QuantiGene® ViewRNA Plate-Based Assay
Contents
1. 000000ee KK 6 Using New Probe Sets 0 0 kk kk kk kk kk kk kk kk Ek kk kK kk kk kk kk kk 7 Optimizing Assay Conditions for a New Cell Type 2 7 Optimizing Duplex Assay Formats 8 Overview 9 Using a Residual Volume na nn kk kk kk kk kk kK KK KK KI KK KK ere 9 Important Procedural Notes kk kk kK KK KK KK KK KK KK KIRI KK eee 10 Preparing Reagents kk k kk kk ka kk kk kk kk kk kk kk kk kk kk kk kk kk kk kk a 10 Fixing Cells and Treating with Protease 0 0 0 KK KK ERK KK KK 12 Performing Hybridizations kk kk kk KK KK KK KK KK KK eae 13 Section V Troubleshooting eee eR see eee 18 Troubleshooting No or Weak Signals liiis eese 18 Variable Staining Within a Sample llle ees 19 High Background on Entire Plates llle ee 19 High Background Within Cells l llis 20 High Number of Dots Appear where Cells are Absent 20 No DAPI Or Dots our RR er ERR ER E E ee ad 20 l gt SEE seh een 5 SEY Lic E dud MIL E ue 21 Fluidic Handling System Minimum Requirements kk kk kk kk kk KK KK KK KK KK KK eee eee 21 Setup Recommendations kk kk kk kk KK KK KK KK KK KK KK KK KK KK KK KK Ke 21 Wash Protocol Settings for BioTek ELx405 Select XL 21 QuantiGene ViewRNA Plate Based Assay User Manual iii Table of Contents Procul n th an ea OU aetna ee dha aed ae Da etal a 23 Compatible Plates i kk kk kk kK KK KK K2. Aspiration rate 01 3 0 mm sec 1 8 0 mm sec Aspiration delay 0000 msec 0 Crosswise aspiration No No Final aspiration Yes Yes Final aspiration delay 0000 msec 0 Dispense 384 Well Plate 96 Well Plate Dispense volume 80 uL well 150 uL well Dispense flow rate 01 01 Dispense height 120 15 240 mm 120 Horizontal disp pos 15 0 686 mm 35 1 600 mm Horiz Y disp pos 00 0 000 mm 00 Bottom wash first No No Prime by start No No QuantiGene ViewRNA Plate Based Assay User Manual Appendix Il Procedure for Dehydrating Cells for Storage at 20 C or for Shipping Appendix II Procedure for Dehydrating Cells for Storage at 20 C or for Shipping About Dehydration Use this procedure if you want to temporarily stop the assay after fixing the cells Procedure Following dehydration store the cells at 20 C for up to 2 months or until ready to use When ready to use follow the procedure for rehydrating the cells before continuing with the permeabilization step IMPORTANT Following the dehydration rehydration steps plates with Matrigel but not poly a lysine or collagen coatings might have a significant increase in background Verify that this will not be an issue before preparation and storage of multiple plates Procedure The 50 uL well volume in this procedure is specific for 384 well plates If you are using 96 well plates increase
3. 15 No Probe Set 1 1 000 for well columns 1 6 1 2 000 for well columns 7 12 1 4 000 for well columns 13 18 1 8 000 for well columns 19 24 Positive control Probe Set New TYPE 1 Probe Set only New TYPE 2 Probe Set only Both TYPE 1 and 2 Probe Sets 30 No Probe Set Positive control Probe Set New TYPE 1 Probe Set only New TYPE 2 Probe Set only Both TYPE 1 and 2 Probe Sets 45 No Probe Set Positive control Probe Set New TYPE 1 Probe Set only New TYPE 2 Probe Set only Both TYPE 1 and 2 Probe Sets TIO nim Ol w gt Oz Z xc QuantiGene ViewRNA Plate Based Assay User Manual 27 Appendix V Alternative Assay Procedure Manual Processing Appendix V Alternative Assay Procedure Manual Processing Overview Important Procedural Notes 28 Preparing Reagents The following procedures can be completed in one long day or broken down between 2 days Cells must be plated in 96 well format and allowed to adhere at least 1 day before starting the Assay Procedure IMPORTANT This alternative procedure is optimized for using multichannel pipettor for manual microplate manipulation IMPORTANT Before starting this procedure carefully review Section lll Recommendations for Experimental Design and Assay Optimization on page 6 Assay Workflow Prepare cells by fixing permeabilizing and di
4. or equivalent Filter sets for Ex 358 nm Em 461 nm DAPI Ex495 nm Em 519 nm FITC Ex 556 nm Em 573 nm Rhodamine Imaging system with the following Applied Precision Inc CellWoRx femmes Thermo Scientific Cellomics 20x or 40x objective Array Scan Uses standard 96 or 384 well plates GE Healthcare InCell 1000 Accepts Filter sets for DAPI MDS Analytical ImageXpress Fluorescein and Rhodamine Mi or equivalent Icro wavelengths Autofocus Broad excitation wavelength 350 600 nm Cooled CCD A fluidic dispenser and plate washer sold separately or together as outlined below Fluidic handling system 96 or Thermo Scientific Matrix PlateMate 2 x 3 384 channel pipet that meste or exceeds Beckman Coulter Biomek FX or the following specifications NX 15 70 uL 5 volume dispensing Agilent Technologies Bravo 5 20 uL sec rate of Velocity11 aspiration dispense or equivalent Replace or wash tip between dispensing 4 QuantiGene ViewRNA Plate Based Assay User Manual Table 1 Required materials not provided Section II Required Materials Part Number Required Material Source or Model Plate washer that meets or exceeds the BioTek ELx405 model following specifications with high 30 200 pL 5 volume throughput pump option 96 or 384 channels Angle dispensing tip Plate stacker Automation capable Minimal dead volume Combination washe
5. Part Number Required Material Source or Model Nuclease free water Major laboratory supplier 100 Ethanol optional Sigma Aldrich or equivalent 459844 10X PBS Ambion or equivalent AM9625 37 Formaldehyde WARNING Formaldehyde is a poison and irritant Avoid contact with skin and mucous membranes Fisher Scientific F79 1 96 or 384 well imaging compatible tissue culture plates See Appendix III 96 and 384 Well Imaging Compatible Plates on page 24 QuantiGene Incubator Temperature Validation Kit Panomics QS0517 QuantiGene ViewRNA Plate Based Assay User Manual s eui9je y pounboy Required Materials Section Il Required Materials Table 1 Required materials not provided Part Number Required Material Source or Model Incubation oven with the following Panomics QS0700 120V specifications QS0710 220V QS0701 120V 40 0 5 C that does not deviate by QS0711 220V more than 1 C when opening closing Uniform temperature throughout the or entire incubator Liconic or Thermo Cytomat Prefer 90 humidity models recommended for automation Automation friendly DAPI DNA stain solution Invitrogen D21490 WARNING DAPIis a possible or equivalent mutagen Avoid contact with skin and mucous membranes Epi fluorescence microscope that meets Olympus IX71 the following specifications 20 40X objective 100 Watt mercury bulb source
6. Aspirate the Formaldehyde Solution and gently wash three times with 1X PBS 1X PBS Vol 8 Formaldehyde Per Well Residual Vol uL uL Solution Vol pL 96 well plate 30 150 30 384 well plate 15 80 15 Note Optional At this point the assay can be stopped by dehydration and storage at 20 C See Appendix II Procedure for Dehydrating Cells for Storage at 20 C or for Shipping on page 23 for a procedure QuantiGene ViewRNA Plate Based Assay User Manual Section IV Assay Procedures for Automated or Batch Processing Residual Volume Method To fix and treat cells continued Step Action 2 Permealize cells with Detergent Solution a Dispense Detergent Solution and incubate for 3 minutes at room temperature b Aspirate Detergent Solution and wash twice with 1X PBS Residual Vol Per Well uL Detergent Vol uL 1X PBS Vol uL 96 well plate 30 30 150 384 well plate 15 15 80 3 Digest with Protease IMPORTANT The Protease dilution used in this example should be sufficient for a majority of cell lines If you observe high cell loss or no signal the optimal dilution needs to be determined Refer to Appendix IV Optimizing Assay Conditions for a New Cell Type on page 25 for more information a Aspirate the 1X PBS from the plate dispense Protease Working Solution cover plate with plate lid and incubate for 10 minutes at room temperature A
