RNAscope 2-Plex Detection Kit (Chromogenic) User Manual
Contents
1. Hybridize Amp 3 Hybridize Amp 4 Hybridize Amp 5 ASD 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 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 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 Briefly spin down the contents of AMP 4B to be sure content is at the bottom of the tube before opening cap Depending on the size of your hydrophobic barrier make AMP4 WORKING SOLUTION per section by using a 1 50 ratio of Amp 4B to Amp 4A For example for a 0 75 x 0 75 barrier add 2 4 uL of AMP 4B to 120 uL of AMP 4A into a tube Mix well 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 Pipette 120 uL of AMP 4 onto each section Ensure sections are covered Place the HybEZ Slide Rack in the HybEZ Humidity Control Tray Close tray and insert into the oven for 15 MIN at 402. Assay The color channels for the RNAscope 2 Plex Detection Kit Chromogenic Assay are shown in the following table Probe Chromogenic Labels C1 GREEN C2 RED gt I U I TU Default channel Channel C1 target probes are Ready To Use RTU while channel C2 probes are shipped as a 50X concentrated stock To independently detect two target RNAs in a 2 Plex 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 6 RNAscope 2 Plex Detection Kit Chromogenic User Manual 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 Fr Reagent Cat No Content Quantity Storage RNAscope Kit 2 Plex Target Various Ready To Use RTU probe for color 3 mL x 1 bottle Probe species gene channel 1 RNAscope Kit 2 Plex Target Various 50X probe for color channel 2 60 uL x 1 tube Probe species gene C2 Control Probes Fr Reagent Cat No Content Quantity Storage RNAscope Kit Positive Various RTU probe targeting a common 3 mL x 1 bottle 4 C Control Probe housekeeping gene Each detection cha
3. Assays are compatible with a variety of sample types You must use both an RNAscope Detection 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 for FFPE Tissue see Catalog No 320511 Visit www acdbio com support technical doc to download a sample preparation user guide Product description Background The RNAscope 2 Plex Chromogenic Assay uses a novel and proprietary method of in situ hybridization ISH to simultaneously visualize two RNA targets in samples mounted on slides 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 two different genes within a cellular context Overview The RNAscope 2 Plex Chromogenic Assay procedure is illustrated in Figure 1 on page Error Bookmark not defined The procedure can be completed in 8 10 hours or conveniently divided over two days 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 RNA specific probes are hybridized to target RNA The RNAscope 2 Plex Chromogenic Assay employs two independent signal amplificatio
4. C Remove the HybEZ Control Tray from the oven and remove HybEZ Slide Rack IMPORTANT Do not insert tray into the HybEZ Oven for the rest of the procedure One slide at a time 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 8 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 5 to entirely cover each section Place the HybEZ Slide Rack in the HybEZ Humidity Control Tray Seal tray and incubate for 30 MIN at RT Remove the HybEZ Slide Rack from the HybEZ Humidity Control Tray 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 RNAscope 2 Plex Detection Kit Chromogenic User Manual 17 AED Hybridize Amp 6 1 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 6 to entirely cover each section 2 Place the HybEZ Slide Rack with the
