RNAscope Fluorescent Multiplex Kit User Manual
Contents
1. ALD Chapter 1 Product Information Before using this product read and understand the information in Appendix B Safety on page 22 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 1 Sample Preparation and Pretreatment Guide see Catalog No 320513 for Fresh Frozen Tissue or Catalog No 320511 for FFPE Tissue Visit www acdbio com support technical doc to download a sample preparation user guide Product description Background Overview 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 The RNAscope Multiplex Fluorescent Assay procedure is illust2. USER MANUAL ADVANCED CELL DIAGNOSTICS INC RNAscope Fluorescent Multiplex Kit User Manual PART 2 Catalog Number 320293 For Part 1 Sample Preparation and Pretreatment Guide see Catalog Number 320513 for Fresh Frozen Tissue or Catalog Number 320511 for FFPE Tissue For Molecular Biology Applications not intended for diagnosis Rev Date 20131104 For Molecular Biology Applications not intended for diagnosis Trademarks RNAscope and HybEZ are trademarks of Advanced Cell Diagnostics Inc All other trademarks belong to their respective owners Citing RNAscope 2 0 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 L C Bui S Nielson A Wu X Vo H T Ma X J and Luo Y RNAscope A Novel In Situ RNA Analysis Platform for Formalin Fixed Paraffin Embedded 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 2013 Advanced Cell Diagnostics Inc All rights reserved ASD Conte
3. 220 VAC Cat No 310013 is designed for the hybridization and incubation steps in the RNAscope Assays Incubation steps in the RNAscope Assay require humid conditions to prevent sections from drying out For instructions on how to use the HybEZ Hybridization System refer to the HybEZ Hybridization System User Manual available at www acdbio com support technical doc and view the training video at www acdbio com support online training videos The system contains the following components A Component Quantity Cat No HybEZ Oven 110 or 220 VAC 1 oven 310010 or 310013 HybEZ Humidity Control Tray with lid 1 tray 310012 HybEZ Slide Rack 20 slide capacity 1 rack 310014 HybEZ Humidifying Paper 2 sheets ES HybEZ Humidifying Paper Pack 15 sheets 310015 RNAscope Fluorescent Multiplex Kit ASD User supplied materials Fr Description Supplier Cat No Fluorescent mounting medium Invitrogen MLS P36930 Tissue Tek Vertical 24 Slide Rack American Master Tech Scientific MLS LWSRA24 Tissue Tek Staining Dish American Master Tech Scientific MLS LWT4457EA oe Clearing Agent Dish xylene American Master Tech Scientific MLS LWTA4456EA Cover glass 24 x 50 mm Fisher Scientific MLS 12 545 F Carboy gt 3L MLS Water bath or incubator capable of MLS holding temperature at 40 1 C Distilled water MLS Tubes various sizes MLS Paper towel or absorbent pape
4. 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 22 for more information RNAscope Fluorescent Multiplex Kit 11 D ADVANCED CELL DIAGNOSTICS INC 12 RNAscope Fluorescent Multiplex Kit User Manual ASD eM Chapter 3 RNAscope Fluorescent Multiplex Assay IMPORTANT For Part 1 Sample Preparation and Pretreatment Guide see Catalog No 320513 for Fresh Frozen Tissue or Catalog No 320511 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 i Run the assay 3 hours 40 MIN Hybridize probe 2 HRS Hybridize Amp 1 FL 30 MIN Hybridize Amp 2 FL 15 MIN Hybridize Amp 3 FL 30 MIN Hybridize Amp 4 FL 15 MIN Counterstain and mount the slides 10 MIN Evaluate the samples RNAscope Fluorescent Multiplex Kit 13 ASD Materials required for the assay Materials provided by the RNAscope Fluorescent Multiplex Kit Materials provided b
5. 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 C 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 Amp4 Alt A Fluorescent Label Channel ID Excitation Emission Color 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 Default channel C1 target probes are Ready To Use 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 IMPORTANT C1 and C2 probes can be used for either fluorescent or chromogenic detection However 3 plex or higher multiplexing capability is only possible with the fluorescent kit There are 3 options for alternate fluorescent color modules Any fluorescent label combination 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 C FL C3 Atto 647 Alexa 488 RNAscope Fluorescent Multip
6. camera that is 64 um respectively pixel size or smaller with gt 6596 peak quantum efficiency e Common models include Orca Flash 4 0 Hamamatsu and Nuance FX Nuance IMPORTANT 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 multi spectral imaging system such as Nuance FX Nuance Solid tumors such as breast colon kidney and liver have been successfully tested RNAscope Fluorescent Multiplex Kit User Manual PED Control examples Figure 2 is an example of expression in the cerebral cortex of normal mouse brain Figure 2 Npy 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 RNAscope Fluorescent Multiplex Kit 19 ASD A Appendix A Reagent Volume Guidelines Determine reagent volume Before starting your experiment measure the inner edge of the hydrophobic barrier to determine the recommended number of drops needed per slide see table below Size of Recommended Recommended Relative template size hyrophobic number of drops volume per slid