7. 0 12 Total volume 15 30 QuantiGene ViewRNA Plate Based Assay User Manual 11 Bu sseo9o d uojeg 10 pejeuiojny oJnpo2o0Jg Aessy Assay Procedure Automated or Batch Processing Section IV Assay Procedures for Automated or Batch Processing Residual Volume Method 12 To prepare reagents continuea Step Action 8 Prepare Working Amp Mixture Dilute Amp or Amps 1 250 in 40 C prewarmed Hyb B Buffer Vortex briefly to mix HL Per Well uL Per Well Component 384 well plate 96 well plate Hyb B prewarmed 14 88 29 76 Amp 1 0 06 0 12 Amp 2 0 06 0 12 Total volume 15 30 9 Prepare Working Label Probe Mixture Dilute LP or LPs 1 250 in 40 C prewarmed Hyb C Buffer Vortex briefly to mix HL Per Well HL Per Well Component 884 well plate 96 well plate Hyb C prewarmed 14 88 29 76 LP 1 0 06 0 12 LP2 0 06 0 12 Total volume 15 30 Fixing Cells and To fix and treat cells Treating with Protease Step Action 1 Fix cells in Formaldehyde Solution WARNING Formaldehyde is a poison and irritant Avoid contact with skin and mucous membranes a Aspirate medium from cells leaving defined residual volume and gently wash two times with 1X PBS Refer to the table below for volumes b Aspirate the final 1X PBS wash dispense 896 Formaldehyde Solution and incubate for 30 minutes at room temperature c
8. 15 uL well for 384 well plates and include overage to accommodate automation equipment dead volumes Keep working reagents at room temperature until use Working reagents are good for 2 days If unused on the day of preparation store at 4 C overnight and rewarm to 40 C for 30 minutes before use 5 Prepare Protease Working Solution Dilute Protease 1 4 000 or concentration optimized for your cell type with room temperature 1X PBS Vortex briefly to mix IMPORTANT Optimal Protease dilution needs to be determined Refer to Appendix IV Optimizing Assay Conditions for a New Cell Type on page 25 for more information 6 Prepare Working Probe Set Mixture Dilute TYPE 1 and TYPE 2 Probe Sets 1 50 in 40 C prewarmed Hyb A Buffer Vortex briefly to mix WARNING Hyb A Buffer contains formamide a teratogen irritant and possible carcinogen Avoid contact with skin and mucous membranes pL Per Well HL Per Well Component 384 well plate 96 well plate Hyb A prewarmed 14 4 28 8 TYPE 1 Probe Set 0 3 0 6 TYPE 2 Probe Set 0 3 0 6 Total volume 15 30 7 Prepare Working PreAmp Mixture Dilute PreAmp or PreAmps 1 250 in 40 C prewarmed Hyb B Buffer Vortex briefly to mix WARNING Hyb B Buffer contains formamide a teratogen irritant and possible carcinogen Avoid contact with skin and mucous membranes HL Per Well HL Per Well Component 384 well plate 96 well plate Hyb B prewarmed 14 88 29 76 PreAmp 1 0 06 0 12 PreAmp 2 0 06
9. 546 573 For detection of greater than 1000 copies cell Nuclear DNA DAPI 358 461 For focusing and cell identification QuantiGene ViewRNA Plate Based Assay User Manual 17 Bu sseo9o d yo eg 10 pa ewojny e Jnpe9o q Aessy Troubleshooting Section V Troubleshooting Section V Troubleshooting Troubleshooting Troubleshooting no or weak signals No or Weak Signals 18 Probable Cause Recommended Actions Inadequate Protease digestion Optimize Protease digestion conditions as described in Appendix IV Optimizing Assay Conditions for a New Cell Type on page 25 Over fixing cells Do not exceed the recommended fixation time for your cell type Over under permeabilization Perform time titration of permeabilization step for 1 10 minutes Inappropriate hybridization temperature Hybridization reactions must be carried out at 40 C 1 C Use a QuantiGene Incubator Temperature Validation Kit to verify and monitor the temperature Incorrect Use of Probe Sets PreAMP AMP and or LP Ensure that Working Solutions are prepared properly and used in the correct order If running singleplex assays use Type 1 and 2 Probe Sets with PreAMP AMP LP 1 and 2 respectively If running multiplex assays include Type 1 and 2 Probe Sets and PreAMP AMP LP 1 and 2 in the appropriate Working Solutions Photo bleaching of fluorescent signals Cover samples with foil duri
10. 96 well plate Component Vol uL Per Well 96 well plate Working Hyb C 59 76 LP 1 0 12 LP2 0 12 Total volume 60 QuantiGene ViewRNA Plate Based Assay User Manual Appendix V Alternative Assay Procedure Manual Processing Fixing Cells and To fix and treat cells Treating with Protease SteP Action 1 Fix cells in 4 Formaldehyde Solution WARNING Formaldehyde is a poison and irritant Avoid contact with skin and mucous membranes a Invert plate containing cells over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel for 1 2 seconds Gently wash 2 times with 150 uL well 1X PBS b Remove as above the final 1X PBS wash add 60 pL 4 Formaldehyde Solution cover plate with plate lid and incubate for 30 minutes at room temperature c Remove as above the Formaldehyde Solution and gently wash 3 times with 150 uL well 1X PBS Note Optional At this point the assay can be stopped by dehydration and storage at 20 C See Appendix II Procedure for Dehydrating Cells for Storage at 20 C or for Shipping on page 23 for a procedure Permealize cells with Working Detergent Solution a Add 60 pL well Working Detergent Solution and incubate for 3 minutes at room temperature b Invert plate over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel f
11. imaging system Low Background with the QuantiGene ViewRNA Assay Plates must have acceptable background levels to achieve required sensitivity and assay precision Some plate coatings may induce non specific binding of the QuantiGene ViewRNA amplification system resulting in high backgrounds To assess plate background run a no cells control described below in Assessing Background for New Projects on page 6 Automation friendly Use a plate type that follows the ANSI recommended specifications The plate should also have a lid to control evaporation Perform all assays in duplicate or triplicate It is important to assess the source of background signals when running a new assay The results can be evaluated using either a microscope or imaging system We recommend running the following controls No Cells Designate at least 2 3 wells that contain no cells and undergo the entire assay procedure This control enables you to assess the background associated with non specific binding of signal amplification reagents to the well and or well coating There should be a low uniform intensity across the well The intensity for these wells should be lower than wells that contain cells only and wells that contain no probe sets The appearance of patchy areas with enhanced intensity or many strong QuantiGene ViewRNA Plate Based Assay User Manual Using New Probe Sets Optimizing Assay Conditions for a New Cell Type QuantiGene ViewRNA P
