RNAscope 2.0 HD Detection Kit (RED) User Manual
Contents
1. 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 Pretreat 1 4 mL x 2 bottles 4 10X Pretreat 2 70 mL x 4 bottles Room temperature 20 25 C Pretreat 3 4 5 mL x 1 bottle 4 C 2 0 HD Detection Kit RED Cat No 310036 Wash Buffer Kit Cat No 310091 Comes in a separate box t Comes in two boxes 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 though they have the same name RNAscope 2 0 HD Detection Kit RED User Manual ASD Required materials and equipment The following materials and equipment are needed to perform the RNAscope Assay HybEZ Hybridization System 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 out2. Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation Repeat Step 5 with fresh 1X Wash Buffer 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 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 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 0 HD Detection Kit RED User Manual 17 ACD Detect the signal Briefly spin down the contents of the RED B to be sure content is at the bottom of the tube before opening the cap 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 Take each slide one at a time from the Tissue Tek Slide Rack and tap and or flick to remove the exce
3. rennnnnnnnonnnnnrnnnnnnnnnennnnevnnnnnnnnnennnnr 5 fat ere MIS GUIE RE EN ERE 5 PrOOUC COS CUO HON EE EN EE 5 BECK NO UG EE EE EE PE NE RER 5 SETE REE EEE ES RE FEET JERE ERR SNE SED ENDERE FORESE eneste 5 Kit contents and StOLAGe cccccsseeeccecceeeseceeeceeeeeeeesueeeeeesseeaeeeeesseaeeeeeessaseeeees 6 RNAscope Probes csececesesessesececeseseecevevececececcecevavavavavereevevevavavveneesevevacates 6 RNAscope 2 0 HD Detection Kit ccccccssssesesceseseceseseresceveveceeesereesevevecaees 7 Required materials and equipment mumsrsssssssrrrrsesrrrrreorrrrrrenrrrrrrnrrrrrrrr rr rr rn nr rr eran 8 HybEZ Hybridization System s mmesusressressrnssrnrsrnrrsrrersen ense en renen enar e nen enn 8 User supplied materials cccccccccccccsssseceeeceeeeeeeeceeeeseeeseeseseeeesseaeeeesssaaass 8 Chapter 2 Before You Begin sccivccsccsteccnscciccwicessecsaveutvecsvssivsassesiveact 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 rrrnnnnnnnnrnnnnnrornnnnnennnnnnrnnnnnnrennnnnrennnnnnrennnnesennnnssennnnn 14 Prepare 1X Wash Buffer wcicineescdtsvessaxivcasscciamneidasiendsrcite vanesnbinastcdsveuehsnsindebels 14 Prepare counterstaining reagents cccseecccc
4. Probes The RNAscope Probes consist of the user specified Target Probe and the Positive and Negative Control Probes Visit www acdbio com products target probes search product to find a gene specific probe from a searchable catalog of gt 27 000 predesigned Target Probes or order a custom probe Visit www acdbio com products target probes controls housekeeping to find appropriate Control Probes Each probe is sufficient for staining 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 The probes have a shelf life of six months from the shipment date when stored as indicated in the following table Target Probes Content Storage Reagent Quantity Probe targeting specific 3 mL x 1 bottle RNA Various RNAscope Singleplex Target Probe species gene 6 RNAscope 2 0 HD Detection Kit RED User Manual ASD Control Probes Fr Reagent Cat No Content Quantity Storage RNAscope Positive Control Probe Various Probe targeting common 3 mL x 1 bottle species PPIB housekeeping gene RNAscope Negative Control Probe 310043 Probe targeting bacterial 3 mL x 1 bottle 4 C DapB gene dapB RNAscope 2 0 HD Detection Kit Each RNAscope 2 0 HD Detection Kit provides enough reagents to stain 20 tissue sections each with an area of approximately 20 mm x 20 mm 0 75 x 0 75
5. Carboy gt 3L Fume hood Xylene 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 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 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 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 14 RNAscope 2 0 HD Detection Kit RED User Manual PED 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
6. 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 0 HD Detection Kit RED User Manual 21 PED 22 Species Human Tissue Microarray Tissue type Pathology Pretreatment Condition Normal Extended Normal Standard Tumor Standard Tumor Standard Normal Standard Tumor Standard Normal Standard Tumor Standard Normal Standard Normal Mild Normal Mild Normal Standard Cancer Standard Normal Standard Abnormal Standard Tumor Standard Normal Standard Cancer Standard Cancer Standard Tumor Standard Normal Standard Benign Standard Normal Standard Tumor Standard Cell pellets fixed with 10 Mild NBF Standard HeLa cells fixed with 10 Formaldehyde PBS ACD Control Zl Zl Z Zi ZIO HETE TETEN 5138 312 51213 o RNAscope 2 0 HD Detection Kit RED User Manual ASD CELL DIAGNOSTI Appendix B Reagent Volume Guidelines Determine reagent volume Before starting your experiment measure the inner edge of the hydroph
