RNAscope® Fluorescent Multiplex Kit User Manual
Contents
1. ADVANCED CELL DIAGNOSTICS Control examples Figure 2 is an example of expression in the cerebral cortex of normal mouse brain Figure 2 Noy red and Fezf2 green expression in the cerebral cortex of normal mouse brain stained using the RNAscope Fluorescent Multiplex Kit 63X oil lens confocal image Troubleshooting For troubleshooting information please contact technical support at support acdbio com 18 RNAscope Fluorescent Multiplex Kit User Manual ADVANCED CELL DIAGNOSTICS A Appendix A Reagent Volume Guidelines Determine reagent volume Before starting your experiment measure the inner edge of the hydrophobic barrier to determine th recommended number of drops needed per slide see table below Size of hyrophobic Recommended Recommended Relative template size barrier in number of drops volume per slide per slide 0 75 x 0 75 t 0 75 x 1 0 0 75 x 1 25 Hydrophobic barrier measured at inner edge References in this user manual are for the 0 75 x 0 75 hydrophobic barrier size t Recommended hydrophobic barrier size is0 75 x 0 75 With this barrier size each proke is sufficient for staining 20 sections Larger tissue sections will result in fewer tests RNAscope Fluorescent Multiplex Kit User Manual 19 f Lie ADVANCED CELL DIAGNOSTICS Chemical safety ZN WAR Appendix B Safety NING GENERAL CHEMICAL HANDLING To minimize hazards ensure2. Here are a few fluorescent imaging recommendations Viewing Image capture is the recommended digital capturing option Fluorescence viewing is the recommended viewing option IMPORTANT colon kidney and liver have b Microscope with camera and fluorescence options Multispectrum microscope camera system recommended eg Nuance FX Fluorescence detection requires a high resolution and high sensitivity cooled CCD camera that is 64 pm pixel size or smaller with gt 65 peak quantum efficiency Common models include Orca Flash 4 0 Hamamatsu and Nuance FX Nuance Leica DM series or equivalent Zeiss Axio Imager or equivalent Inverted microscope is okay if optics and condenser meet requirements ADVANCED CELL DIAGNOSTICS Optics 20X N A 0 75 air 40X N A 0 8 air 40X N A 1 3 oil 63X N A 1 3 oil and 100X N A 1 4 oil 20X and 40X objective can be used for visualization of high expression genes and low expression genes respectively The RNAscope Fluorescent kits are primarily targeted for fresh frozen and cultured cells This is mainly due to imaging and analysis challenges with interference from tissue autofluorescence You can run the RNAscope Fluorescent kit on solid tumor FFPE tissues if you have access to a multispectral imaging system such as Nuance FX Nuance Solid tumors such as breast n successfully tested RNAscope Fluorescent Multiplex Kit User Manual
3. yewear face shield clothi ious diseases Follow all applicable local state provincia ations Wear appropriate protective equipment which inc ucted in properly quipped facilities using the appropriate ndividuals should be ts and blood of humans and other animals have the potential to uids infectious transmit and or national udes but is not limited ng lab coat and gloves Al safety work should be equipment for rained according to potentially infectious materials Read and follow the applicable regu atory requirements in the following ore working with guide ines and or RNAscope Fluorescent Multiplex Kit User Manual In the U S In the EU N DA ADVANCED CELL DIAGNOSTICS U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cde gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara dr waisidx_01 2029ctr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www edegov Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO labora