5. Human Tumor Standard Tumor Standard Normal Standard Tumor Standard Tumor Standard Normal Standard Normal Standard Cancer Standard Abnormal Standard Tumor Standard Cancer Standard Tumor Standard Normal Standard Benign Standard Normal Standard Tumor Standard Cell pellets fixed with 10 Mild NBF HeLa cells fixed with 10 Standard Formaldehyde PBS ACD control ZIZ z Zl Zl Ol OO Z Z Z Z AE S es S 2 2 3 3 3 16 31318183 3 3 3 3 Sv v 5 EEE v v D D Tissue Microarray 22 RNAscope 2 Plex Detection Kit Chromogenic User Manual ASD CELL DIAGNOSTI Appendix B 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 slide barrier in per slide uL 7 E 6 180 0 75 x 1 25 Em 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 RNAscope 2 Plex Detection Kit Chromogenic User Manual 23 AED ADVANCED CELL DIAGNOSTICS INC 24 RNAscope 2 Plex
6. 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 RNAscope 2 Plex Detection Kit Chromogenic User Manual 21 Headquarters 3960 Point Eden Way Hayward CA 94545 Phone 1 510 576 8800 Toll Free 1 877 576 3636 2 For support email support acdbio com NL ADVANCED CELL DIAGNOSTICS INC www acdbio com
7. 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 to entirely cover each section 2 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 Hybridize Amp 2 16 1 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 to entirely cover each section 2 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 RNAscope 2 Plex Detection Kit Chromogenic User Manual
8. bottle 4 C 2 Plex Amp 2 4 5 mL x 1 bottle 4 C 2 Plex Amp 3 3 mL x 1 bottle 4 C RNAscope 2 Plex Detection Kit Chromogenic User Manual AED Wash Buffer Kit Cat No 310091 Fr Reagent 50X Wash Buffer Comes in a separate box t Comes in two boxes Quantity 60 mL x 4 bottles Storage Room temperature 20 25 IMPORTANT RNAscope Detection Kits share the same Pretreatment Kit and Wash Buffer but have unique Detection Kits Do not interchange the reagent components of the Detection 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 The HybEZ Hybridization System 110 VAC Cat No 310010 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 Fr Component Quantity Cat No HybEZ Oven 110 or 220 VAC 1 oven 310010 or 31
9. page 25 for more information RNAscope 2 Plex Detection Kit Chromogenic User Manual 11 AED ADVANCED CELL DIAGNOSTICS INC 12 RNAscope 2 Plex Detection Kit Chromogenic User Manual ASD Chapter 3 RNAscope 2 0 Assay IMPORTANT For Part 1 Sample Preparation and Pretreatment Guide for FFPE Tissue see Catalog No 320511 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 HRS Hybridize probe 2 HRS Hybridize Amp 1 30 MIN Hybridize Amp 2 15 MIN Hybridize Amp 3 30 MIN Hybridize Amp 4 15 MIN Hybridize Amp 5 30 MIN Hybridize Amp 6 15 MIN Detect the signal 40 MIN Counterstain the slides 2 MIN Bake Slides 15 MIN Mount samples 5 MIN 4 Review results RNAscope 2 Plex Detection Kit Chromogenic User Manual 13 ACD Materials required for the assay Materials provided by the RNAscope 2 Plex Detection Kit Chromogenic Materials provided by RNAscope Probes Other materials and equipment e 50X Wash Buffer e 2 Plex Amp 1 e 2 Plex Amp 2 e 2 Plex Amp 3 e 2 Plex Amp 4A e 2 Plex Amp 4B e 2 Plex Amp 5 e 2 Plex Amp 6 e Red A e Red B e Green A e Green B Prepare the materials C1 Target Probe 50X C2 Target Probe 2 Plex Positive Control Probe Negative Control P
10. slides in the HybEZ Humidity Control Tray Close tray and incubate for 15 MIN at RT Remove the HybEZ Slide Rack from the HybEZ Humidity Control Tray One slide at a time 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 Detect the red signal 1 Briefly spin down the contents of the RED B tube to be sure content is at the bottom of the tube before opening the cap 2 Depending on the size of your hydrophobic barrier make RED WORKING SOLUTION per section by using a 1 60 ratio of Red B to Red A For example for a 0 75 x 0 75 barrier add 2 uL of RED B to 120 uL of RED A into a tube Mix well IMPORTANT Use the RED solution within 15 MIN Do not expose to direct sunlight or UV light 3 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 Pipette 120 uL RED solution onto each tissue section Ensure sections are covered Place the HybEZ Slide Rack with the slides in the HybEZ Humidity Control Tray Seal tray and incubate for 30 MIN at RT 6 Remove the HybEZ Slide Rack from the HybEZ Humidity Control Tray To remove the RED solution from the slides tilt each slide one at a time over a waste container and ta