12. minutes e e e 5 Proceed to step 2 on page 13 and continue with the permeabilization of the cells QuantiGene ViewRNA Plate Based Assay User Manual 23 Appendix III 96 and 384 Well Imaging Compatible Plates Appendix III 96 and 384 Well Imaging Compatible Plates Compatible Plates Below is a table of plates that have been shown to work with our assay Please refer to Selecting the Appropriate Plate type on page 6 for criteria to consider when selecting a 24 plate Format Vendor Cat Bottom Coating 384 well Greiner 781946 Polystyrene Poly D lysine MatriCal MGB101 1 2 Glass None LG L Perkin Elmer 6007439 Polystyrene None CellCarrier TC 384 96 well Nunc 165305 Polymer None Nunc 164588 Glass None BD Biocoat 35 4640 or Polystyrene Poly D lysine 35 6640 QuantiGene ViewRNA Plate Based Assay User Manual Appendix IV Optimizing Assay Conditions for a New Cell Type Appendix IV Optimizing Assay Conditions for a New Cell Type Initial Optimization In the initial experiment we recommend you start with a single plex assay 80 95 cell and Assessment density 30 minute fixation time and a 10 minute incubation time with 4 different Protease of Assay dilutions Refer to Assessing Background for New Projects on page 6 for details on Backgrounds commended controls for assessing backgrounds The table below is a recommended plate map for an initial optimizatio
13. IB ACTB HeLa cells were cultured fixed permeabilized and protease treated in a 96 well clear bottom black wall plate The cells were then probed using a TYPE 2 Probe Set for ACTB red color and TYPE 1 Probe Sets for HCV Her2 HPRT and PPIB green color After QuantiGene ViewRNA branched DNA amplification and DAPI nuclear stain the HeLa cells in the plate were scanned on a CellWoRx scanner using the 20X objective lens The exposure time and the intensity settings used for each channel are summarized below Typical results of HeLa positive control No signal is observed in the negative controls DAPI and No Probe Green but not red signal can be observed in the panel of HCV ACTB Increasing number of green dots are observed in panels representing expression of Her2 HPRT and PPIB respectively whereas ACTB signal remains the same Channel Exposure Time seconds Min Max Intensity Setting DAPI 0 2 4 000 50 000 Fluorescein 3 750 2 000 Rhodamine 3 1 000 10 000 26 QuantiGene ViewRNA Plate Based Assay User Manual Appendix IV Optimizing Assay Conditions for a New Cell Type Additional If acceptable data is not obtained during the initial optimization assay it may be Optimization necessary to evaluate additional cell densities and or fixation times A recommended plate map is provided below Sample plate map for a 384 well plate Fixation Protease Dilution Time min Row Control Type
14. K KK KK KK ree 24 Initial Optimization and Assessment of Assay Backgrounds 25 Examples of Optimized Conditions 00000 eee eee eee 26 Additional Optimization kk kk kk kK kk KK eee 27 OVetViGW su cle d puce ge es RUE RATER qu ee ek hdd aU RERO E ea dog 28 Important Procedural Notes kk kk kk kk kk KK KK KK KIR KI KIRI sees 28 Preparing Reagents 02 0224 kaka xwa kaka a R alal aka ee kul k ak anan la E 28 Fixing Cells and Treating with Protease 0 0 RR RR KK KK eee 31 Performing Hybridizations kuz cesis kla eee 31 QuantiGene ViewRNA Plate Based Assay User Manual Section I Introduction Section I Introduction About This Manual The QuantiGene ViewRNA Reagent System is an n situ hybridization method for visualization of one target RNA at single transcript detection level and one housekeeping or higher expressing gt 1 000 transcripts target RNA simultaneously within individual cells This manual provides complete instructions for performing QuantiGene ViewRNA assays for adherent cells in either Anautomated format for 96 or 384 well plates or A manual format for 96 well plates QuantiGene Panomics QuantiGene ViewRNA assay is a novel RNA in situ hybridization solution ViewRNA Assay based on patent pending Probe Set design and proprietary signal amplification Basics technology QuantiGene ViewRNA offers single copy RNA sensitivity in individual cells in a mult
15. Plate Based Assay User Manual 1 uononpoJdju Required Materials Section Il Required Materials Section Il Required Materials QuantiGene ViewRNA Reagent System QuantiGene ViewRNA Plate based Assay Kit Components QuantiGene ViewRNA Plate Based Signal Amplification Kits The QuantiGene ViewRNA Plate Based Reagent System is comprised of 3 modules each sold separately QuantiGene ViewRNA Plate Based Assay Kit QuantiGene ViewRNA Plate Based Signal Amplification Kit QuantiGene ViewRNA Probe Sets The components of the QuantiGene ViewRNA Plate Based Assay Kit and their recommended storage conditions are listed below This Assay Kit contains the universal reagents for running a QuantiGene ViewRNA assay This Assay Kit must be used in conjunction with a QuantiGene ViewRNA Plate based Signal Amplification Kit and appropriate QuantiGene ViewRNA probe sets The QuantiGene ViewRNA Plate based Assay Kit is available in multiple sizes Refer to the product insert for quantities of individual components supplied Kits have a shelf life of 6 months from date of receipt Component Description Storage Protease Enzyme in aqueous buffered solution 2 8 C Hybridization Buffer A Aqueous solution containing formamide and 2 8 C Hyb A detergent Hybridization Buffer B Aqueous solution containing formamide and 2 8 C Hyb B detergent Hybridization Buffer C Aqueous solution contain
16. Prepare Working Hyb B Buffer by diluting stock Hyb B Buffer prewarmed to 40 C 1 1 with Working PreHyb Buffer and then vortexing briefly to mix WARNING Hyb B Buffer contains formamide a teratogen irritant and possible carcinogen Avoid contact with skin and mucous membranes Prepare enough for the preparation of Working PreAmp Mixture and Working Amp Mixture 16 mL is sufficient for one 96 well plate 13 Prepare Working Hyb C Buffer by diluting stock Hyb C Buffer prewarmed to 40 C 1 1 with Working PreHyb Buffer and then vortexing briefly to mix 8 mL is sufficient for one 96 well plate 14 Prepare Working PreAmp Mixture by diluting PreAmp or PreAmps 1 500 with Working Hyb B Buffer and then vortexing briefly to mix 8 mL is sufficient for one 96 well plate Component Vol uL Per Well 96 well plate Working Hyb B 59 76 PreAmp 1 0 12 PreAmp 2 0 12 Total volume 60 15 Prepare Working Amp Mixture by diluting Amp or Amps 1 500 with Working Hyb B Buffer and then vortexing briefly to mix 8 mL is sufficient for one 96 well plate Component Vol uL Per Well 96 well plate Working Hyb B 59 76 Amp 1 0 12 Amp 2 0 12 Total volume 60 16 Prepare Working Label Probe Mixture by diluting LP or LPs 1 500 with Working Hyb C Buffer and then vortexing briefly to mix 8 mL is sufficient for one
17. QuantiGene ViewRNA Plate Based Assay for adherent cells User Manual Panomics Panomics Inc QuantiGene ViewRNA Plate Based User Manual Copyright Copyright 2009 Panomics Inc All rights reserved Trademarks QuantiGene is a registered trademark exclusively licensed to Panomics Inc All other trademarks belong to their respective owners Citing QuantiGene ViewRNA in Publications When describing a procedure for publication using this product please refer to it as the QuantiGene ViewRNA assay If a paper cites a QuantiGene ViewRNA product and is published in a research journal the lead author s may receive a travel stipend for use at a technology conference or tradeshow by sending a copy of the paper to our technical support group at techsupport panomics com or via fax at 510 818 2610 Disclaimer Panomics 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 Panomics Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Contacting Panomics U S Corporate Headquarters Panomics Inc 6519 Dumbarton Circle Fremont CA 94555 Toll Free 877 PANOMICS 1 877 726 6642 Direct 1 510 818 2600 Fax 1 510 818 2610 Email info panomics com Email