4. 1 on page 6 and can be completed in 6 hours Most of the RNAscope Assay reagents are available in convenient Ready ToUse RTU dropper bottles and provide a simple nearly pipettefree workflow Starting with properly prepared samples sections are first pretreated and then RNAspecific probes designed for different fluorescent detection channels are hybridized to multiple target RNAs After a series of highly effective and specific signal amplifications single RNA transcripts for two or more target genes appear as punctate dots in two or more distinctly fluorescent channels These dots are visibl using a common fluorescent microscope with the appropriate filters RNAscope Fluorescent Multiplex Kit User Manual 5 A ADVANCED CELL DIAGNOSTICS Compatible sample types The RNAscope Multiplex Fluorescent Assay is compatible with fresh frozen FF tissue cultured adherent cells on chamber slides formalinfixed parattinembedded FFPE tissue fixed frozen tissue and peripheral blood mononuclear cells PBMC Use the guide below to determine the appropriate pretreatment reagent from the Universal Pretreament Reagents Kit Cat No 322380 or RNAscope Protease Ill and IV Reagents Cat No 332340 Fluorescent Detection Pretreatment Guide Tissue Type Pretreatment Cat No Cultured Adherent Cells 320045 Fresh Frozen 322336 Fixed Frozen 322335 Peripheral oo Cells 320045 RNAscope Target Retrieval is not
5. Slide Rack submerged in the TissueTek Staining Dish filled with 1X Wash Buffer 5 Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation Repeat step 5 with fresh 1X Wash buffer Counterstain and mount the slides IMPORTANT Do this procedure with no more than 5 slides at a time 1 Remove excess liquid from the slides and add 4 drops of DAPI to each section 2 Incubate for 30 SEC at RT 3 Remove DAPI from slides and immediately place 1 2 drops of the fluorescent mounting medium onto each section 4 Caretully place a 24 mm x 50 mm coverslip over the tissue section Avoid trapping air bubbles Store slides in the dark at 4 C IMPORTANT Image the slides after 8 hours or within a few days Evaluate the samples For an example of successful staining see Figure 2 on page 18 Examine tissue sections under a standard fluorescent microscope at 20 40X magnification A confocal microscope may also be used e Assess tissue and cell morphology e Assess positive control signal strength Positive control signal should be visible as punctuate dots within cell with 20X magnification e Assess negative control background Five dots in every10 cells displaying background staining per microscope field is acceptable with 20X magnification e Evaluate target probe signal using the scoring guidelines in the next section 16 RNAscope Fluorescent Multiplex Kit User Manual Fluorescent Imaging Recommendations
6. included in the RNAscope Fluorescent Multiplex Reagent Kit Please order it separately Cat No 320043 Note For FFPE tissue preparation follow the sample preparation guide Document No 322452 USM except for the following differences e RNAscope Hydrogen Peroxide is not required e RNAscope Target Retrieval before creating the hydrophobic barrier e Replace RNAscope Protease Ill with RNAscope Protease IV this will reduce potential issues with autofluorescence during imaging Please contact technical support at support acdbio com if you have any questions 6 RNAscope Fluorescent Multiplex Kit User Manual AED ADVANCED CELL DIAGNOSTICS ZZ Target RNA Specific Oligo Probes J PreAMP AMP gt Label Probe Z Zz Z rr ed z z zs z e z gt Se I y mRNAI n mRNA2 1 Tissue section 2 Hybridize to target RNA 3 Amplify signal Start with properly prepared Hybridize multiple sets of Use up to four signal amplification systems to Visualize target RNA tissue sections and pretreat to gene specific probe pairs to target detect multiple target RNAs Probes are hybridized using a standard allow access to target RNA mRNAs to a cascade of signal amplification molecules fluorescent microscope culminating in binding of dye labeled probes visible in different fluorescent chanels Figure 1 Procedure overview Kit contents and storage The RNAscope Multiplex Fluorescent Assay requ
7. laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions e Read and understand the Safety Data Sheets SDSs provided before you store handle or work with any chemicals or hazardous materials To obtain SDSs http www acdbio com technical support user manuals e Minimize contact with chemicals Vear appropriate personal protectiv safety glasses gloves or protective clothing quipment when handling chemicals for exampl e Minimize the inhalation of chem adequa e Charac reagents and substrates used in e Ensur e ve erize hat the waste is stored ntilation for examp by analysis if nec icals Do not leave chemical containers open Use only with e fume hood ssary the waste generated by the particular applications your laboratory local s e IMPORTANT Radioactive or bioh disposa ate provincial and or ransferred transported and disposed of according to all national regulations limitations may apply Biological hazard safety AN 20 WAR agen infec regu to p cond example physical containment devices icable regulatory and company institution requirements b appl azardous materials may require special handling and NING BIOHAZARD Biological samples such as tissues body f rotectiv
8. with properly prepared sections Refer to our sample preparation and pretreatment user guides available at www acdbio com technical sup port user manuals Use only samples mounted on SuperFrost Plus Slides Fisher Scientific Cat No 12 550 15 e Follow the recommended pretreatment conditions for your sample Refer to our sample preparation and pretreatment user guides available at www acdbio com technical support user manuals e Always run positive and negative control probes on your sample to assess sample RNA quality and optimal permeabilization e Do not substitute required materials Assay has been validated with these materials only e Follow the protocol exactly for best results e Do not let your sections dry out during the procedure e Use good laboratory practices and follow all necessary safety procedures Refer to Appendix B Safety on page 20 for more information RNAscope Fluorescent Multiplex Kit User Manual 11 ADVANCED CELL DIAGNOSTICS 3 Chapter 3 RNAscope Fluorescent Multiplex Assay IMPORTANT For Part I Sample Preparation and Pretreatment Guide see Document No 320513 for Fresh Frozen Tissue or Document No 322452 USM for FFPE Tissue This procedure flows directly from sample preparation and pretreatment Refer to the appropriate sample preparation and pretreatment user guide for your specific sample type Workflow Prepare the materials 30 MIN 4 Run the assay 5 5 HR
9. 2 USM for FFPE Tissue IMPORTANT En sure probes are prewarmed and cooled to RT prior to use 1 Tap and or flick to remove excess liquid from slides and place in the HybEZ Slide Rack Add 4 drops of the appropriate probe to entirely cover each section Note Refer number of d appropriate 2 Place th to Appendix A Reagent Volume Guidelines on page 19 to determine the recommended rops needed per slide For example for a 0 75 x 0 75 barrier add 4 drops of the probe e HybEZ Slide Rack in the HybEZ Humidity Control Tray removed from the HybEZ Oven Close tray and insert back into the oven for 2 HRS at 40 C IMPORTANT To prevent evaporation make sure the turn nob is completely turned to lock position Remove the HybEZ Control Tray from the oven and remove HybEZ Slide Rack 4 One slide at a time quickly remove excess liquid by decanting and place slide in a Tissue Tek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1X Wash Buffer 5 Wash slides in 1X Wash Buffer for 2 MIN at RT Agitate slides by moving the slide rack up and down in the dish 6 Repeat Hybridize Amp 1 FL Step 5 with fresh 1X Wash Buffer 1 Take each slide one at a time from the TissueTek Slide Rack and tap and or flick to remove the excess liquid before placing in the HybEZ Slide Rack Add 4 drops of Amp 1FL to entirely cover each section 2 Place the Hy