11. 0013 HybEZ Humidity Control Tray with lid 1 tray 310012 HybEZ Slide Rack 20 slide capacity 1 rack 310014 HybEZ Humidifying Paper 2 sheets HybEZ Humidifying Paper Pack 15 sheets 310015 RNAscope 2 Plex Detection Kit Chromogenic User Manual ASD IMPORTANT Do not substitute other materials for the EcoMount listed in the following table User supplied materials Fr Description Supplier Cat No 100 ethanol EtOH American Master Tech Scientific MLS ALREAGAL Gill s Hematoxylin I American Master Tech Scientific MLS HXGHE1LT Tissue Tek Vertical 24 Slide Rack American Master Tech Scientific MLS LWSRA24 Tissue Tek Staining Dish 3 required American Master Tech Scientific MLS LWT4457EA econ Clearing Agent Dish xylene resistant American Master Tech Scientific MLS LWT4456EA Cover Glass 24 x 50 mm Fisher Scientific MLS 12 545 F Ammonium hydroxide 28 30 Sigma Aldrich MLS 320145 500mL Water bath or incubator capable of holding MLS temperature at 40 1 C Se GS Graduated cylinder Microcentrifuge Microscope and accessories Drying oven capable of holding temperature at 60 1 C Major Laboratory Supplier in North America For other regions please check Catalog Numbers with your local lab supplier RNAscope 2 Plex Detection Kit Chromogenic User Manual AED ADVANCED CELL DIAGNOSTICS INC 10 RNAscope 2 Plex D
12. Detection Kit Chromogenic User Manual C ALD Appendix C Safety Chemical safety A 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 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 In the U S WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of human
13. NTING reagents mnuessnssressrrrersrresrrrrnrrrrerrrrerrrrrrr rr nns r rr rr rr ren rr rann 15 Equilibrate reagents vmnsmnssrsesssrrsssrressrrrorrrrerrrrrnr renen rr rr ner rr rr rna rr rr nr ERE LEE nro nn 15 RUN the ASSAY ccccccccsescecceseeeceeseecseuseeccsaeeecsaeeecseueeeeseuseeessageeessageeeessags 15 Hybridize probe REE 15 Hybridize AMP eee 16 Hybridize AMP EEE EEE 16 Hybridize AMP Ooosmnresssrrsssrresssraessrrenrrrrrrrrrrrrrrrn rr rr arr rr rar rr nr RR KARA RR SRK KKR nr rn nn 17 Hybridize AMP 4 rrrnnnnvnnnnnrvvnnnnnevrnnnnrvnnnnnrennnnnrennnnnnsennnnnrennnnenennnnnnsennnnnnen 17 Hybridize AMP D rrannnnnnnnnrvnnnnnnennnnnnrvnnnnnrennnnnnennnnnnsnnnnnnrennnnssennnnnerennnnnsen 17 Hybridize AMP 6 EE EEE 18 Detect the red signal cccccssesecccccesseeeeeeceeeeeceeeeeeeeeeeeeeeaaeeeessseeseeeeseaas 18 Detect the green signal annnrnnnnnnrnnnnnnnvnnnnnnvnnnnnnrnnnnnnsnnnnnnnrnnnnnnrnnnnnnsene 18 Counterstain the slides rrrrnnnnrnrrrrnnnnrvrnrrnnnnrvrnvnnnnnrennnrnnnnnsnnnnnnnsnsnnnnnnn 19 Mount the samples saken 19 Evaluate the samples ccccccccsesececseeeeeeeeeeceeeeeeeeesseeeeeesseueeessaneeeesaeeeeneass 19 Scoring guidelines rrrrrrrrrrnnnnnrvvvrnnnnrvenrrnnnnrsennnnnnnrsrnnnnnnnrsennnnnnsssennnnnn 20 Quantitative Image Analysis sssssssssssssssssssssssssseeessseeeeeeeeees 20 Control examples ccccccccsessececceeeeeeecceeeeeeeeecseauseeeeseaeseeeeessaageee
14. PE tissue types e Samples prepared differently than the sample preparation protocol found in Part 1 Sample Preparation and Pretreatment Guide for FFPE Tissue Cat No 320511 Tissue pretreatment recommendation 1 Stain representative samples using the positive and negative control probes 2 Fix sample in fresh 10 NBF for 16 32 HRS at RT Note Perform tissue fixation step using the recommended amount of time Over or under fixation will result in significant signal loss when performing the RNAscope Assay 3 Depending on your tissue type see section below vary the PRETREAT 2 and or PRETREAT 3 TIME Reagent Mild Standard Extended Pretreat 2 15 