution to low. Enure that p value i between 7.0 and 7.5 of olution. FITC fluorophore are enitive to p below 7. Reduce DAPI/antifade concentration. DAPI intenity i too high reulting in crotalk to FITC filter. If the recommended meaure do not olve the problem, or your problem i not lited, pleae contact etasytem. etasytem Gmb Repreentative USA etasytem Group, Inc. Robert-Boch-Str Bridge Street, Suite 100 D Altluheim Newton, A etasytem Gmb Robert-Boch-Str. 6 D Altluheim Tel.: toxic toxique Soft tiue arcoma, uch a fibroarcoma and chronic lymphocytic leukemia contain: Formamide R 61: ay caue harm to the unborn child. S 45: In cae of accident or if you feel unwell, eek medical advice immediatly (how the label where poible). S 53: Avoid expoure --- obtain pecial intruction before ue. Only for indutry conumer. contient: Formamide R 61: Rique pendant la groee d'effet néfate pour l'enfant. S 45: En ca d'accident ou de malaie, conulter immédiatement un médecin (i poible, lui montrer l'étiquette). S 53: Éviter l'expoition --- e procurer de intruction péciale avant l'utiliation. Uniquement pour le utiliateur profeionnel. Product Label Order No. Pack Size XT ET amp orange/green D OG 100µl -18 C g xxxxx xxxx-xx XT ALT1 Break Apart Probe 100µl (X10) h D OG Product Label Order No. Pack Size FIS ProTaq Cutomer Support jellyfis Detect amplification in the ematologic XT ALT1 orange/green D OG FIS 100µl 80 µl FIS ET Pleae contact Probe etasytem Gmb in or etasytem Group, Inc. long in the USA arm by telephone of or chromoome (contact detail, ee below). 7, diorder, etasytem diclaim any proprietary interet in the mark and name of other. The XT ALT1 probe i deigned a a break apart probe. It orange labeled part hybridize proximal to the ALT1 gene at 18q21, the green olid tumor labeled probe hybridize to the dital region of ALT1. Thi probe i intended for Tiue-FIS application and ha been optimized for to the ET locu at 7q31, green and deriving labeled probe hybridize to the metatae, uch etasytem combined ue with etasytem pretreatment kit. Analytical and performance characteritic are not etablihed. Gmb Repreentative USA etasytem Group, Inc. Robert-Boch-Str. 6 TES 7p11-q11 7cen region 70 Bridge and Street, function Suite 100 a a a carcinoma of D7S486 D Altluheim control probe Newton, A tomach, head D7S522 and neck, liver, 395 kb ovar, lung, thyroid Troublehooting ET D7S2460 7q31 Problem Fax: Potential +49 (0)6205 Caue() Recommended Solution Fax: No FIS ignal can be detected in the Reflected light hutter cloed / top Open hutter / move top lider out of the light probe@metaytem.de upport@metaytem.org microcope. lider in light path. path. URL: Fluorecent lamp i off. Switch on fluorecent lamp. URL: Wrong fluorecence filter in light path. ove correct filter into light path. Reviion: : Rev Objective out of poition. Swing objective into light path. FIS ProTaq Phototube jellyfis in camera poition. Direct light Break path to eyepiece. apart probe, orange Lymphoma ybridization ignal become weak Symbol after Ued Immerion oil oaked in-between Replace coverlip and DAPI/antifade. Ue 24 x FIS ALT1 Probe labeled part hybridize proximal a while. lide and coverlip. 32 mm² coverlip even if only a mall region i Symbol Symbol Decription hybridized. to the ALT1 gene at 18q21, D18S887 Diffue ignal. Preparation not adequately Check Analyte -pecific reagent h Reference optical no pathway of microcope. Adjut NEDD4L illuminated. the UV to light the properly. dital Check the region lifetime of the of ALT kb Analytical and performance characteritic UV lamp. are Focu not etablihed. plane cannot be adjuted Ue enough immerion oil. Do not mix different ALPK2 aximum torage temperature properly. immerion oil. Ue immerion oil uitable for D18S881 fluorecence. Antifade layer i too thick for focuing. Do not ue too much DAPI/antifade. 10 µl per ALT1 anufacturer g lide Lot (24 number mm x 32 mm coverlip) are ufficient. 