10. GNOSTICS IMPORTANT C1 and C2 probes can be used for either fluorescent or chromogenic detection However 3plex or higher multiplexing capability is only possible with the fluorescent kit There are 3 options for alternate fluorescent color modules Any fluorescent label combinations Amp 4 Alt A B or C can be selected based on your experiment design Color Module Options Probe Channel ID Amp 4 Alt A FL Amp 4 Alt B FL Amp 4 Alt C FL Cl Alexa 488 Atto 550 Atto 550 C2 Atto 550 Alexa 488 Atto 647 C3 Atto 647 Atto 647 Alexa 488 Each probe is sufficient for staining 20 sections each with an area of approximately 20 mm x 20 mm 0 75 x 0 75 larger tissue sections will result in fewer tests The probes have a shelf lif of two years from the date of bulk manufacturing when stored as indicated in the following table Target Probes Target Probe species gene Various Ready To Use RTU probe for color 3 mL x 1 bottle 4 C channel 1 species gene 50X probe for color channel 2 60 pL x 1 tube 4 C C2 Target Probe species gene 50X probe for color channel 3 60 pL x 1 tube 4 C C3 Target Probe species gene 50X probe for color channel 4 60 pL x 1 tube 4 C cat Control Probes Positive Control Probe Various RTU probe targeting a common 3 mL x 1 bottle 4 C housekeeping gene Each detection channel has its own positive control probe 3 Plex Positive Control Probe 320861 RTU mixtur
11. S Hybridize probe 2 HRS Hybridize Amp 1 FL 30 MIN Hybridize Amp 2 15 MIN Hybridize Amp 3 30 MIN Hybridize Amp 4 15 MIN Counterstain the slides 2 MIN Evaluate the samples RNAscope Fluorescent Multiplex Kit User Manual N DO ADVANCED CELL DIAGNOSTICS Materials required for the assay Materials provided by the RNAscope Materials provided by RNAscope Other materials and equipment Fluorescent Multiplex Kit Probes e 50X Wash Buffer e Cl Target Probe Prepared sections e Amp 1 FL 50X C2 Target Probe Distilled water e Amp 2 FL 50X C3 Target Probe Carboy gt 3L e Amp 3 FL 3 Plex Positive Control Probe Tissue Tek Staining Dish e Amp 4 FL AltA Amp 4 FL AltB or Amp 4 amp Negative Control Probe Tissue Tek Clearing Agent Dish FL AltC xylene resistant e DAPI HybEZ Humidifying System Water bath or incubator Tissue Tek Vertical 24 Slide Rack Tubes various sizes Paper towel or absorbent paper Fluorescent mounting medium Cover Glass 24 mm x 50 mm Prepare the materials You may prepare the reagents at the same time you prepare pretreatment reagents Refer to a sample preparation and pretreatment user guide available at www acdbio com technical support user manuals Some of the materials may be prepared in advance and stored at room temperature Prepare 1X Wash Buffer e Prepare 3 L of 1X Wash Buffer by adding 2 94 I distilled water to 1 bottle 60 ml in a large carboy Mix well Note f precipitat
12. USER MANUAL ACP ADVANCED CELL DIAGNOSTICS RNAscope Fluorescent Multiplex Kit User Manual PART 2 Document Number 320293 For Part I Sample Preparation and Pretreatment Guide see Document Number 320513 for Fresh Frozen Tissue or Document Number 322452 USM for FFPE Tissue Research Use Only Not for diagnostic use Rev Date 11052015 For Research Use Only RUO Not for diagnostic use Trademarks RNAscope and HybEZ are trademarks of Advanced Cell Diagnostics Inc All other trademarks belong to their respective owners Citing RNAscope in Publications When describing a procedure for publication using this product please refer to it as the RNAscope 2 0 Assay and cite Wang F Flanagan J Su N Wang LC Bui S Nielson A Wu X Vo HT Ma XJ and Luo Y RNAscope A Novel n Situ RNA Analysis Platform for FormalinFixed ParaffinEmbedded Tissues J Mol Diagnostics 2012 14 22 29 Disclaimers Advanced Cell Diagnostics Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Advanced Cell Diagnostics Inc assumes no liability for any errors omissions or for any damages resulting from the use of this information Copyright 2015 Advanced Cell Diagnostics Inc All rights reserved ADVANCED CELL DIAGNOSTICS Contents Chapt