7. dark at 4 C Evaluate the samples For an example of successful staining see Figure 2 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 e Assess negative control background One dot in every 10 cells displaying background staining per microscope field is acceptable Evaluate target probe signal using the scoring guidelines in the next section RNAscope Fluorescent Multiplex Kit 17 ASD Fluorescent Imaging Recommendations Here are a few fluorescent imaging recommendations 18 Viewing Detection Microscope Optics e Image capture is e Microscope with camera e Leica DM series or e 20X N A 0 75 air the recommended and fluorescence equivalent 40X N A 0 8 air 40X digital capturing options Multispectrum N A 1 3 oil 63X e Zeiss Axio Imager or option microscope camera N A 1 3 oil and Eliorescance system recommended eden 100X N A 1 4 oil e viewing is the eg Nuance FX ee Cope e 20X and 40X objective l is okay if optics and recommended e Fluorescence detection can be used for due condenser meet l dise l viewing option requires a high visualization of high l requirements resolution and high d expression genes and sensitivity cooled CCD low expression genes
8. e barrier in per slide uL B E 6 180 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 is 0 75 x 0 75 With this barrier size each probe is sufficient for staining 20 sections Larger tissue sections will result in fewer tests 20 RNAscope Fluorescent Multiplex Kit User Manual RNAscope Fluorescent Multiplex Kit AED ADVANCED CELL DIAGNOSTICS INC 21 ASD Appendix B Safety Chemical safety AN WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure 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 Read and understand the Material Safety Data Sheets MSDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain MSDSs see Documentation and support in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Characterize by analysis if necessary the waste generated by the
9. ize probe ee ee 15 IVORIOIZS AMP T F iesca ai Nirea 16 Hybridize Amp 2 FL errnnnnonnnnnnonrnnnnnvnnnnnnennnnnnennnnnnnennnnenennnnsnrnnnnnnnennnnnne 16 Hybridize Amp I TlLL KG eksemet 16 Hybridize Amp 4 FL rrrrnnnnonnnnnronnnnnnrvnnnnnnennnnnnennnnnnrennnnenennnnnnrnnnnnnnennnnene 17 Counterstain and mount the slides seeeeeseeeeeeeeses 17 Evaluate the samples cccccccsescecceeeeeecceeseecseseeecsaseeeseaseeessageeessaneessseass 17 Fluorescent Imaging Recommendations eesssssse 18 Control examples ssseeesesseseeee nennen nnne nennen nnns 19 Appendix A Reagent Volume GuidelinesError Bookmark not defined Determine reagent volume sese 20 Appendix B Safety Lu te secus ax VessadUs ducas Rav csSk d nud qa x EE nde US dvinuS 22 Chemical safety ccccccccccccccsssseeeeeeeeeseeeeeeeeeseeeeseeeeeeeesseeeeeesssaaeeeesessaeeeees 22 Biological hazard safety sss nnne 22 Documentation and support eee eese eee eee 24 DEN WIS DSS mt 24 RNAscope Fluorescent Multiplex Kit User Manual AD ADVANCED CELL DIAGNOSTICS INC ODNIO SUID OM EEE 24 Contact information 2 seceko se sextese susti rie pata voe eeu w Ens ee be ve RIED Peres tese EDXN EP De seo ak 24 Limitea prod ct Waranty EEE EE EE 24 4 RNAscope Fluorescent Multiplex Kit User Manual
10. lex Kit ASD 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 life of six months from the shipment date when stored as indicated in the following table Target Probes Reagent Cat No Content Quantity Storage Target Probe species gene Various Ready To Use RTU probe for color 3 mL x 1 bottle 4 C channel 1 Target Probe species gene 50X probe for color channel 2 60 uL x 1 tube 4 C C2 Target Probe species gene 50X probe for color channel 3 60 uL x 1 tube 4 C C3 Target Probe species gene 50X probe for color channel 4 60 uL x 1 tube 4 C C4T Control Probes Fr Reagent Cat No Content Quantity Storage Positive Control Probe Various RTU probe targeting a common 3 mL x 1 bottle 4 housekeeping gene Each detection channel has its own positive control probe 3 Plex Positive Control Probe Various RTU mixture of three probes targeting 3 mL x 1 bottle 4 POLR2A in channel C1 PPIB in channel C2 and UBC in channel C3 Negative Control Probe dapB Various RTU probe targeting a bacterial gene 3 mL x 1 bottle 4 Each detection channel has its own positive control probe Blank Probe C1 300041 RTU Target Probe diluent 3 mL x 1 bottle 4 C T Available only for custom orders RNAscope Multiplex Fluore
11. n 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 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 3 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 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 Re