MIN 15 MIN 30 MIN Pretreat 3 15 MIN 30 MIN 30 MIN Note Sample types such as certain Xenografts and Cell Pellets require less time For these tissue types vary the PRETREAT 2 TIME to 8 min and PRETREAT 3 TIME to 15 min If you have a tissue type not listed contact support at support acdbio com Tissue specific pretreatment conditions If your sample fixation is successful in fresh 10 NBF Step 2 above then refer to the following table for tissue specific pretreatment conditions For information about species or tissue type not listed here contact support at support acdbio com Species Tissue type Pathology Pretreatment Condition RNAscope 2 Plex Detection Kit Chromogenic User Manual 21 PED Species Tissue type Pathology Pretreatment Condition
15. USER MANUAL WE ADVANCED CELL DIAGNOSTICS ING RNAscope 2 Plex Detection Kit Chromogenic User Manual PART 2 Catalog Number 320494 For Part 1 Sample Preparation Pretreatment Guide for Formalin Fixed Paraffin Embedded FFPE see Catalog Number 320511 For Molecular Biology Applications not intended for diagnosis Rev Date 20131220 For Molecular Biology Applications not intended for diagnosis Trademarks a 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 ALD Contents Chap
16. eessagass 20 TLOUDIGSNOOTING cccccceccseeeeceeecaeeseeeeeeaeeeeeeeseeeeceeessaeeeeeeesaeaeeeeeessaeeeeeeeas 20 Appendix A Tissue Pretreatment Recommendation 21 Tissue pretreatment reCOMMENaAtiON ccccccseeeeeeceeeeeeeeeeeeeseeeeeeeseeeeeeens 21 RNAscope 2 Plex Detection Kit Chromogenic User Manual Tissue specific pretreatment conditions cccccecseeeeeeseeeeeeeaeeeeeeaeeeees 21 Appendix B Reagent Volume Guidelines scenerne 23 Determine reagent Volume ccccccssseecccesececceeseecceauseecseuseeessaseeecsaeeeesseass 23 Append C Safely ssssrsssrpsvnssissvansnspennissk skianss nsr Nkkesn nndessss nssERA NER 25 Chemical EE NINNA 25 Biological hazard SafCtY sssseossrrreosrrrrresrrrrrrrrrrrrrenr rr rr rr rar rr rens rr rr nr RR RR rn rn nn 25 Documentation and SUpport nnrnnnennnnnnnnnnnnnennnennnnnnnnnnnnnennnnennnennnunnn 27 DEE vr 27 Obtaining SUpport eee 27 Contact information EE EE 27 Limitea prod ct War ANA EEE EEE 27 RNAscope 2 Plex Detection Kit Chromogenic User Manual ASD Chapter 1 Product Information Before using this product read and understand the information in Appendix C Safety on page 25 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 2 Plex Detection Kit Chromogenic Cat No 310035 RNAscope
17. etection Kit Chromogenic User Manual ACD Chapter 2 Before You Begin IMPORTANT For Part 1 Sample Preparation and Pretreatment Guide for FFPE Tissue see Catalog No 320511 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 Run the assay on FFPE RNAscope Control Slides Cat No 310045 using the Positive and Negative Control Probes Important procedural guidelines e Start with properly fixed and prepared sections Refer to Appendix A Tissue Pretreatment Recommendation on page 21 and 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 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 C Safety on
18. hird 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 2 Plex Detection kit Chromogenic User Manual PED ANCED CELL DIAGNOSTICS IN 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 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 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