13. bEZ Slide Rack in the HybEZ Humidity Control Tray removed from the HybEZ Oven Seal tray and insert back into the oven for 30 MIN at 40 C RNAscope Fluorescent Multiplex Kit User Manual Hybridize Amp 2 FL l 6 Hybridize Amp 3 FL iP Hybridize Amp 4 FL le ADVANCED CELL DIAGNOSTICS Remove the HybEZ Control Tray from the oven and remove HybEZ Slide Rack One slide at a time quickly remove excess liquid and place slide in a Tissue Tek Slide Rack submerged in the TissueTek Staining Dish filled with 1X Wash Buffer Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation Repeat Step 5 with fresh 1X Wash Buffer Take each slide one at a time from the Tissue Tek Slide Rack and tap and or flick to remove the excess liquid before placing in the HybEZ Slide Rack Add 4 drops of Amp 2 FL to entirely cover each section Place the HybEZ Slide Rack in the HybEZ Humidity Control Tray Close tray and insert into the oven for 15 MIN at 40 C Remove the HybEZ Control Tray from the oven and remove HybEZ Slide Rack One slide at a time quickly remove excess liquid and place slide in a Tissue Tek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1X Wash Buffer Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation Repeat Step 5 with fresh 1X Wash Buffer Take each slide one at a time from the TissueTek Slide Rac
14. cdbio com Limited product warranty Advanced Cell Diagnostics Inc and or its affiliate s warrant their products as set forth in the ACD General Terms and Conditions of Sale found on the ADC website at www acdbio com technical support user manuals f you have any questions please contact Advanced Cell Diagnostics at www acdbio com about contact 22 RNAscope Fluorescent Multiplex Kit User Manual 3960 Point Eden Way Hayward CA 94545 Phone 1 510 576 8800 Toll Free 1 877 576 3636 For support email support Qacdbio com Headquarters gr AV ADVANCED CELL DIAGNOSTICS www acdbio com
15. e Mien 13 Equilibrate reagents cccccccesceesceeseeseceeecnseceeseecseecueeneeesseeeaes 14 ee 14 Hybridize pre be se 14 Hybridize Amp MEL ics varemesse 14 Hybridize Amp luuesssssmeeesresreeseeeaeavl 15 Hybridize Amp 3 Flisnes iiec niaren ariei aiSia 15 Hybridize AnpAPluunegsassormveeseatn me 15 Counterstain and mount the slides ccceeseeseeeteeeteesreeeetteeneeteens 16 Evaluate the samples cc s 3 cy ccasheemaovarscesasneeddpanie Neeme Gp 16 Fluorescent Imaging Recommendations c ccccccesseesseeesseeeseeeneeeenes 17 Control sompleavvvvedesmauiamenmr menn de 18 Tronbleshoofngssuusamsaretsaneeanmnldnesbieddu ind 18 Appendix A Reagent Volume Guidelines ccccccccccccceceeeees 19 Determine reagent VolUMe ccccccccccceceseceesceeeeecseeceseecaeeeseeenseecseeeseenaeees 19 RNAscope Fluorescent Multiplex Kit User Manual ADVANCED CELL DIAGNOSTICS Appendix B Safety cccccceceeeseeesssssssssnsaaeeeeeeeeeeeeeeeeeeeees 20 Gal 111200 ee ENE E E EEEE ETE 20 Biological hazard sale vag ero aeGe NG Ande 20 Documentation and support srrrrrrrrorrrrnnnnnnnnnnnnnnnrnrnnnnnnenne 22 Obtaining MSDSs 1vavvedeseee AGE 22 G 119 l gi are Ke RE eee ee ee eer 22 GE 90000115 EEE REE 22 Limited product vir x c01scanieierceiencstce ieee 22 4 RNAscope Fluorescent Multiplex Kit User Manual Live Fr Na ADVANCED CELL DIAGNOSTICS Chapter 1 Product Information Before using t
