Ready Set Go Getting Started with
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1. HybEZ oven 15 required as it provides both temperature humidity control necessary to obtain optimal nz b ACCESSORIES FOR WASHING STEPS 1155 washing tray EZ Batch for slide processing TIP 2 Batch slide processing tray is easy and convenient for loading multiple slides for washing steps USING CONTROLS 6 IMAGE ANALYSIS RNASCOPE SEMI QUANTITATIVE SCORING Score 3 Score 3 4 10 dots cell J QUALIFY YOUR SAMPLES USING CONTROLS o Control e slides QC check Sample QC check e g Hela PPIB gt 2 PPIB gt 2 PASS DapB 41 DapB 41 z Run your Negative control Negative control target probes probe e g DapB probe e g DapB gt FAIL Verify technique Positive control Positive control Check RNA quality with probe e g PPIB probe e g PPIB gt new samples Technique check Sample RNA quality check SHOR ASSAY OPUMIZANON ED TIP Always start with standard conditions OPTIMIZE YOUR ASSAY SAMPLE QUALIFICATION QC STEP 1 STAIN SLIDES WITH RNASCOPE ASSAY TEST INVALID Run your samples with Hela or 3T3 Control slides Retest with fresh slides Positive control probe POLR2A UBC s Negative dapB probe Technique check FAIL QC CHECK Hela Control spec POLR2A PPIB gt 2 UBC gt 3 dapB lt 1 TEST FAIL2. READY SET GO VEO E S GETTING STARTED WITH RNASCOPE lt lt Advanced Gell Diagnosties b TOPICS How Does RNAscope Work Getting Started with RNAscope in your Laboratory Tips and Tricks on Running the Assay Frequently Asked Questions Time for Q amp A RNASCOPE WORKFLOW VISUALIZE gt QUANTIFY single cell expression PERMEABILIZE HYBRIDIZE AMPLIFY gt to target gt signal gt with morphology cells or tissue TISSUE CELLS SINGLE MOLECULE SEEN AS PUNETATE DOT V BREAKTHROUGH PLATFORM SINGLE Molecule Detection Genome Gene or Tissue SIGNAL UNIQUE Amplificat Back d mplification Backgroun Probe Design Suppression in Single Cells b RNASCOPE TECHNOLOGY ZZ PROBE DESIGN PROBE DESIGN OVERVIEW Customer a Gene of interest gt i Access on No e g ENSG00000097007 i ACD Alignment Algorithm Identify Low Homology Avg GC Region 1 000 nucleotides Standard Probe Configuration 20 Z oligo pairs 1 000 nt p Target mRNA transcript E HOW DOES RNASCOPE 9 WORK SIGNAL AMPLIFICATION abel Probes rr eo Wie BEE SI 7 A MW 4 E w 4 WE a A 4 47 P AW E XA y 4 Y a AW A AW aay AW AW rn 6 gt gt a d 3 lav dl Target RNA
3. TEST PASS Sample FAIL RNA quality check SAMPLE CHECK SAMPLE FAIL OPTIMIZATION araya M 7 OPTIMIZE YOUR ASSAY lt SAMPLE PASS 55 RUN YOUR TARGET MARKERS Repeat Steps 1 2 amp 3 with optimized RNAscope assay SAMPLE FAIL Contact ACD support amp acdbio com TIP Refer to the Troubleshooting Guide OPTIMIZE YOUR SAMPLE IN EASY STEPS STEP 1 START WITH STANDARD CONDITIONS STEP2 STEP 3 ADJUST PRETREATMENT 2 BOILING TIME ADJUST PRETREATMENT 3 4 PROTEASE TIME Under fixed ar special sample types Ovar fixed samples and special tissues 15 15 PRETREAT 2 BOILING TIME MIN PRETREAT 3 PROTEASE TREATMENT TIP For cultured cells protease is diluted 1 15 in 1X PBS For fresh frozen samples only protease pretreatment is required and is performed at room temperature 4 TROUBLESHOOTING OVERDIGESTION EE control dapB Positive control E A Sample FFPE Xenograft Assay RNAscope 2 0 HD Red Issue Destroyed morphology ghost 3 nuclei high nuclear background weak El hematoxylin staining nu Optimization Decrease pretreatment 2 E conditions B E Result Strong staining for positive e controls with no negligible background E intact morphology strong hematoxylin 3 staining E TIP Visit http www acdbio com technical support downloads rnascop