19. n systems each using a different chromogenic enzyme Single RNA transcripts for two target genes appear as punctate dots of two distinctly colored chromogen precipitates visible using a common bright field microscope at 40 100X magnification RNAscope 2 Plex Detection Kit Chromogenic User Manual AED ADVANCED CELL DIAGNOSTICS INC 1 Tissue section 2 Hybridize to target RNA 3 Amplify signal Start with properly prepared Hybridize two sets of Use two independent signal amplification Visualize target RNA tissue sections and pretreat gene specific probe pairs to systems to detect both target RNAs Probes are using a standard to allow access to target target MRNA hybridized to a cascade of signal amplification bright field RNA molecules culminating in binding of HRP or microscope AP labeled probes Add two chromogenic substrates to detect RNAs Figure 1 Procedure overview Kit contents and storage The RNAscope 2 Plex Assay requires the RNAscope Probes and the RNAscope 2 Plex Detection Kit Chromogenic which are available separately RNAscope Probes The RNAscope Probes consist of user specified Target Probes and Positive and Negative Control Probes Each Target Probe contains a mixture of short oligonucleotides designed to bind to a specific target RNA and detectable in one of two color channels C1 or C2 Note Different colors are assigned to the C1 and C2 color channels depending on the particular RNAscope
20. nification e Assess tissue and cell morphology e Assess positive control signal strength Positive control signal should be visible as punctuate dots within cell nuclei at 20 0X magnification e Assess negative control background One dot to every 10 cells displaying background DAB staining per 20X microscope field is acceptable RNAscope 2 Plex Detection Kit Chromogenic User Manual 19 ASD ADVANCED CELL DIAGNOS e Evaluate target probe signal using the scoring guidelines in the next section Scoring guidelines The RNAscope Assay can enhance the value of in situ hybridization results by enabling a semi quantitative scoring guideline utilizing the estimated number of punctate dots present within each cell boundary An example of how to develop such a guideline for semi quantitative assessment of RNAscope staining intensity is presented below for a gene with expression level varying between 1 to gt 10 copies per cell If your gene expression level is higher or lower than this range you may need to scale the criteria accordingly Categorize staining into five grades 0 1 2 3 and 4 according to the following table Staining score Microscope objective scoring 0 No staining or less than 1 dot in every ten cells 40X magnification 1 1 3 dots cell visible at 20 40X magnification 2 4 10 dots cell Very few dot clusters visible at 20 40X magnification 3 gt 10 dots cell Less than 10 po
21. nnel has its own positive control probe RNAscope Kit 2 Plex Positive Various RTU mixture of two probes targeting 3 mL x 1 bottle 4 Control Probe POLR2A in channel C1 and PPIB in channel C2 RNAscope Kit Negative 310043 RTU probe targeting a bacterial gene 3 mL x 1 bottle 4 Control Probe dapB Each detection channel has its own negative control probe Blank Probe C1 300041 RTU Target Probe diluent 3 mL x 1 bottle 4 C No C1 in label RNAscope 2 Plex Detection Kit Chromogenic Each RNAscope 2 Plex Detection Kit Chromogenic Cat No 310035 provides enough reagents to stain 20 tissue 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 310020 Fr Reagent Quantity Storage 1X Pretreat 1 endogenous enzyme blocker 4 mL x 2 bottles 4 C 10X Pretreat 2 70 mL x 4 bottles Room temperature 20 25 C 1X Pretreat 3 protease 4 5mL x 1 bottle 4 C 2 Plex Detection Kit Cat No 320701 Fr Reagent Quantity Storage 2 Plex Amp 1 3 mL x 1
22. ore 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 320511 for FFPE Tissue RNAscope 2 Plex Detection Kit Chromogenic User Manual 15 AED IMPORTANT Ensure probes are prewarmed to dissolve any precipitation 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 B 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 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 and place slide in a Tissue Tek Slide Rack submerged in the Tissue Tek Staining Dish filled with 1X WASH BUFFER 9 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 Hybridize Amp 1 1 Take each slide one at