16. e of three probes targeting 3 ml x I bottle 4 C POLR2A in chanrel C1 PPIB in channel C2 and UBC in channel C3 Negative Control Probe dapB Various RTU probe targeting a bacterial gene 3 mL x1 bottle 4 C Each detection channel has its own negative control probe Blank Probe C1 300041 RTU Target Probe diluent 3 mL x I bottle 4 C t Available only for customorders 8 RNAscope Fluorescent Multiplex Kit User Manual ADVANCED CELL DIAGNOSTICS RNAscope Multiplex Fluorescent Reagent Kit Each RNAscope Multiplex Fluorescent Reagent Kit Cat No 320850 provides enough reagents to stain 20 tissue sections 20 sections each with an area of approximately 20 mm x 20 mm 0 75 x 0 75 larger tissue sections will result in fewer tests Each kit contains three subkits a Pretreatment Kit a Detection Kit and a Wash Buffer Kit IMPORTANT Directions to use the Pretreatment Kit are included in separate sample preparation and pretreatment user guides The reagents have a shelf life of nine months from the date of bulk manufacturing when stored as indicated in the following table Pretreatment Kit Cat No 322340 or Universal Pretreatment Reagents Cat No 322380 1X RNAscope Protease III 4 5 mL x1 bottle 4 C 2X RNAscope Protease IV 4 5 mL x 2 bottles 4 C Detection FL Kit Cat No 320851 Reagent Storage Amp 2 FL 4 C Wash Buffer Kit Cat No 310091 50X Wash Buffer 60 mL x 4 bot
17. er 1 Product Information ccccccccccccceeeeeseeseesesesssssaaees 5 About his EE EEE 5 Product description zsinor renni a ANG 5 Background EE ar eraa i ee nee ae ea 5 OVNEN Wipe EEE A E E 5 Compatible sample By O54 cadvvdvvacivadasiectavnepscvedansasdtoamessanhannernnedeccureann 6 Kit contents and SBI Sessa sian dcarwesbasacasadiaabaunanasverhadicadanaabaununsigeedderaex 7 RNAscope Probes cxinuess cacy cts aus ssipeaateas eazeecuaacatsstimeadideraisetentaieetr deeds 7 RNAscope Multiplex Fluorescent Reagent Kit cccescecseereeteeteeeteens 9 Required materials and equipment ccccccceccesceeceessecseeecsseeeteeeeseeeseeesaes 9 HybEZ Hybridization System c ccccccssecssecsseceeseeeseeeeseecseeeneeseeens 9 User supplied materials ji2 vjcsssersesanignsaadvemsadersonasduieseatuemradesseeeeupee 10 Chapter 2 Before You Begin ccccccccccccceeeessseseseessstsstaaeees 11 Important procedural guidelines ccccccccescecesecesseeeseeesseeeseeeseeeteeetseeens 11 Chapter 3 RNAscope Fluorescent Multiplex Assay sssceeee 12 EEE EEE 12 Materials required for the assay ccscccscesecssecssecsseceeeesteseeeseecseeeseeseeaeens 13 Prepare the materials c cccccccccessceeseeeseeceeeseeeeseeeeeeeeseeensesneseteeenseeens 13 Prepare 1X Wash Buffer ss c6scczcconsdacidccsanoiccsacaraccgatacdstasadenantassiarseeasie 13 Prepare PG seisen ennenen Eea naea EEr PAAA BeTa tmrminsaedcenn
18. h xylene resistant American Master Tech Scientific MLS Cover glass 24 x 50 mm Fisher Scientific MLS Water bath or incubator capable of holding temperature at 40 1 C Distilled water Tubes various sizes Paper towel or absorbent paper Fluorescent microscope with filter set Ex 358 nm Em 461 nm DAPI Ex 501 nm Em 523 nm FITC Ex 554 nm Em 576 nm Cy3 Ex 644 nm Em 669 nm Cy5 Ex 740 nm Em 764 nm Cy7 t n MLS contains the Cat No 310010 or 310013 310012 310014 310015 Cat No P36930 LWSRA24 LWT4457EA LWT4456EA 12 545 F Major Laboratory Supplier in North America For other regions please check Catalog Numbers with your local lab supplier t For custom 4 plex assays only RNAscope Fluorescent Multiplex Kit User Manual ADVANCED CELL DIAGNOSTICS Chapter 2 Before You Begin IMPORTANT For Part I Sample Preparation and Pretreatment Guide see Document No 320513 for Fresh Frozen Tissue or Document No 322452 USM for FFPE Tissue Prior to running the RNAscope Assay on your samples for the first time we recommend that you e View the video demonstrations available at www acdbio com technical support learn more e Use an RNAscope Multiplex Fluorescent Channel Assessment Slide Cat No 310022 to nsure that the fluorescent microscope is properly equipped with the correct excitation and emission filter set Important procedural guidelines e Start