12. nts Chapter 1 Product Information 5 ADOUL lg qo Pe RERO 5 FOOU raS IDU e EE T o 5 BACO UNO EE NE 5 NAN 5 Compatible sample types cccccsssescccecsseeceeeceeeeeeeeeseeaeeceeesseaeeeeeseaaaees 6 Kitcontents and Storage eee 7 RNAscope Probes csescssesesececesesescevevecececevereecevevecacevenersevevavaveveneneeeevens 7 RNAscope Multiplex Fluorescent Reagent Kit ee 8 Required materials and equipment rrrrrnnnnnnorrrnnnnnnvnnrnnnnnrennrnnnnnsennnnnnsnsennnnnn 9 HybEZ Hybridization System enn 9 User supplied Mater ialS ccccccccsssseeeeceessseeecceeseeeeeseaseeeeessagseeeeseas 10 Chapter 2 Before You Begin ss 11 Important procedural guidelines rrrrrrrnnnnnrrnrrnnnnrrenrrnnnnnrrnnnnnnnnsennnnnnsnsenn 11 Chapter 3 RNAscope Fluorescent Multiplex Assay 13 NNN 13 Materials required for the ASSAY rrrrrrrnnnnnnvrrrnnnnnrrnnrnnnnnrrnnnnnnnnsennnnnnnsnnnnnnn 14 Prepare the materials rrrrnnnnnrrrrnnnnronnnnnnennnnnnrnnnnnnrennnnnennnnnnrennnnesennnnsennnnn 14 Prepare 1X Wash Buffer sees 14 Prepare probes sssesssssseseeee nennen nennen nnne nnn nnn nnn nnn nnns 14 Equilibrate reagents siiciiisinanscvdewacannsencsh axsieweensssdeaesaddheacentheneshaivarbensixinancane 15 RUN INE Se EE S aE 15 Hybrid
13. particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply Biological hazard safety AN 22 WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following RNAscope Fluorescent Multiplex Kit User Manual In the U S In the EU PED U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogen
14. peat Step 5 with fresh 1X Wash Buffer RNAscope Fluorescent Multiplex Kit User Manual Hybridize Amp 4 FL AGD 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 4 FL to entirely cover each section Note There are 3 options for alternate fluorescent color modules Any fluorescent label combination Amp 4 FL Alt A B or C can be selected For tissue the default recommended module is Amp 4 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 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 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 Incubate for 30 SEC at RT Remove DAPI from slides and immediately place 1 2 DROPS of the fluorescent mounting MEDIUM onto each section Carefully place a 24 mm x 50 mm coverslip over the tissue section Avoid trapping air bubbles Store slides in the
15. r MLS Fluorescent microscope with filter set MLS 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 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 10 RNAscope Fluorescent Multiplex Kit User Manual ALD Chapter 2 Before You Begin IMPORTANT For Part 1 Sample Preparation and Pretreatment Guide see Catalog No 320513 for Fresh Frozen Tissue or Catalog No 320511 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 support online training videos e Use an RNAscope Multiplex Fluorescent Channel Assessment Slide Cat No 310022 to ensure that the fluorescent microscope is properly equipped with the correct excitation and emission filter set Important procedural guidelines e Start with properly prepared sections Refer to our sample preparation and pretreatment user guides available at www acdbio com support technical doc e 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 support technical doc e
16. rated in Figure 1 on page 6 and can be completed in 6 hours Most of the RNAscope Assay reagents are available in convenient Ready To Use RTU dropper bottles and provide a simple nearly pipette free workflow Starting with properly prepared samples sections are first pretreated and then RN A specific 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 visible using a common fluorescent microscope with the appropriate filters RNAscope Fluorescent Multiplex Kit AED ADVANCED CELL DIAGNOSTICS INC Compatible sample types The RNAscope Multiplex Fluorescent Assay is compatible with fresh frozen FF tissue cultured adherent cells on chamber slides formalin fixed paraffin embedded FFPE tissue fixed frozen tissue and peripheral blood mononuclear cells PBMC Use the guide below to determine the appropriate pretreatment reagent Fluorescent Detection Pretreatment Guide Tissue Type Pretreatment Kit Pretreatment Cat No Cultured Adherent Cells 320842 Fresh Frozen 320842 Fixed Frozen 320043 Peripheral Blood Mononuclear 320842 Cells PBMC Pretreat 2 is not included in the RNAscope Fluorescent Multiplex Reagent Kit Please order it separately Cat No 320043 No
17. s 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 2029cfr1910a_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 cdc gov Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory 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 europa eu LexUriServ LexUriServ do uri OJ L 2010 133 0001 0043 EN PDF RNAscope Fluorescent Multiplex Kit 23 ASD Documentation and support Obtaining MSDSs Material Safety Data Sheets MSDSs are available at www acdbio com support technical doc category msds 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 support 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 ci