18. V Assay Procedures for Automated or Batch Processing Residual Volume Method To perform hybridizations continued Step Action 6 Hybridize Amplifiers AMPs a Aspirate Wash Buffer dispense PreHyb Buffer prewarmed to 40 C and pipet up and down twice to mix Aspirate PreHyb Buffer and dispense Working AMP Mixture Cover plate with plate lid and incubate at 40 1 C for 60 minutes Residual Vol PreHyb Buffer Working Amp Per Well uL Vol uL Vol uL 96 well plate 30 30 30 384 well plate 15 15 15 Aspirate the Working AMP Mixture and wash 3 times with Wash Buffer Include a 30 seconds soak at the last wash step Per Well Residual Vol uL Wash Buffer Vol uL 96 well plate 30 150 384 well plate 15 80 IMPORTANT Protect samples from light during this and all subsequent steps Hybridize Label Probes LPs a Dispense PreHyb Buffer prewarmed to 40 C and pipet up and down twice to mix Aspirate PreHyb Buffer and dispense Working LP Mixture Cover plate with plate lid and incubate at 40 1 C for 60 minutes PreHyb Buffer Working LP Vol Per Well Residual Vol uL Vol pL uL 96 well plate 30 30 30 384 well plate 15 15 115 Wash three times with Wash Buffer a Aspirate the Working LP Mixture and wash 3 times with Wash Buffer Include a 30 second soak at the last wash step b Dispense 1X PBS At this
19. d with cells only and without Probe Set PreAmp Amp and Label Probe Incorrect filter setup Replace filters used in the imaging system QuantiGene ViewRNA Plate Based Assay User Manual 19 Bu oousejqno Troubleshooting Section V Troubleshooting High Background Troubleshooting high background within cells Within Cells High Number of Dots Appear where Cells are Absent Probable Cause Recommended Actions Samples were allowed to dry Once the in situ part of the assay is started do not allow the cells to dry at any stage Do not use less than the recommended volumes of any hybridization solution for your sample type Use residual volume method or leave recommended volume after each wash step This will minimize the chance of a well drying out completely Insufficient washing Add an additional wash step to all washes Non specific binding of Probe Set s Hybridization reactions must be carried out at 40 C 1 C Use a QuantiGene Incubator Temperature Validation Kit to verify and monitor the temperature Insufficient Protease digestion Optimize Protease digestion conditions as described in Appendix IV Optimizing Assay Conditions for a New Cell Type on page 25 Troubleshooting a significant number of dots outside the cells Probable Cause Recommended Actions Non specific binding of Probe Set PreAmp Amp or Label Probe to
20. g 96 or 384 well multichannel pipettor and microplate washer It is not ideal for manual microplate manipulation See Appendix V Alternative Assay Procedure Manual Processing on page 28 IMPORTANT Before starting this procedure carefully review the recommendations in Section III Recommendations for Experimental Design and Assay Optimization on page 6 Assay Workflow Sample preparation Adherent cells must be plated in 96 or 384 well format and allowed to firmly adhere to the plate before starting the Assay Procedure Preparing reagents Prewarm reagents Prepare working reagents Fix cells optional stop point Permeabilize cells and digest with Protease Perform hybridizations Hybridize Probe Sets optional stop point Sequentially hybridize PreAmp s Amp s and LP s Counter stain with DAPI optional stop point Analyze samples on imaging system Using a Residual To minimize cell detachment from the bottom of the plate the following procedures leave Volume a residual volume in each well at each step At the end of each step there is always a minimum volume of 15 uL well in a 384 well format and 30 uL well in a 96 well format See Appendix I Liquid Handling Recommendations on page 21 for more information This figure illustrates what residual volume is the volume remaining after aspiration Dispense Aspirate Residual Volume QuantiGene ViewRNA Plate Based Assay User Manual 9 As
21. gesting with Protease Perform hybridizations Analyze plate Hybridization reactions must be carried out at 40 1 C Verify and monitor oven temperature using the QuantiGene Incubator Temperature Validation Kit Protect samples from light during the Label Probe hybridization and all subsequent steps Before opening reagents supplied in microfuge tubes briefly centrifuge to collect contents at the bottom of the tube All Hyb Buffers stored at 4 C must be prewarmed to 40 C for 30 minutes to redissolve any precipitates Precipitation will form at room temperature To prepare reagents for alternative processing procedure Step Action 1 Thaw warm the following reagents Prewarm PreHyb Hyb A Hyb B and Hyb C Buffers to 40 C for 30 minutes Thaw Probe Sets PreAmp or PreAmps Amp pr Amps and LP or LPs and place on ice 2 Prepare the appropriate volume of the following reagents 1XPBS 250 mL is sufficient for one 96 well plate 496 Formaldehyde Solution in 1X PBS prepare daily 7 2 mL is sufficient for one 96 well plate WARNING Formaldehyde is a poison and irritant Avoid contact with skin and mucous membranes 3 Prepare Wash Buffer For example to a 1 000 mL graduated cylinder add the following in this order 800 mL nuclease free water 3mL Wash Buffer Comp 1 5mL Wash Buffer Comp 2 Adjust to 1 liter with nuclease free water Scale preparation according to the
22. ides in 20 C LP2 aqueous buffered solution In addition to the Assay Kit and Signal Amplification Kit QuantiGene ViewRNA Probe Sets specific to your target and housekeeping or higher expressing RNAs of interest must be purchased separately Probe Sets are available in multiple sizes Refer to the product insert for details on Probe Set design and specificity QuantiGene ViewRNA Probe Sets should be stored at 20 C and have a shelf life of 12 months from date of receipt Component Description Storage QuantiGene ViewRNA TYPE 1 Probe Set RNA specific oligonucleotides for use with the 20 C PreAMP1 Amp1 LP1 Signal Amplification Kit for visualization of single copy RNAs 1 copy cell QuantiGene ViewRNA TYPE 2 Probe Set RNA specific oligonucleotides for use with the 20 C PreAMP2 Amp2 LP2 Signal Amplification Kit for visualization of high abundance RNAs such as 18S ACTB or GAPD more than 1000 copies cell Visit www panomics com for the most up to date listing of available QuantiGene ViewRNA Probe Sets By Request QuantiGene ViewRNA Probe Sets can be designed and synthesized at no additional cost Please provide the accession number including version or gi number or RNA sequence with your By Request order Other materials required to perform the QuantiGene ViewRNA assay that are not included in the Plate Based Assay Kit are listed here Table 1 Required materials not provided