7. 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 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 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 Buffe
8. see Catalog No 320511 Visit www acdbio com support technical doc to download a sample preparation user guide Product description Background Overview The RNAscope Assays use a novel and proprietary method of in situ hybridization ISH to visualize single RNA molecules per cell in samples mounted on slides RNAscope Assays do not require the RNA free environment used for traditional ISH The assays are based on ACD s patented signal amplification and background suppression technology Compared with the RNAscope 1 0 Assay the 2 0 Assay incorporates an additional signal amplification step which enhances the signal for low expressing genes and RNA present in archived samples and partially degraded specimens The RNAscope Assay procedure is illustrated in Figure 1 on page 6 The procedure can be completed in 7 8 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 tissue samples sections are first pretreated and then RNA specific probes are hybridized to target RNA The signal is amplified using a multi step process followed by hybridization to horseradish peroxidase HRP or alkaline phosphatase AP labeled probes and detected using a chromogenic substrate Each single RNA transcript appears as a distinct dot of chromogen precipitate visible using a
9. sensitive Do not dehydrate the slides in alcohol Cool the slides for 5 MIN at RT Briefly dip one slide into fresh pure xylene and immediately place 1 2 DROPS of EcoMount on the slide before the xylene dries WARNING Use the EcoMount mounting medium only Carefully place a 24 mm x 50 mm coverslip over the tissue section Avoid trapping air bubbles Repeat steps 2 and 3 for each slide Air dry slides for 5 MIN RNAscope 2 0 HD Detection Kit RED User Manual ASD CED CELL DIAGNOSTICS IN Evaluate the samples Examine tissue sections under a standard bright field microscope at 20 40X magnification e Assess tissue and cell morphology e Assess positive control signal strength Positive control signal should be visible as punctate dots within cells at 20 40X magnification e Assess negative control background One dot to every 10 cells displaying background DAB staining per 20X microscope field is acceptable 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
10. 6 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 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 2 0 HD Detection Kit RED 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 web
11. 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 Component Quantity Cat No EE 77 EE ME OE EE TJ le JE User supplied materials IMPORTANT Do not substitute other materials for the EcoMount listed in the following table Description Supplier Cat No Gill s Hematoxylin American Master Tech Scientific MLS HXGHE1LT 00 MLS i hentes Clearing Agent Dish xylene resistant American Master Tech Scientific MLS LWT4456EA LS Tissue Tek Vertical 24 Slide Rack American Master Tech Scientific MLS LWSRA24 Tissue Tek Staining Dish 3 required American Master Tech Scientific MLS LWT4457EA Water bath or incubator capable of holding M temperature at 40 1 C Tubes various sizes RNAscope 2 0 HD Detection Kit RED User Manual Fr Description Supplier Cat No vs as z as E as as as i 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 0 HD Detection Kit RED User Manual AED ADVANCED CELL DIAGNOSTICS INC 10 RNAscope 2 0 HD Detection Kit RED User Manual A
12. LD 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 for Human control slide Hela Catalog No 310023 for Mouse control slide 3T3 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 guidelines 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 proced
13. USER MANUAL GP ADVANCED CELL DIAGNOSTICS INC RNAscope 2 0 HD Detection Kit RED User Manual PART 2 Catalog Number 320487 For Part 1 Sample Preparation and Pretreatment Guide for FFPE Tissue see Catalog Number 320511 For Molecular Biology Applications not intended for diagnosis Rev Date 20131113 For Molecular Biology Applications not intended for diagnosis Trademarks HS 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 Contents Chapter 1 Product Information