19. his product read and understand the information in Appendix B Safety on page 20 in this document IMPORTANT We recommend reading the entire user manual before beginning any protocols About this guide This user manual provides guidelines and protocols to use the RNAscope Fluorescent Multiplex Reagent Kit Cat No 320850 You must use both an RNAscope Reagent kit user manual and a sample preparation and pretreatment user guide to perform the entire assay IMPORTANT For Part I Sample Preparation and Pretreatment Guide see Document No 320513 for Fresh Frozen Tissue or Document No 322452 USM for FFPE Tissue Visit www acdbio com technical support user manuals to download a sample preparation user guide Product description Background The RNAscope Multiplex Fluorescent Assay uses a novel and proprietary method of in situ hybridization ISH to simultaneously visualize up to three different RNA targets per cell in samples mounted on slides Simultaneous detection of four different RNA targets is possible and requires a custom kit order The assay is based on ACD s patented signal amplification and background suppression technology and incorporates multiplexed signal amplification systems which enable users to investigate expression as well as positional relationship between multiple genes within a cellular context Overview The RNAscope Multiplex Fluorescent Assay procedure is illustrated in Figure
20. ion occurs in 50X Wash Buffer warm it up at 40 C for 10 20 MIN before making 1X Wash Buffer 1X Wash Buffer may be prepared ahead of time and stored at room temperature for up to one month Prepare probes 1 Warm probes for 10 MIN at 40 C in a water bath or incubator then cool to ROOM TEMPERATURE RT 1 Briefly spin the C2 and C3 probes to collect the liquid at the bottom of the tubes 2 Mix 1 1 50 ratios of C2 C3 and C1 probes by pipetting 1 volume of C2 and 1 volume of C3 probes to 50 volumes of C1 probe into a tube Invert the tube several times Note Do not mix probes of the same channel The mixed Target Probes can be stored at 4 C for up to 6 months RNAscope Fluorescent Multiplex Kit User Manual 13 A amp N A le PAN LS ADVANCED CELL DIAGNOSTICS Equilibrate reagents e Place Amp 1 4 FL reagents at RT e Ensure HybEZ Oven and prepared Humidity Control Tray are at 40 C Run the assay IMPORTANT Do NOT let sections dry out between incubation steps Work quickly and fill barrier with solutions IMPORTANT View the wash step video at www acdbio com technical support learn more before proceeding Note VVe recommend running control probe on your sample before running any of your specific target probes to Hybridize probe IMPORTANT Pri optimize the protocol or to this step ensure you have pretreated your samples See Catalog No 320513 for Fresh Frozen Tissue or Catalog No 32245
21. ires the RNAscope Probes and the RNAscope Multiplex Fluorescent Reagent Kit Probes and Reagent Kits are available separately RNAscope Probes The RNAscope Probes consist of userspecified Target Probes and Positive and Negative Control Probes Visit www acdbio com products target probes search product to find a gene specific Target Probe Visit http www acdbio com control slides and probes to order appropriate Control Probes Each Target Probe contains a mixture of short oligonucleotides designed to bind to a specific target RNA and detectable in one of three color channels C1 C2 and C3 using the Amp 4 amplification step Note Different colors are assigned to the C1 C2 and C3 color channels depending on the particular RNAscope Assay The color channels for the RNAscope Multiplex Fluorescent Assay are shown in the following table Probe Amp 4 Alt A Fluorescent Label C1 Alexa 488 nm 540 10 nm GREEN C2 Atto 550 nm 580 10 nm ORANGE C3 Atto 647 nm 690 10 nm FAR RED Detault channel C1 target probes are Ready ToUse RTU while C2 and C3 probes are shipped as a 50X concentrated stock To independently detect different target RNAs in a multiplex assay each target probe must be in a different color channel and there must be a C1 probe in the mixture A Blank Probe C1 Cat No 300041 can be used in place of a specific target probe RNAscope Fluorescent Multiplex Kit User Manual 7 ADVANCED CELL DIA