23. ignal of a dot and dividing by the average background signal outside of the cell When using a Probe Set for the first time we recommend running the Probe Set in both a singleplex and multiplex assay format When working with a new sample type optimization of cell density formaldehyde fixation and Protease digestion are key factors In the initial experiment we recommend that you start with a singleplex assay 80 9596 cell density a 30 minute fixation time and a 10 minute incubation time with Protease dilutions 1 1 000 1 2 000 1 4 000 and 1 8 000 This approach should work for most sample types In some cases it may be necessary to evaluate additional cell densities and fixation times of 15 and 60 minutes Under fixation and over treatment with Protease will result in high cell loss Cell loss can be checked using a bright field microscope See Appendix IV Optimizing Assay Conditions for a New Cell Type on page 25 for sample plate layouts for the optimization conditions The selection criteria for the optimal fixation and Protease treatment conditions are the balance of cell loss and maximum signal to background When visualizing the plate under a fluorescent microscope or in an imaging platform the following criteria should be used to select the optimal assay conditions Least cell lost The majority of cell loss typically occurs after the Protease treatment We recommend that you verify the cell density after the Protease treatment u
24. ing detergent 2 8 C Hyb C Protease Stop Buffer Aqueous buffered solution 15 30 C Storage Buffer Aqueous buffered solution 15 30 C Detergent Solution Aqueous buffered solution containing detergent 15 30 C Wash Buffer Component 1 Aqueous solution containing detergent 15 30 C Wash Comp 1 Wash Buffer Component 2 Aqueous buffered solution 15 30 C Wash Comp 2 Pre Hybridization Buffer Aqueous buffered solution 15 30 C PreHyb Buffer The components of the QuantiGene ViewRNA Plate Based Signal Amplification Kit and their recommended storage conditions are listed below There are multiple options for signal amplification in the QuantiGene ViewRNA assay Depending on the need for single or multiplex assays the items supplied will differ PreAmp 1 Amp 1 LP 1 are compatible with TYPE 1 Probe Sets and PreAmp 2 Amp 2 LP 2 are compatible with TYPE 2 Probe Sets Refer to the product insert for quantities of individual components supplied Kits have a shelf life of 6 months from date of receipt Storage 20 C Component Description PreAmplifier PreAmp 1 and or PreAmp 2 DNA in aqueous buffered solution QuantiGene ViewRNA Plate Based Assay User Manual QuantiGene ViewRNA Probe Sets Required Materials Not Provided Section Il Required Materials Amplifier Amp 1 and or DNA in aqueous buffered solution 20 C Amp 2 Label Probe LP1 and or Fluorescent dye conjugated oligonucleot
25. iplex assay format Signal amplification is predicated on specific hybridization of adjacent Probe Set oligonucleotides to a target RNA see How It Works below resulting in excellent signal to noise ratios Independent but compatible signal amplification systems enable simultaneous detection of two RNAs in a single assay How It Works Target mRNA specific Probe Sets Z TYPE 1 blue Z TYPE 2 green PreAmp 1 PreAmp 2 2z 2 gt eZ 2 m Ampi z zz 1 Amp 2 z P 4 oLabel Probe 1 cS C Eo Label Probe 2 1 Step 1 Prepare Sample Step 2 Hybridize Probe Sets Step 3 Amplify Signal Step 4 Image Step 1 Prepare Sample Adherent cells on a solid surface are fixed and permeabilized Step 2 Hybridize Probe Sets Gene specific Probe Sets hybridize to target MRNAs For clarity only single oligonucleotide pairs are shown however a typical Probe Set contains 20 or more oligonucleotide pairs Each Probe Set TYPE for example TYPE 1 interacts specifically with a corresponding signal amplification system for example PreAmp 1 Amp 1 Label Probe 1 to generate signal for visualization Step 3 Amplify Signal Independent but compatible signal amplification systems enable simultaneous detection of multiple RNAs in a single assay Distinct sets of PreAmp Amp and Label Probe molecules are used to detect different target RNAs Step 4 Image Target mRNAs are visualized using standard fluorescence microscopy QuantiGene ViewRNA
26. izations Step Action 2 Invert plate over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel for 1 2 seconds Wash 3 times with 150 uL well Wash Buffer 3 Optional Plates can be stored at 4 C for up to 24 hours To continue without storage proceed to step 4 Prepare for storage a Invert plate over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel for 1 2 seconds Add 60 uL well Working Storage Buffer b Plate is now ready for storage Cover with plate lid wrap with parafilm and store at 4 C c After storage remove as above Working Storage Buffer and wash the plate 2 times with 150 uL well Wash Buffer before hybridizing to the preamplifier IMPORTANT Ensure that the refrigeration is at 4 C Lower temperatures will cause precipitation of Storage Buffer resulting in significant cell loss 4 Hybridize Pre Amplifiers PreAMPs If continuing from step 3 following storage make sure working reagents stored at 4 C have been prewarmed and are ready See Preparing Reagents on page 28 a Invert plate over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel for 1 2 seconds Add 60 uL well Working PreAmp Mixture b Cover plate with plate lid and incubate at 40 C 1 C for 60 minutes 5 Invert plate over appropriate receptacle and gently expel contents then inver
27. l Residual Vol uL Wash Buffer Vol uL 96 well plate 30 150 384 well plate 15 80 3 Optional Plates can be stored at 4 C for up to 24 hours To continue without storage proceed to step 4 Prepare for storage a Aspirate Wash Buffer and dispense Storage Buffer b After storage aspirate Storage Buffer and wash the plate twice with Wash Buffer IMPORTANT Ensure that the refrigeration is at 4 C Lower temperatures will cause precipitation of Storage Buffer resulting in significant cell loss Residual Vol Storage Buffer Wash Buffer Vol Per Well uL Vol uL uL 96 well plate 30 30 150 384 well plate 15 15 80 4 Hybridize PreAmplifiers PreAMPs If continuing from step 3 make sure working reagents stored at 4 C have been prewarmed and are ready see Preparing Reagents on page 10 a Aspirate Wash Buffer dispense PreHyb Buffer prewarmed to 40 C and pipet up and down twice to mix b Aspirate PreHyb Buffer and dispense Working PreAMP Mixture Cover plate with plate lid and incubate at 40 C 1 C for 60 minutes Residual PreHyb Buffer Vol Working PreAmp Per Well Vol uL uL Vol uL 96 well plate 30 30 30 384 well plate 15 15 15 5 Aspirate the Working PreAMP Mixture and wash 3 times with Wash Buffer Include a 30 second soak at the last wash step Per Well Residual Vol uL Wash Buffer Vol uL 96 well plate 30 150 384 well plate 15 80 QuantiGene ViewRNA Plate Based Assay User Manual Section I
28. late Based Assay User Manual 7 Section Ill Recommendations for Experimental Design and Assay Optimization individual dots indicates a problem with the assay See Section V Troubleshooting on page 18 for more information Cells Only Designate at least 2 3 wells that undergo only the cell staining with DAPI This control enables you to assess the amount of autofluorescence the cells are contributing to assay background No Probe Set s Designate 2 3 wells that undergo the entire assay procedure with the probe sets omitted This control enables you to assess the non specific binding of amplification reagents to the cell There should be less than 1 strong dot 10 cells Negative Control Probe Set Designate 2 3 wells that contain Probe Set targeting a gene that is not expressed in the target cell This control enables you to assess the specificity of the assay For example using a TYPE 1 Probe Set designed to detect the sense strand of 18S RNA There should be less than 1 strong dot 10 cells Positive Control Probe Set Designate 2 3 wells that contain a Probe Set targeting a housekeeping gene such as GAPD or another gene that is expected to be expressed There should be distinct dots in the cytosol of the cells The background should be comparable to the no Probe Set control Note A strong dot is defined as exhibiting a measurement of 2 5X signal to background ratio Signal to background is calculated by taking the maximum s