18q21 Weak ignal. Chromoome lide preparation too Slide hould not be older than two week. 330 kb No old. of tet Aging, baking or further fixation may inhibit the ZNF532 X Denaturation of chromoome not hybridization and i not recommended. adequate. D18S529 igh diffue background in green color p value of wahing olution to low. Enure that p value i between 7.0 and 7.5 of channel. olution. FITC fluorophore are enitive to p DAPI intenity i too high reulting in below 7. FIS ProTaq crotalk jellyfis to FITC filter. Reduce Break DAPI/antifade apart concentration. probe, orange Product Lymphoma V Label Chromoome Order No. 18 Pack Size FIS If the recommended meaure Bcl-2 do not olve Probe the problem, or your problem i not lited, pleae contact labeled etasytem. part hybridize proximal Cutomer Support to the Bcl-2 gene at 18q21, XT JAK2 amp orange/green D OG 100µl Pleae contact etasytem Gmb in or etasytem Group, Inc. in the USA by telephone or (contact detail, ee below). etasytem diclaim any proprietary interet in the mark and name of other. to the dital region of Bcl-2. The XT JAK2 probe detect amplification in the hort arm of chromoome 9. The orange labeled probe i deigned to hybridize to the JAK2 locu at 9p24. A to the 9cen region and function a a control probe. Thi probe i intended for Tiue-FIS application and ha been optimized for combined ue with etasytem pretreatment kit. Analytical and performance characteritic are not etablihed. etasytem Gmb Robert-Boch-Str. 6 D Altluheim Tel.: toxic toxique contain: Formamide R 61: ay caue harm to the unborn child. S 45: In cae of accident or if you feel unwell, eek medical advice immediatly (how the label where poible). S 53: Avoid expoure --- obtain pecial intruction before ue. Only for indutry conumer. contient: Formamide R 61: Rique pendant la groee d'effet néfate pour l'enfant. S 45: En ca d'accident ou de malaie, conulter immédiatement un médecin (i poible, lui montrer l'étiquette). S 53: Éviter l'expoition --- e procurer de intruction péciale avant l'utiliation. Uniquement pour le utiliateur profeionnel. -18 C g xxxxx xxxx-xx 100µl (X10) h D OG XT JAK2 amp Deletion/Amplification Fax: +49 (0) Fax: probe@metaytem.de FIS FIS URL: JAK2 Probe Reviion: : Rev Symbol Ued Symbol Symbol Decription Analyte -pecific reagent Analytical and performance characteritic are not etablihed. h Reference no aximum torage temperature upport@metaytem.org Detect amplification in the hort URL: arm of chromoome 9, to the JAK2 locu at 9p24, to a region in 9q22 and function a a control probe. yeloproliferative neoplam anufacturer g Lot number X No of tet V Chromoome 9 230

5 Preatreatment and FIS on Paraffin Tiue Pretreatment Kit i recommended for pretreatment of FFPE tiue ection. The kit i optimized for probe. The kit and additional reagent are performed to increae the permeabilization of the cell membrane to facilitate penetration of the probe. Cat.-No. Format Application Quantity FIS001 Pretreatment Kit Concentrate FIS Pretreatment of FFPE tiue Tet Pretreatment Kit contain Pretreatment Buffer, Proteae and Proteae Buffer. Pleae find below all additionally required reagent. Cat.-No. Synonym Format p Application Quantity ProTaq Alcohol 100 % Ethanol Ready-to-ue FIS: Deparaffinization 1 l ProTaq Xylene 100 % Ready-to-ue FIS: Deparaffinization 1 l ProTaq Clear Xylene Alternative Ready-to-ue FIS: Deparaffinization 5 l SSC Buffer (2x) Saline Sodium Citrate Buffer Ready-to-ue 7.0 FIS: Pretreatment, Pot-ybridization 1 l SSC Buffer (0.4x) Saline Sodium Citrate Buffer Ready-to-ue 7.0 FIS: Pot-ybridization 1 l SSC Buffer (2x) w % Tween ProTaq ount Fluor Anti Fading Saline Sodium Citrate Buffer Ready-to-ue 7.0 FIS: Pot-ybridization 1 l Decription: ProTaq ount Fluor Anti Fading contain 500 ng/ml DAPI for countertain tep. Ready-to-ue FIS: Countertain 15 ml 5.2. Wah Solution and Buffer Cat-No. Synonym Delivered/ Stock Solution Working Solution Format p Application Pot ybridization Wah Solution 6 x 20 ml (20x) 6 x 400 ml Concentrate IS SSC Buffer Concentrate Saline Sodium Citrate Buffer 6 x 20 ml (20x) 6 x 400 ml Concentrate 7.0 Southern Blot, IS, DNA icroarray, Northern Blot Decription: SSC buffer i ued a a hybridization buffer TdT Buffer (TUNEL) Terminal Deoxynucleotidyl Tranferae Buffer 6 x 20 ml (5x) 6 x 100 ml Concentrate 7.2 olecularbiology TB Buffer (TUNEL) Termination Buffer 6 x 20 ml (5x) 6 x 100 ml Concentrate olecularbiology Decription: TUNEL technology (Terminal deoxynucleotidyl Tranferae Biotin-dUTP Nick End Labeling) i ued for detection and quantification of apoptoi at ingle cell level, baed on labeling of DNA trand break TES Wahbuffer Concentrate N-[TRIS(hydroxymethyl)methyl]- 2-aminoethaneulfonic Acid Decription: It i one of the Good' buffer and can be ued to make buffer olution in the p range x 20 ml (25x) 6 x 500 ml Concentrate 7.5 olecularbiology 231