14. ceeeeeeceeseeeeseeeeeeaeeeeeeneeeeeesaaes 14 Prepare mounting FEAGENIS ccccseeceecseeeeeeeeeeeeeceaseeeeseeeeeeseeeeeseeeeessess 15 Equilibrate reagents NE 15 RUNIE ASSAY EE S aiea 15 Hybridize probe eee 15 PYDd AMD Tieg E 16 Hybridize AMP eee 16 Hybridize AMD J PE EE ENE 16 Hybridize AMP Gbossossosssrrsssaresssraessrrenrrrrrrrrrrnrrrrnr ERE ERE rn rr rr RR RKA RR KKR RKA KARA Kron Ann 17 Hybridize AMP 5 oosseeesssrressrresssrressrrnorrrrerrrrrnrrrrnn rr rr nr rann rr rr nr RAKA R KKR RR RR KKR RR Ronna 17 Hybridize AMP 6 oosssessssrressrresssrressrrenrrrrerrrrrrrr rr nns r narr rr rr rr rr RR RKA ERE SEERE RR RR nn 17 Detect the EEE ED 18 Counterstain the slides msssmssssesssrrrsesrrrrrreorrrrrrenrrrrrrrrrrrrr rr rr enn r rr rr nr RAR Kran nn nn 18 Mount the samples ae ean KRKA KKR RR Rn nn 18 Evaluate the samples mnvosssesesssresrsrressrrenrsrrerrrrernrrrrrr rr rr nr rr ren rr rr nar RR nr RR KRK RKA Rn nonan 19 Scoring guidelines SR 19 Quantitative Image AnalysiS rrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrnnnnnnnnne 19 Control examples sosseeeeseeessrrsrrreeesserrrrrrrenrrrrrrrrs nns skr rr rr nn rr RR ARR RAR R RR RAR RR RR Rn 20 Troubleshooting EE 20 Appendix A Tissue Pretreatment Recommendation 21 Tissue pretreatment reCOMMENAtION ccccccseeeeeeceeseeeeueeeeesseeeeeeeaeeeeens 21 Tissue specific pretreatment conditions nmssmsmssrsessssrressrsrrrnsrsrrrnnrrrrronnrnr rr anna 21 RNAsco
15. 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 to every 10 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 positive 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 obtain 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 RNAscope 2 0 HD Detection Kit RED User Manual 19 ED ADVANCED CELL DIAGNOSTICS INC Control examples Figure 2 is an example of HeLa cell pellet sections using dapB Negative Control Probe and PPIB Positive Control Probe at 40X magn
16. common bright field microscope at 40 100X magnification The RNAscope 2 0 Assay has additional amplification RNAscope 2 0 HD Detection Kit RED User Manual AED ADVANCED CELL DIAGNOSTICS INC steps that allow observable results under 10 20X magnification RNAscope 2 0 Assays offer the choice of two Detection Kits Brown DAB and Red Fast Red which enable RNA molecules to be visualized as brown or red chromogenic dots respectively The procedure can be automated using the Ventana DISCOVERY XT or ULTRA Systems Refer to the RNAscope VS Assay User Manual available at www acdbio com support technical doc for more details r 9 i ZZ Target RNA Specific Oligo Probes PreAMP AMP Label Probe zZz Z z 1 Tissue section 2 Hybridize to target RNA 3 Amplify signal Start with properly prepared Hybridize gene specific probe Probes are hybridized to a cascade of signal Visualize target sections and pretreat to pairs to the target mRNA amplification molecules culminating in binding RNA using a allow access to target RNA of HRP or AP labeled probes The 2 0 Assay standard bright enhances signal further with additional field microscope amplification steps Add DAB or Fast Red substrate to detect target RNA Figure 1 Procedure overview Kit contents and storage The RNAscope 2 0 Assay requires the RNAscope Probes and the RNAscope 2 0 HD Detection Kit Probes and Detection Kits are available separately RNAscope
17. er 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 RNAscope 2 0 HD Detection Kit RED User Manual 15 AED Hybridize Amp 1 Hybridize Amp 2 Hybridize Amp 3 16 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 Agitate slides by moving the Slide Rack up and down in the dish 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 1 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
18. ification 7 2 a dapB Figure 2 RNAscope 2 0 Assay RED performed on FFPE RNAscope Control Slides Cat No 310045 using the dapB Negative Control Probe Cat No 310043 and PPIB Positive Control Probe Cat No 313901 40X magnification Slides contain HeLa cell pellet sections Troubleshooting For troubleshooting information please contact technical support at support acdbio com 20 RNAscope 2 0 HD Detection Kit RED 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 FFPE 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
19. obic 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 i 6 180 0 75 x 1 25 a 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 0 HD Detection Kit RED User Manual 23 AED ADVANCED CELL DIAGNOSTICS INC 24 RNAscope 2 0 HD Detection Kit RED User Manual C ASD Appendix C Safety Chemical safety N 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 cl