29. n assay Sample plate map for a 384 well plate Row Control Type Protease Dilution A B No cells 1 1 000 for 1 2 000 for 1 4 000 for 1 8 000 for C D Cells only well well well well columns columns columns columns19 E F No Probe Set 1 6 7 12 13 18 24 G H Negative control Probe Set l J Positive control Probe Set K L New TYPE 1 Probe Set only M N New TYPE 2 Probe Set only O P Both TYPE 1 and 2 Probe Sets From each optimization experiment select the optimal conditions based on the following criteria Least cell loss Lowest background inside and outside cells in the negative control wells no dots and low uniform background Highest signal background ratio of dots inside the cell Best assay precision between replicate wells and plates for cell number low background and high signal background ratio When running both a TYPE 1 and TYPE 2 Probe Set weight your selection for optimal assay conditions for the TYPE 1 Probe Set In some cases finding the optimal conditions may require an additional experiment See the following section for a description of a plate map for further optimization QuantiGene ViewRNA Plate Based Assay User Manual 25 Appendix IV Optimizing Assay Conditions for a New Cell Type Examples of The images below show representative examples of optimized assay conditions Optimized Conditions DAPI No Probe HCV ACTB Her2 ACTB HPRT ACTB PP
30. ng LP hybridization and all subsequent steps Store samples in the dark Inappropriate microscope set up or operation Ensure that your microscope is in good working order and that your light source objectives filters and exposure time are selected properly Poor cell retention Titrate cell numbers fixation and Protease treatments Try using different plate type to ensure good cell adhesion Plastic plates work better but tend to exhibit 2 4X higher background Try coating the plating surfaces with different extracellular matrices such as MatriGel or Poly D lysine Use 5 0 uL sec dispensing aspiration speed to minimize cell detachment Dispense and aspirate near the edge of the well and image on the opposite edge of the wells Use residual volume protocol If necessary increase amount of residual volume The volume of all reagents in all steps must be increased proportionally For example if there is 60 uL of residual volume then 60 uL of reagents must be used Use adherent cells and ensure cells are firmly adhered to the plate prior to the assay QuantiGene ViewRNA Plate Based Assay User Manual Section V Troubleshooting Variable Staining Troubleshooting variable staining within a sample Within a Sample Probable Cause Recommended Actions Samples were allowed to dry Once the samples have been rehydrated do not allow the cells to dry at any stage Do not use less than the
31. nterstain nuclei with DAPI solution WARNING DAPI is a possible mutagen Avoid contact with skin and mucous membranes IMPORTANT Protect from light a Prepare DAPI Working Solution by diluting DAPI Stock Solution 10 mg mL 1 10 000 in 1X PBS and briefly vortexing to mix well For example add 1 uL of stock DAPI solution to 10 mL 1X PBS 7 2 mL is sufficient for one 96 well plate b Invert plate over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel for 1 2 seconds Add 60 uL well DAPI Working Solution c Incubate at room temperature for 1 minute d Remove as above the DAPI Working Solution and wash once with 150 uL well 1XPBS e Add 150 uL well fresh 1X PBS The plate is ready for imaging Alternatively the plate can be stored at 4 C for several days Seal the plate with an adhesive seal and protect from light when storing at 4 C Note that after 2 days the signal will drop by 50 QuantiGene ViewRNA Plate Based Assay User Manual 33 Appendix V Alternative Assay Procedure Manual Processing To perform hybridizations Step Action 11 Scan the plate on an imaging system by Using DAPI fluorescein and rhodamine channels or by Visualizing under a fluorescent microscope using appropriate filter sets We recommend the following Magnify the image by 200 400 fold through the combined use of a 10X eyepiece and a 20 40X fluorescence objec
32. number of plates to be processed 200 mL is sufficient for processing one 96 well plate QuantiGene ViewRNA Plate Based Assay User Manual Appendix V Alternative Assay Procedure Manual Processing To prepare reagents for alternative processing procedure continued Step Action 4 For steps 5 16 prepare an appropriate volume of working reagents Plan for 60 uL well for 96 well plates and include overage to accommodate the use of reagent reservoirs Volumes sufficient for one 96 well plate are provided throughout the procedures Keep working reagents at room temperature until use Working reagents are good for 2 days If unused the day of preparation store at 4 C overnight and rewarm to 40 C for 30 minutes before use Swirl working reagents to visually verify there are no precipitates 5 Prepare Working Detergent Solution by diluting stock Detergent Solution 1 1 with 1X PBS and then vortexing briefly to mix 8 mL is sufficient for one 96 well plate 6 Prepare Working Protease Solution by diluting Protease 1 8 000 or optimized concentration for your cell type with room temperature 1X PBS and then vortexing briefly to mix 8 mL is sufficient for one 96 well plate 7 Prepare Working Protease Stop Buffer by diluting stock Protease Stop Buffer 3 1 with 1X PBS and then vortexing briefly to mix Prepare enough for the assay and also for the preparation Working Hyb A Buffer 13 mL 9 75 mL stock P
33. or 1 2 seconds c Add 150 pL well 1X PBS Digest with Working Protease Solution IMPORTANT The Protease dilution used in this example should be sufficient for a majority of cell lines If you observe high cell loss or no signal the optimal dilution should be determined Refer to Appendix IV Optimizing Assay Conditions for a New Cell Type on page 25 for more information a Invert plate over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel for 1 2 seconds Add 60 uL well Working Protease Solution cover plate with plate lid and incubate for 10 minutes at room temperature b Remove as above the Working Protease Solution and gently wash 3 times with 150 uL well 1X PBS c Remove as above 1X PBS and add 60 uL well Working Protease Stop Buffer d Proceed to hybridization procedure Samples may sit in Working Protease Stop Buffer for up to 30 minutes Performing To perform hybridizations Hybridizations Step Action 1 Hybridize target Probe Sets a Invert plate over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel for 1 2 seconds Add 60 uL well Working Probe Set Mixture b Cover plate with plate lid and incubate plate for 3 hours at 40 C 1 C QuantiGene ViewRNA Plate Based Assay User Manual 31 Appendix V Alternative Assay Procedure Manual Processing To perform hybrid