22. k and tap and or flick to remove the excess liquid before placing in the HybEZ Slide Rack Add 4 drops of Amp 3FL to entirely cover each section Place the HybEZ Slide Rack in the HybEZ Humidity Control Tray Close tray and insert into the oven for 30 MIN at 40 C Remove the HybEZ Control Tray from the oven and remove HybEZ Slide Rack One slide at a time quickly remove excess liquid and place slide in a Tissue Tek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1X Wash Buffer Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation Repeat Step 5 with fresh 1X Wash Buffer Take each slide one at a time from the TissueTek Slide Rack and tap and or flick to remove the excess liquid before placing in the HybEZ Slide Rack Add 4 drops of Amp 4FL to entirely cover each section Note There are 3 options for alternate fluorescent color modules Any fluorescent label combination Amp 4FL Alt A B or C can be selected For tissue the default recommended module is Amp 4FL Alt B Place the HybEZ Slide Rack in the HybEZ Humidity Control Tray Close tray and insert into the oven for 15 MIN at 40 C Remove the HybEZ Control Tray from the oven and remove HybEZ Slide Rack RNAscope Fluorescent Multiplex Kit User Manual 15 ASD ADVANCED CELL DIAGNOSTICS 4 One slide at a time quickly remove excess liquid and place slide in a TissueTek
23. tles Room temperature 20 25 C IMPORTANT Do not interchange the reagent components of the Reagent Kits even those having the same name Required materials and equipment The following materials and equipment are needed to perform the RNAscope Assay HybEZ Hybridization System IMPORTANT The RNAscope Assay has been validated using this system only RNAscope Fluorescent Multiplex Kit User Manual 9 ADVANCED CELL DIAGNOSTICS 4 th H e RNAscope Ass For instructions on he HybEZ Hybridization System 110 VAC Cat No 310010 220 VAC Cat designed for the hybridization and incubation steps in the RNAscope Assays Incubation steps in ay require humid conditions to prevent sections from drying out how to use the HybEZ Hybridization System refer to the HybEZ No 310013 is ybridization System User Manual available at www acdbio com technical support user manuals and Vv iew the training v ideo at www acdbio com technical support learn more The system following components HybEZ Oven HybEZ Humidity Control Tray with lid 110 or 220 VAC HybEZ Humidifying Paper Pack 15 sheets User supplied materials a Spal Fluorescent mounting medium Invitrogen MLS Tissue Tek Vertical 24 Slide Rack American Master Tech Scientific MLS Tissue Tek Staining Dish American Master Tech Scientific MLS Tissue Tek Clearing Agent Dis
24. tory Biosafety Manual third edition found at www who int csr resources publications biosafety who cds csr lyo 2004 11 en Information about the Registration Evaluation Authorisation and Restriction of Chemicals REACH can be found at eur lex europu eu LexUriServ LexUriServ do uri 0J L 2010 133 0001 0043 EN PDF RNAscope Fluorescent Multiplex Kit User Manual 21 f f gt f D ADVANCED CELL DIAGNOSTICS Documentation and support Obtaining MSDSs Safety Data Sheets SDSs are available at www acdbio com technical support user manuals For the MSDSs of chemicals not distributed by Advanced Cell Diagnostics contact the chemical manufacturer Obtaining support For the latest services and support information go to www acdbio com technical sup port support overview At the website you can e Access telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Search for user documents MSDSs application notes citations training videos and other product support documents e Find out information about customer training events Contact information Advanced Cell Diagnostics Inc 3960 Point Eden Way Hayward CA 94545 Toll Free 1 877 576 3636 Direct 15105768800 Fax 1 5 10576 8801 Information info acdbio com Orders orders acdbio com Support Email support Qa
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