18. scent 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 sub kits 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 six months from the shipment date when stored as indicated in the following table Pretreatment Kit Cat No 310842 Reagent Quantity Storage 1X Pretreat 3 protease 4 5 mL x 1 bottle 4 C 2X Pretreat 4 protease 4 5 mL x 2 bottles 4 C 8 RNAscope Fluorescent Multiplex Kit User Manual ASD CELL DIAGNOSTI Detection FL Kit Cat No 320851 Reagent Quantity Storage Wash Buffer Kit Cat No 310091 Reagent Quantity Storage 50X Wash Buffer having the same name Required materials and equipment 60 mL x 4 bottles IMPORTANT Do not interchange the reagent components of the Reagent Kits even those Room temperature 20 25 C 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 The HybEZ Hybridization System 110 VAC Cat No 310010
19. tations 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 1 510 576 8800 Fax 1 510 576 8801 Information info acdbio com Orders orders acdbio com Support Email support acdbio 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 ACD website at www acdbio com tos terms and conditions of sale If you have any questions please contact Advanced Cell Diagnostics at www acdbio com support 24 RNAscope Fluorescent Multiplex Kit User Manual Headquarters 3960 Point Eden Way Hayward CA 94545 Phone 1 510 576 8800 Toll Free 1 877 576 3636 For support email support acdbio com www acdbio com
20. te For FFPE tissue preparation follow the sample preparation guide Catalog No 320511 1 Tissue section Start with properly prepared tissue sections and pretreat to allow access to target RNA except for the following differences 1 Pretreat 1 is not required Apply Pretreat 2 after creating the hydrophobic barrier Replace Pretreat 3 with Pretreat 4 this will reduce potential issues with autofluorescence during imaging Please contact technical support at support acdbio com if you have any questions ZZ Target RNA Specific Oligo Probes PreAMP AMP Label Probe z z z Zz z z ez Zz Z Z 2 Hybridize to target RNA 3 Amplify signal Use up to four signal amplification systems to detect multiple target RNAs Probes are hybridized to a cascade of signal amplification molecules culminating in binding of dye labeled probes visible in different fluorescent chanels Hybridize multiple sets of gene specific probe pairs to target mRNAs Figure 1 Procedure overview RNAscope Fluorescent Multiplex Kit User Manual Visualize target RNA ASD LL DIAGNOSTI j Kit contents and storage The RNAscope Multiplex Fluorescent Assay requires the RNAscope Probes and the RNAscope Multiplex Fluorescent Reagent Kit Probes and Reagent Kits are available separately RNAscope Probes The RNAscope Probes consist of user specified Target Probes and Positive and Negative Control Probes Each Target
21. the appropriate probe 2 Place the 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 3 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 Step 5 with fresh 1X Wash Buffer RNAscope Fluorescent Multiplex Kit 15 ASD Hybridize Amp 1 FL Hybridize Amp 2 FL Hybridize Amp 3 FL 16 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 1 FL to entirely cover each section Place the HybEZ 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 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 i
22. tom of the tubes 3 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 14 RNAscope Fluorescent Multiplex Kit User Manual PED 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 support online training videos wash slides before proceeding Note We recommend running controls before running any of your samples to optimize the protocol Hybridize probe IMPORTANT Prior to this step ensure you have pretreated your samples See Catalog No 320513 for Fresh Frozen Tissue or Catalog No 320511 for FFPE Tissue IMPORTANT Ensure 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 to Appendix A Reagent Volume Guidelines on page Error Bookmark not defined to determine the recommended number of drops needed per slide For example for a 0 75 x 0 75 barrier add 4 drops of
23. y RNAscope Probes Other materials and equipment e 50X Wash Buffer e Amp 1 FL e Amp 2 FL e Amp 3 FL e Amp 4 Alt A FL Amp 4 Alt B FL or Amp 4 Alt C FL e DAPI Prepare the materials C1 Target Probe 50X C2 Target Probe 50X C3 Target Probe 3 Plex Positive Control Probe Negative Control Probe Prepared sections Distilled water Carboy gt 3L Tissue Tek Staining Dish Tissue Tek Clearing Agent Dish xylene resistant 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 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 support technical doc 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 L distilled water and 1 bottle 60 mL of 50X Wash Buffer to a large carboy Mix well Note Warm 50X Wash Buffer up to 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 2 Briefly spin the C2 and C3 probes to collect the liquid at the bot
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