23. p the corner on the edge of the container Immediately insert the slide into a Tissue Tek Slide Rack submerged in a 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 8 with fresh 1X Wash Buffer Detect the green signal 1 Briefly spin down the contents of the GREEN B tube to be sure content is at the bottom of the tube before opening the cap 2 Depending on the size of your hydrophobic barrier make GREEN WORKING SOLUTION per section by using a 1 50 ratio of Green B to Green A For example for a 0 75 x 0 75 barrier add 2 4 uL of GREEN B to 120 uL of GREEN A into a tube Mix well IMPORTANT Use the GREEN solution within 15 MIN Do not expose to direct sunlight or UV light 18 RNAscope 2 Plex Detection Kit Chromogenic User Manual ALD 3 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 Pipette 120 uL GREEN solution onto each tissue section Ensure sections are covered 5 Place the HybEZ Slide Rack with the slides in the HybEZ Humidity Control Tray Seal tray and incubate 10 MIN at RT 6 Remove the HybEZ Slide Rack from the HybEZ Humidity Control Tray To remove the GREEN WORKING SOLUTION from the slides tilt each slide one at a time over a waste container and tap the corner on the edge of the container Immedia
24. petting 1 volume of C2 probe to 50 volumes of C1 probe into a tube Invert the tube several times Note Do not mix probes of the same color The mixed Target Probes can be stored at 4 C for up to 6 MONTHS Prepare counterstaining reagents e Inthe fume hood prepare 50 HEMATOXYLIN staining solution by adding 100 mL Gill s Hematoxylin I to 100 mL distilled water in a Staining Dish Note 50 Hematoxylin staining solution can be reused for up to 1 week e Inthe fume hood prepare 0 02 w v AMMONIA WATER bluing reagent by adding 1 43 mL of IN ammonium hydroxide to 250 mL distilled water in a graduated cylinder or other container e Seal the cylinder with parafilm Mix well 3 5 TIMES Note For assay quantitation it is critical to use Ammonium Hydroxide Prepare mounting reagents IMPORTANT Do not reuse deparaffinization reagents for dehydration of the slides after the assay e Inthe fume hood add 200 mL XYLENE to a Clearing Agent Dish Note Reagents may be prepared ahead of time Ensure all containers remain covered Equilibrate reagents e Place AMP 1 6 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 Werecommend running controls bef
25. robe Prepared sections Distilled water Carboy gt 3L Fume hood Xylene 100 ethanol Tissue Tek Staining Dish 3 Tissue Tek Clearing Agent Dish xylene resistant 1 Gill s Hematoxylin Ammonium hydroxide 28 30 Graduated cylinder Parafilm HybEZ Humidifying System Water bath or incubator Tissue Tek Vertical 24 Slide Rack Tubes various sizes Paper towel or absorbent paper Pipettors and tips 1 1000 UL Microcentrifuge Dry oven EcoMount 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 to 1 bottle 60 mL in a large carboy Mix well Note If precipitation 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 14 RNAscope 2 Plex Detection kit Chromogenic User Manual ACD 1 Warm probes for 10 MIN at 40 C in a water bath or incubator then cool to ROOM TEMPERATURE RT Prepare probes Briefly spin the C2 probe to collect the liquid at the bottom of the tubes Mix 1 50 ratio of C2 probe to C1 probe by pi
26. s 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 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 Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 2029cfr1910a_01 html RNAscope 2 Plex Detection Kit Chromogenic User Manual ACD 26 In the EU 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 t