4. Brown dot Red dot HRP DAB AP Fast Red TIP Do not interchange reagents within Brown Red assays or across similar 2 0 HD Assays By default 2 0 assays require C1 probes that are ready to use no further dilution is required b 9 2 PLEX AMPLIFICATION SCHEMATIC Amplification Amp6 Brown Enzyme HRP Amplification Amp6 Red Enzyme ALP steps Substrate Green steps Substrate FastRed e e e Pre Amp AMP1 Pre Amp AMP1 mRNA 2 Green dot Red dot HRP DAB AP Fast Red TIP By default C1 probes are 1X concentration while C2 probes are 50X To make 2 plex probe mixture at 1X concentration mix C2 probes 1 50 with C1 probes To view C2 probes only use the blank probe C1 as a diluent mix at a 1 50 dilution hl RNASCOPE MULTIPLEX FLUORESCENT SCHEMATIC Amp3 C1 Amp3 C3 7 Alexa 488 3 Atto 550 Atto 647 Amp4 Alt A Amp4 Alt Amp4 Alt O Q Q a A 5 KR 2 lt lt lt mRNA 1 2 mRNA3 Alexa 488 Atto 550 Atto 647 Ex Em 495nm 520nm 555nm 575nm 645nm 670m Amp4 Alt A GREEN Alexa 488 ORANGE Atto 550 FAR RED Atto 647 Amp4 Alt B ORANGE Atto 550 GREEN Alexa 488 Amp4 Alt C ORANGE Atto 550 FAR RED Atto 647 GREEN Alexa 488 TIP By default C1 probes are 1X concentration while C2 and C3 probes are 50X To make 3 plex pr
5. Y Storage in desiccant FFPE for RNA integrity v Always use fresh reagents v Warm probes and wash buffer at 40 C precipitation occurs during storage v Remember to optimize pretreatment conditions when you switch tissues TIP Refer to the Troubleshooting Guide http www acdbio com technical support downloads rnascope ish guide troubleshooting Dg SUMMARY 1 Reviewed RNAscope technology ZZ probe design and the workflow for manual assays 2 Tips for getting started with RNAscope in your lab 3 easy steps to getting started in your lab 3 Tips for success with RNAscope assay when you switch tissues Pretreatment 2 and 3 optimization Under fixed or special sample types Over fixed samples and special tissues Under fixed or special sample types Over fixed samples and special tissues PRETREAT 2 BOILING TIME MIN PRETREAT 3 PROTEASE TREATMENT VISIT THE SUPPORT PAGE TO LEARN MORE BED in situ hybridization technology Support tab Advanced Cell Diagnostics Technology Applications Science Diagnostics Services OPTIMIZE YOUR ASSAY Technical Support Overview Training Webinars 2 Downloads User Manual Selection Guide Manuals MSDS Positive amp Negative Control Images Tissue Requirements Troubleshooting Tips Getting Started Frequently Asked Questions FAQs SUPPORT OVERVIEW B Product Literature Technical Support Overview We value yo
6. gt 32 hrs Refer to Table 1 for Colon Tumor under over fixed tissue pretreatment guidelines Vary PRETREAT 2 and or PRETREAT 3 TIME based your tissue type see Table 2 Lung NOTE Certain Xenografts and Cell Prostate Tumor Pellets require very mild pretreatment PRETREAT 2 for 8 min PRETREAT 3 for 15 min Lymph node Tumor Table 1 Tissue Pretreatment Guidelines Tonsil Normal Protreat 2 13 min 25 min 30 min Pretrect 3 15 min 30 min 30 min Cervical Cancer For information about species or tissue type not listed here contact support at support acdbio com Bn TIP Refer to the user manual for tissue specific pretreatment guidelines TROUBLESHOOTING TIPS MULTIPLEX FLUORESCENT ASSAY 41 IMPACT OF FIXATION CONDITIONS 2 plex Mouse Positive Control Probe Mm POLR2A ETS we W 72 GAL E LIS A n 4 14 DT 4 2 X 4 PFA os 4 HE PN HA a 21 Fresh Frozen Mouse Brain TIP Sample fixation can have a great effect on the success of your assay Solution Use prechilled 10 NBF at 4 C 1 A 7 a 1 Y BH EH WI PA 20 2 ON ER 1 a Im 1 4 WNN l B W a g i AW 1 mu J Y E Daum 1 MN 7 a H E A 27 1 p 40 C protease pretreatment 4 Over digested RT protease pretre