34. orders panomics com Email techsupport panomics com European Headquarters Panomics Srl Via Sardegna 1 20060 Vignate Milano Italy Tel 39 02 95 360 250 Fax 39 02 360 992 Email info_europe panomics com Email order_europe panomics com Email techsupport_europe panomics com Asia Pacific Headquarters Panomics Inc 16F Gemdale Plaza Tower A No 91 Jiango Road Beijing 100022 P R China Tel 86 10 59208157 Fax 86 10 59208111 Email info_asia panomics com Email order_asia panomics com Email techsupport_asia panomics com Contents Section Introduction ss ey aye see KI DIK nto co SEDE 1 About This Manual sse hs 1 QuantiGene ViewRNA Assay Basics l llle 1 How It Works iux ee AR RR EXE esa due ES RESET ERAI 1 Section llsRequiredMaterialst IE ET US M UU SEA OS 2 QuantiGene ViewRNA Reagent System KK KK KK KK KK KK KK KK gt 2 QuantiGene ViewRNA Plate based Assay Kit Components 2 QuantiGene ViewRNA Plate Based Signal Amplification Kits 2 QuantiGene ViewRNA Probe Sets kk kK KK KK KK KK KK KK ee 3 Required Materials Not Provided 20200 0c KK KK RR RR KK KK 3 OVGV SW iius selk deg di nav dk etd RR aOR edid de n ok Wik abe dee ed 6 Selecting the Appropriate Plate type 20 0c eee eee ee 6 ReplicateS i e ci ea ease a eee ean eee a ee ee een 6 Assessing Background for New Projects
35. ote Signals drop by approximately 50 after 2 days Residual 1X PBS 1X PBS Final Per Well Vol uL DAPI uL uL uL 96 well plate 30 30 150 150 384 well plate 15 15 80 80 QuantiGene ViewRNA Plate Based Assay User Manual Section IV Assay Procedures for Automated or Batch Processing Residual Volume Method To perform hybridizations continued Step Action 11 Scan the plate on an imaging system by Using DAPI fluorescein and rhodamine channels or by Visualizing under a fluorescent microscope using appropriate filter sets We recommend the following Magnify the image by 200 400 fold through the combined use of a 10X eyepiece and a 20 40X fluorescence objective with numeric aperture equal to or greater than 0 75 Set exposure time to obtain optimal signal to background ratio Normally DAPI stain requires 10 50X less exposure time than for target RNA 1 and 2 Use the following table to determine the multi bandpass fluorescence microscope filter sets to visualize signals For the best viewing signal set the gray scale or intensity level as follows min 2X above background outside of cell max 4X above background outside of cell Adjust as necessary Peak Peak Nucleic Excitation Emission Acid Fluorophore nm nm Purpose Target RNA 1 Fluorescein 495 519 For detection of 1 copy cell Target RNA 2 Rhodamine
36. plate Verify by running the assay without cells in wells High cell loss and cell damage from excess detergent Protease treatment or fixation Optimize detergent Protease and fixation conditions No DAPI or Dots Troubleshooting imaging problems 20 Probable Cause Recommended Actions Incorrect filter setup Verify that the correct filters are installed See the Technical Note Verification of microscope suitability for ViewRNA Plate and imaging system incompatible Verify by scanning for nuclear DAPI stain The signal should be strong and sharp If it is not switch to a plate type that is suitable for the imaging system or adjust the imaging to match the plate type This involves the adjustment of the Z axis QuantiGene ViewRNA Plate Based Assay User Manual Appendix Liquid Handling Recommendations Appendix I Liquid Handling Recommendations Fluidic Handling Pipetting Stations Systems 4 Platemate 2x3 Biomek FX or NX Velocity11 Bravo Plate Washers BioTek ELx405 Select Pipetting Station and Plate Washer Combination BioTek EL406 Minimum 96 or 384 channel pipette head Requirements go uL 5 pipette capacity 30 uL is acceptable but will result in increased processing time Tips can be replaced or washed between dispensings Setup The goal of these recommendations is to minimize cell loss Recommendations Start with a dispense a
37. point the plate is stable at room temperature for several hours Wash Buffer Vol Per Well Residual Vol uL uL 1X PBS Vol uL 96 well plate 30 150 150 384 well plate 15 80 80 QuantiGene ViewRNA Plate Based Assay User Manual 15 Bu sseo9o d uojeg 10 pejeuiojny e Jnpe9o q Aessy Assay Procedure Automated or Batch Processing Section IV Assay Procedures for Automated or Batch Processing Residual Volume Method 16 To perform hybridizations continued Step Action 10 Counterstain nuclei with DAPI solution WARNING DAPI is a possible mutagen Avoid contact with skin and mucous membranes IMPORTANT Protect from light a Prepare DAPI Working Solution by diluting DAPI Stock Solution 10 mg mL 1 5 000 in 1X PBS and briefly vortexing to mix well For example 1 uL DAPI stock in 5 mL of 1X PBS Scale reagents as appropriate see below Vol uL Per Well Vol uL Per Well Component 384 Well Plate 96 Well Plate 1X PBS 14 997 29 994 10 mg mL DAPI 0 003 0 006 Total Volume 15 30 e 9 pp The plate is ready for imaging Alternatively it can be stored at 4 C for several Aspirate 1X PBS and dispense DAPI Working Solution Incubate at room temperature for 1 minute Aspirate the DAPI Working Solution and wash once with 1X PBS Aspirate the 1X PBS and dispense fresh 1X PBS days Seal the plate with an adhesive seal when storing at 4 C N
38. r dispenser BioTek ELx406 QuantiGene ViewRNA Plate Based Assay User Manual s eui9je y pounboy Experimental Design and Assay Optimization Section IIl Recommendations for Experimental Design and Assay Optimization Section Ill Recommendations for Experimental Design and Assay Optimization Overview Selecting the Appropriate Plate type Replicates Assessing Background for New Projects In this section we provide recommendations for experimental design and optimizing assay conditions Firm adherence of cells to the plate is essential for a successful assay Selection of the assay plate should be based on the following criteria Strong Cell Attachment Better adherence to the plate means that fewer cells will be lost during the processing resulting in better assay precision The goal should be 80 9596 confluence from start to finish of the assay procedures Plastic bottomed cell culture plates are typically better for cell attachment than glass bottomed plates However plastic bottomed plates tend to exhibit higher 2 4X backgrounds Glass bottomed plates typically have superior optical qualities and lower background They can be coated with extracellular matrices such as poly D lysine MatriGel or collagen to enhance cell attachment Imaging System Compatibility Imaging platforms often have limitations on plate types compatible with their systems Please ensure the plates used are compatible with your
39. recommended volumes of any hybridization solution for your sample type Use residual volume method or leave recommended volume after each wash step This will minimize the chance of a well drying out completely Non uniform aspiration dispensing of the multi channel pipettor Correct and verify uniform volume for aspiration and dispense of all pipet tips Insufficient mixing of reagents Pre warm hybridization buffers to re dissolve any precipitates before use Keep PreHyb Buffer at 40 C at all times before adding to the plate Briefly vortex all working hybridization solutions to mix well before use Located outside depth of field of objective lens Adjust focus avoid using non uniform and thick plastic plates High Background Troubleshooting high background on plates on Entire Plates Probable Cause Recommended Actions Samples were allowed to dry Once the in situ part of the assay is started do not allow the cells to dry at any stage Do not use less than the recommended volumes of any hybridization solution for your sample type Leaving recommended residual volume after each wash step will minimize the chance of a well drying completely Insufficient washing Make sure you wash according to the procedure High autofluorescence from the plates or extracellular matrices Select plates or extracellular matrix that exhibit no or low autofluorescence Test backgroun