20. 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 ROOM TEMPERATURE RT e Ensure HybEZ OVEN and prepared Humidity Control TRAY are at 40 C e Before each use warm the Target and or Control PROBES for 10 MIN at 40 C in a water bath or incubator Swirl gently to mix 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 Hybridize probe 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 23 to determine the recommended number of drops needed per slide For example for a 0 75 x 0 75 barri
21. othing 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 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 2 0 HD Detection Kit RED User Manual 25 PED In the U S In the EU 2
22. pe 2 0 HD Detection Kit RED User Manual Appendix B Reagent Volume Guidelines suser ennen 23 Determine reagent Volume ccccccssseeecceeseecceeeecceaeeeceeuseeessageeessageeeeseess 23 Appendix C Safety ane 25 Chemical safety 2 area RKA ARR RR RKS R RR RR ons 25 Biological hazard SafCtY sssseossrrreosrrrrreorrrrrrrrrrrrrrnrrrrrrrr rr rr rr nr rr rna RR RR Rn nn rr nan 25 Documentation and SUpport nnrnnnennnnnnnnnnnnnennnennnnnnnnnnnnnennnnnnnnennnunnn 27 Obtaining MSDOS EEE EEE EE NE 27 Obtaining SUDDO EEE 27 Contact information errrrnnnnnvvrrnnnnnrovnrnnnnnrrnnnnnnnnsnnnnnnnnnsennnnnnnnsnnnnnnsnssnnnnnnsnee 27 Limited product warranty sescccseventedevencnnatnneedenenencedasanecdsnidesanndsseexectedicneetaesaeateas 27 RNAscope 2 0 HD Detection Kit RED User Manual ACD Chapter 1 Product Information Before using this product please read the safety 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 0 HD Detection Kit RED Cat No 310036 RNAscope 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
23. r RNAscope 2 0 HD Detection Kit RED User Manual AG D y Hybridize Amp 4 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 4 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 3 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 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 Wash slides in 1X Wash Buffer for 2 MIN at RT with occasional agitation Repeat Step 5 with fresh 1X Wash Buffer Hybridize Amp 5 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 5 to entirely cover each section 2 Place the HybEZ Slide Rack in the HybEZ Humidity Control Tray Seal tray and incubate for 30 MIN at RT 3 Remove the HybEZ Slide Rack from the HybEZ Humidity Control Tray 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
24. site 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 and other product support documents e Obtain information about customer training and view training videos 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 RNAscope 2 0 HD Detection Kit RED 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
25. ss 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 10 MIN at RT Remove the HybEZ Slide Rack from the HybEZ Humidity Control Tray To remove the RED 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 Immediately 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 4 Mount the samples 1 pn 18 Move the Tissue Tek Slide Rack into the Staining Dish containing the 50 HEMATOXYLIN I staining solution for 2 MIN at RT Slides will be purple 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 Replace distilled water in the Staining Dish with 0 02 AMMONIA WATER Move rack up and down 2 3 TIMES Section should turn blue Replace ammonia water with DISTILLED WATER Wash slides 3 5 TIMES Remove the Slide Rack from the Staining Dish and dry slides in a 60 C dry oven for 15 MIN IMPORTANT The RED substrate is alcohol
26. ures Refer to Appendix C Safety on page 25 for more information RNAscope 2 0 HD Detection Kit RED User Manual 11 AED ADVANCED CELL DIAGNOSTICS INC 12 RNAscope 2 0 HD Detection Kit RED 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 10 30 MIN 4 Run the assay 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 10 MIN Counterstain the slides 2 MIN Bake Slides 15 MIN Mount samples 5 MIN Review results RNAscope 2 0 HD Detection Kit RED User Manual 13 ACD Materials required for the assay Materials provided by the RNAscope 2 0 HD Detection Kit RED Materials provided by RNAscope Probes Other materials and equipment e 50X Wash Buffer e 2 0 Amp 1 e 2 0 Amp 2 e 2 0 Amp 3 e 2 0 Amp 4 e 2 0 Amp 5 RED e 2 0 Amp 6 RED e 2 0 Fast RED A e 2 0 Fast RED B Prepare the materials e Target Probe e Positive Control Probe e Negative Control Probe Prepared sections Distilled water
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