7. Transcript Dig Amplifiers Preamplifier Double Z Target Probes N b DOES RNASCOPE 9 WORK AMPLIFICATION st zr 2 Non specific large 20 Z pairs x 20 Amplifiers x 20 Labels ACD N 4 8000 labelled molecules per 1kb region HOW DOES RNASCOPE 9 WORK leftset 4 4 1 if 1 L 185 i fright set Full Sel No Target Probes Left Set Right Set gt RNASCOPE PUBLICATION AND LITERATURE REFERENCES 180 publications since 2011 Publications by Research Area 2011 2015 Cancer Infectious HPV Inflammation Neuroscience non coding RNA stem Gells Endocrine Other O 50 100 150 Wang et of Publications al 2012 Visit www acdbio com and download a publication of your interest 2014 Advanced Cell Diagnostics Inc acdbio com 10 Chromogenic Diaminobenzene DAB HRP Channel 1 1 Probes Chromogenic Fast Red ALP Channel 1 1 Probes Chromogenic HRP Green Fast Red ALP Channel 1 2 C1 C2 Probes Fluorescent FITC Cy3 Cy5 Channel 1 2 3 C1 C2 C3 Probes 2 0 HD AMPLIFICATION SCHEMATIC Amplification Amp6 Brown Enzyme HRP Amplification Amp6 Red Enzyme ALP steps Label probe Substrate DAB steps Label probe Substrate FastRed A A J e 3 J e re Pre Amp AMP1 Pre Amp AMP1 ZZ mRNA 1 mRNA 1 2 0 HD BROWN 2 0 HD RED
8. atment 4 Optimal Fresh Frozen Mouse Kidney 28104 94044 041002 xejd z Fresh Frozen Mouse Brain TIP Pretreatment temperature has a great effect on the success of your assay Solution Perform pretreatment at RT to avoid over digestion of your sample IMPACT OF SAMPLE THICKNESS 2 plex Mouse Positive Control Probe Mm POLR2A Fresh Frozen Mouse Brain Experiment condition 10 NBF 15 min Fixation Pretreatment 4 RI TIP Sample thickness can signal in your samples Solution Use recommended sample thickness 10 20um AUTOFLUORESCENCE a Mouse Kidney Mouse FFPE Intestine Mouse FFPE Colon Mouse FFPE Brain TIP FFPE sample have inherent autofluorescence Solution Use appropriate background correction software to reduce autofluorescence MULTIPLEX FLUORESCENT ASSAY 101 PROBLEMS AND SOLUTIONS SOURCE PROBLEM SOLUTION Microscopy No weak signal Wrong filter setting longer emission Use correct filter settings Nonspecific signal cut off Do not use using autoexposure at first verify 2 Wrong exposure signal with naked eye 3 Inappropriate imaging enhancing 3 Use known image enhancing software e g with software Nuance Sample No weak signal 1 Compromised RNA quality 1 Use new sample with proven RNA quality 2 Sample preparation high 2 Follow the pretreatment guideline autofluorescence background on recommended the sample 3 Always perform assay with 3 plex positive
9. control and 3 plex negative probes to assess RNA quality 4 Always check signal with naked eye under objective lens first MULTIPLEX FLUORESCENT ASSAY 101 TIPS AND TRICKS Be aware of the suggested filter settings for your microscope Use the suggested pretreatment condition Use the sample preparation protocol PART 1 for your samples for optimal results Always run a 3 plex positive control and negative control to assess quality and to verify microscope setting are appropriate Always evaluate the results by eye first before capturing images FREQUENTLY ASKED QUESTIONS 48 b FREQUENTLY ASKED QUESTIONS e HNAscope assay