27. sitive cells have dot clusters visible at 20X magnification 4 gt 10 dots cell More than 10 positive cells have dot clusters visible at 20X magnification Discount cells with artificially high nuclear background staining Quantitative Image Analysis RNAscope Spot Studio Software is designed for pathologists with no prior training in image analysis This intuitive software allows users to get statistical results with complete information of cell count region and number of spots cell Simply load any image select a region of interest define settings and run analysis followed by a quality control review before results are exported Further information is available on our website at www acdbio com Control examples Figure 2 is an example of the detection of PECAM1 and EGFR in lung FFPE tissue at 40 X magnification 7 MEE A i er 2 N gt vi Ty PG ig y i i 4 2 sh dk AN o gt a is a 2 3 PG y HQ g ik Va p oo Ce Vv PETA KA i i TER H on 3 k Figure 2 RNAscope 2 Plex detection of PECAM1 and EGFR mRNA in lung FFPE tissue Troubleshooting For troubleshooting information please contact technical support at support acdbio com 20 RNAscope 2 Plex Detection Kit Chromogenic User Manual ASD A Appendix A Tissue Pretreatment Recommendation Follow the recommended pretreatment conditions based on your tissue type for e Any new or previously untested FF
28. tely insert the slide into a Tissue Tek Slide Rack submerged in a Tissue Tek Staining Dish filled with DISTILLED WATER Rinse again with fresh distilled water Counterstain the slides 1 Move the Tissue Tek Slide Rack into the Staining Dish containing the 50 HEMATOXYLIN I staining solution for 30 SEC at RT Slides will be purple 2 Immediately transfer the Slide Rack back into the Staining Dish containing distilled water and WASH slides 3 5 TIMES by moving the rack up and down Keep repeating with fresh distilled water until the slides are clear while sections remain purple 3 Replace distilled water in the Staining Dish with 0 02 AMMONIA WATER Move rack up and down 2 3 TIMES Section should turn blue 4 Replace ammonia water with DISTILLED WATER Wash slides 3 5 TIMES Mount the samples 1 Remove the Slide Rack from the Staining Dish and dry slides in a 60 C dry oven for 15 MIN IMPORTANT The GREEN AND RED SUBSTRATES are alcohol sensitive Do not dehydrate the slides in alcohol 2 Briefly dip one slide into fresh pure xylene and immediately place 1 2 DROPS of EcoMount on the slide before the xylene dries IMPORTANT Use the EcoMount mounting medium only 3 Carefully place a 24 mm x 50 mm coverslip over the tissue section Avoid trapping air bubbles 4 Repeat steps 2 and 3 for each slide 9 Air dry slides for 5 MIN Evaluate the samples Examine tissue sections under a standard bright field microscope at 20 40X mag
29. ter 1 Product Information rennnnnnnnonnnnnrnnnnnnnnnennnnevnnnnnnnnnennnnr 5 fat ele lg SUNG Sect RE EE EN 5 Frou SSC N T EE EN EE 5 BAGO UII PE EE ENE ERE BES NERE 5 GE EEE EEE 5 Kit contents and StOLAGE cccccsssesccccccesseeeeeceeeseeeeeceeeeceeesseaeeeeesssaaeeeeessaeeass 6 RNAscope PrOobeS sesecscesesessssececececereecevevececececersevevavavavevereevevevavarenenen 6 RNAscope 2 Plex Detection Kit Chromogenic 22 7 Required materials and equipment rrrnrnnnnnnornrnnnnnrennrnnnnnrennrnnnnnsennnnnnsnsennnnnn 8 HybEZ Hybridization SYSteMa smmssssessressrnssrnsrsrnrser ense eran eran rena nen 8 User supplied materials ccccccccsssseceeeceeeseceeeceeesececeseeeeeeeseseaeeeeeesaaaes 9 Chapter 2 Before You Begin ssssssssssssssrssssrrarsnnrnnnrnnn nn nn nn nn 22 11 Important procedural guidelines mosmsmsssrrrssrrrrresrrrrrrsrrrrrrrrnrrrrrrrnr rr rr rr nr rr rr ennen 11 Chapter 3 RNAscope 2 0 ASSAY ssssrrsrsssrsrsrrsrenrnnrnrsnrn rar 13 NN 13 Materials required for the ASSAY rrrrrrnnnnnnrnrrnnnnnrrnrrnnnnnrennrnnnnnsennnnnnnsnnnnnnn 14 Prepare the materials rrrnnnnnnnrnnnnnnronnnnnnennnnnnrnnnnnnrennnnnennnnnnrennnnesennnnsnennnnn 14 Prepare 1X Wash Buffer rrrnrnnnnrnnrnnnrvnrnnnnrnvnnnnrnnnnnnrnnnnnnnennnnnnrnnnnnnsene 14 Prepare probes ee 15 Prepare counterstaining rea0CENMS smn sssressrresrrrrerrrrenrrrrrr renen rr rrnr rr ren rr rann 15 Prepare MOU
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