compatibility with different tissues RNAscope manual assay can be used with FFPE fresh frozen fixed frozen and cultured cells RNAscope automated assays are primarily supported with the FFPE tissue Please refer to the User Manual Selection Guide http www acdbio com technical support downloads Key differences between RNAscope ISH assay and IHC No cooling Is required during Epitope retrieval users should directly put the slides in water at room temperature dehydrate and proceed to Pretreatment 3 step as per the manual Part 1 TIP Visit www acdbio com support for additional FAQs b GUIDELINES TO FOLLOW WITH RNASCOPE 9 ASSAY Y Head the manual and perform RNAscope exactly Y Utilize optional stopping points Y Flicking tapping the slides for adequate drying of slides
10. e ish guide troubleshooting co 3 EXAMPLE OF SUCCESFUL RNASCOPE RESULTS Posi ge tive control PPIB Target probe dr FR uo AENA s 8 S ae E D 4 e KS Bee Y 7 E FA WE RNAscope 2 0 HD Red Assay Human breast cancer tissue Hs PPIB Atto 550 RNAscope Multiplex Fluorescent Assay Human Hela Cell Line Amp 4 ALT TROUBLESHOOTING TIPS CHROMOGENIC ASSAYS 35 FACTORS AFFECTING RNASCOPE ASSAY PERFORMANCE XI Fixation conditions are not optimal RNA is degraded Hybridization conditions not optimal Samples drying during assay THE SOLUTIONS Fix samples as recommended E g for FFPE use 10 NBF RT 16 32 hrs Acquire new samples assess RNA quality M Use the HybEZ hybridization oven only Use Immedge and add adequate reagents to avoid drying NBF Neutral Buffered Formalin IMPACT OF FIXATION CONDITIONS 24 hours fixation Optimal 3 weeks fixation Over fixe Synaptophysin LTE a i k M Sample FFPE brain sample Assay RNAscope 2 0 Brown TIP Sample fixation has a great effect on the success of your assay Solution Increase pretreatment for better target accessibility LENA UNDER FIXATION Sample Flash Frozen followed by FFPE sample preparation Rat intestines Assay RNAscope 2 0 HD Brown Issue Weak
11. ilable conditions for your tissues for publication quality data THE CHECKLIST USE CONTROLS OPTIMIZE TIP Visit www acdbio com go for more information on getting started THE CHECKLIST WHAT YOU NEED RINAscope target probes single plex and multiple Positive and Negative control probe Hot Plate for pretreatment target retrieval step RNAscope reagent kit Detection Kit Pretreatment kits amp Wash Buffer HybEZ Hybridization system EZ Batch slide processing system or Tissue Tek system Ecomount for 2 0 HD Red amp 2 plex chromogenic assay RNAscope control slides Immedge hydrophobic barrier pen User supplied reagents refer to user manual 444444444 lt Read the user manual Part 1 Sample Prep Part 2 Detection Assay Visit www acdbio com go for more information on getting started Checklist is available on the website and in the manual USING A HOT PLATE Hotplate for retrieval boiling TIP When using a hot plate for pre treatment step pay close attention to the TIME and boiling TEMPERATURE AED RNASCOPE 9 REAGENT KIT CONTENTS NEW OLD Contents of the reagent kit 1 Pretreatment reagents 2 RNAscope detection kit 3 Wash buffer TIP Warm probes at 40 C for 10 minutes before use TIP Warm 50x wash buffer at 40 C for 20 minutes if you notice a precipitation USING HYBEZ HYBRIDIZATION OVEN x HyBEZ hybdrization system
12. obe mixture at 1X concentration mix C2 and C3 probes 1 50 with C1 probe If C2 and are all at 50X concentration use the blank probe C1 as a diluent and mix at a 1 50 dilution A FAR RED Atto 647 RNASCOPE WORKFLOW CHROMOGENIC ASSAY RNAscope user workflow Steps Description Time to Completion Deparaffinization Y H O block Pretreat 1 Epiton 1 5 hours we Pretreat Protease Pretreat 3 or 4 a D Label probe hybridization 1 5 hours Stain and detect Detection Download manuals http www acdbio com technical support downloads RNAscope user workflow gt RNASCOPE WORKFLOW FLUORESCENT ASSAY Steps Description Pretreatment conditions will based on sample type Download manuals http www acdbio com technical support downloads Time to Completion Dg ONE DAY TWO DAY ASSAY ONE DAY ASSAY TWO DAY ASSAY Sample preparation Sample preparation DAY 1 Sample pretreatment D RNAscope assay sample pretreatment RNAscope assay Review the User Manuals PART 1 PART 2 for optional stopping points is NE GETTING STARTED WITH RNASCOPE IN YOUR LAB 19 GEI SIARTED BY FOLLOWING 3 EASY STEPS B BI Check that all 8 5 56 required control probes RNAscope materials are and slides assay ava
13. staining destroyed morphology fresh frozen sample is under fixed Positive control Rn PPIB Optimization Fixation according to recommended guidelines for FFPE samples Result Strong staining for positive control PPIB intact morphology TER gt h e Positive control Rn de fo Refer to the Guide http www acdbio com technical support downloads rnascope ish guide troubleshooting VERA RU INA ASSAT ST AAN Sample FFPE Hela pellet Assay RNAscope 2 0 HD Brown me 4 Y Hi aif Issue Tissue dried out high background Optimization Do not allow drying between amplification steps Use the Immedge hydrophobic barrier pen Negative control dapB Result Clean background Negative control dapB TIP Refer to the Guide Al 39 http www acdbio com technical support downloads rnascope ish guide troubleshooting N REFER TO SAMPLE PRETREATMENT GUIDELINE Tissue Pretreatment Guidelines ALD 4 Table 2 Tissue Pretreatment Table based on your tissue type for ua Any new or previously untested FFPE tissues Samples prepared suboptimally Embryo Normal Guidelines for Optimal Tissue Pretreatment Spleen Normal e Fea L Fix sample FRESH 10 NBF for 16 32 Kidney Normal HOURS at ROOM TEMPERATURE NOTE Do not fix at 4 DO NOT fix for ix lt 16 hrs
14. ur RNA research and we recognize you need the b Brochures pport you in getting n L d 3 o O superior results from our RNAscope assays Please start with Probe Lists nks to product tutorial Training Webinars deo Publication List Scientific Posters or an duct inquiries please fill ou tact form t Application Reviews D m For any product inquiries please fill out our contact form to thi App Researcher in the Spotlight For technical assistance with an RNAscope assay please conta Visit www acdbio com technical support support overview rev dii b CONTACT ACD SUPPORT gt Support via email support acdbio com gt oupport via 1 877 376 3636 option 3 gt Time 8 00am 6 00pm PST gt Support Resources available on website www acdbio com Getting m a Product F Started Literature Download manuals simple tips amp tricks for Browse through our View our product Find RNAscope technical notes and you 1o get the best product frequently asked workflow videos on our publication lists gene MSDS RMAscope result from questions or add one of Video page lists and download day your own product brochures Go gt Go Go gt Go gt Go gt Advanced Cell Diagnostics 2013 Advanced Cell Diagnostics Inc Confidential and Proprietary
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