40. rotease Stop Buffer plus 3 25 mL 1X PBS is sufficient for one 96 well plate 8 Prepare Working Hyb A Buffer by diluting stock Hyb A Buffer prewarmed to 40 C 1 1 with Working Protease Stop Buffer and then vortexing briefly to mix WARNING Hyb A Buffer contains formamide a teratogen irritant and possible carcinogen Avoid contact with skin and mucous membranes 8 mL is sufficient for one 96 well plate 9 Prepare Working Probe Set Mixture by diluting TYPE 1 and TYPE 2 Probe Sets 1 100 with Working Hyb A Buffer and then vortexing briefly to mix 8 mL is sufficient for one 96 well plate Vol uL Per Well Component 96 well plate Working Hyb A prewarmed 58 8 TYPE 1 Probe Set 0 6 TYPE 2 Probe Set 0 6 Total volume 60 10 Prepare Working Storage Buffer by diluting stock Storage Buffer 1 1 with nuclease free water and then vortexing briefly to mix 8 mL is sufficient for one 96 well plate 11 Prepare Working PreHyb Buffer by diluting stock PreHyb Buffer prewarmed to 40 C 1 1 with nuclease free water and then vortexing briefly to mix Prepare enough for the preparation of Working Hyb B and C Buffers 13 mL is sufficient for one 96 well plate QuantiGene ViewRNA Plate Based Assay User Manual 29 Appendix V Alternative Assay Procedure Manual Processing 30 To prepare reagents for alternative processing procedure continued Step Action 12
41. say Procedure Automated or Batch Processing Section IV Assay Procedures for Automated or Batch Processing Residual Volume Method Important Procedural Notes 10 Preparing Reagents All quantities of buffers used in these procedures take into account the presence of this residual volume If you must increase the residual volume to further minimize cell disruption adjust the quantities of the assay reagents proportionally so that they match the residual volume Note f making this procedural modification additional assay reagents may need to be purchased IMPORTANT If you are working in 96 well formats and are using manual pipetting please refer to Appendix V Alternative Assay Procedure Manual Processing on page 28 for a procedure that does not utilize residual volumes Hybridization reactions must be carried out at 40 1 C Verify and monitor oven temperature using the QuantiGene Incubator Temperature Validation Kit Protect samples from light during the Label Probe hybridization and all subsequent steps Before opening reagents supplied in microfuge tubes briefly centrifuge to collect contents at the bottom of the tube All Hyb Buffers stored at 4 C must be prewarmed to 40 C for 30 minutes to redissolve any precipitates If necessary Probe Set PreAmp Amp LP reactions can be left in wells at room temperature for 30 minutes before placing at 40 C If necessary Probe Set PreAmp Amp LP reac
42. sing a bright field microscope If the cell confluence is greater than 5096 proceed with the assay Lowest background inside and outside cells in the negative control wells low number of dots less than 1 strong dot 10 cells and low even background Highest signal background ratio of dots inside the cell Best assay precision between replicate wells and plates for cell number low background and signal background ratio uoneziundo Aessy pue uBiseg jejueuiedx3 Section IIl Recommendations for Experimental Design and Assay Optimization Optimizing Duplex To detect two RNAs in a multiplex assay you must use one TYPE 1 Probe Set and one Assay Formats TYPE 2 Probe Set IMPORTANT Never combine two TYPE 1 Probe Sets or two TYPE 2 Probe Sets in a multiplex assay Note TYPE 1 Probe Sets hybridize to the PreAMP AMP LP 1 signal amplification system and can be used to visualize single copy RNAs Note TYPE 2 Probe Sets hybridize to PreAmp Amp LP 2 and can be used to visualize high abundance 1 000 copies cell RNAs such as 18S ACTB or GAPD 8 QuantiGene ViewRNA Plate Based Assay User Manual Section IV Assay Procedures for Automated or Batch Processing Residual Volume Method Section IV Assay Procedures for Automated or Batch Processing Residual Volume Method Overview The following procedures can be completed in one long day or split between 2 days IMPORTANT These procedures are optimized for usin
43. spirate rate to 5 uL sec Slowly increase the flow rate making sure that cells are not detaching in the process If the liquid handling is setup properly 8096 of the cells will remain following the procedure If you use the recommended residual volume protocol dispense aspirate rates of 20 uL sec could be achieved Seta residual volume to remain after each aspiration 15 ul well for 384 well plates and 30 uL well for 96 well plates This amount of residual liquid is accounted for in the assay procedures and will minimize cell detachment If you must increase the residual volume adjust the quantities of the assay reagents proportionally so that they match the residual volume Orientthe pipet tip toward the left or right side of the well and then capture the image on the opposite side Less cell loss should be observed on the opposite side of the pipet tip location Wash Protocol MPORTANT Settings might require some adjustment since different instruments of the same Settings for BioTek model can behave differently ELx405 Select Herod Setting Number of Cycles Per assay protocol Wash format Plate Soak Shake Per assay protocol Aspirate 384 Well Plate 96 Well Plate Asp height 027 3 429 mm 036 4 572 mm Horizontal asp pos 25 1 143 mm 45 2 057 mm Horiz y asp pos 00 00 00 mm 00 QuantiGene ViewRNA Plate Based Assay User Manual 21 Appendix Liquid Handling Recommendations 22
44. spirate the Protease solution and gently wash 5 times with 1X PBS Aspirate 1X PBS dispense Protease Stop Buffer and pipet up and down twice to mix Aspirate Protease Stop Buffer and dispense fresh Protease Stop Buffer Proceed to hybridization procedure Samples may sit in Protease Stop Buffer for up to 30 minutes Diluted Protease Residual Protease Vol 1X PBS Stop Buffer Per Well Vol uL uL Vol uL Vol uL 96 well plate 30 30 150 30 384 well plate 15 15 80 15 Performing To perform hybridizations Hybridizations Step Action 1 Hybridize target Probe Sets a Aspirate Protease Stop Buffer and dispense Working Probe Set Mixture b Cover plate with plate lid and incubate plate for 3 hours at 40 C 1 C Per Well Residual Vol uL Working Probe Set Vol pL 96 well plate 30 30 384 well plate 15 15 QuantiGene ViewRNA Plate Based Assay User Manual 13 Bu sseo9o d u2 eg 10 pejeuiojny e Jnpe9o q Aessy Assay Procedure Automated or Batch Processing Section IV Assay Procedures for Automated or Batch Processing Residual Volume Method 14 To perform hybridizations continued Step Action 2 Aspirate the Working Probe Set Mixture and wash 3 times with Wash Buffer Include a 30 second soak at the last wash step Per Wel
45. t plate on a clean dry paper towel for 1 2 seconds Wash 3 times with 150 uL well Wash Buffer 6 Hybridize Amplifiers Amps a Invert plate over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel for 1 2 seconds Add 60 uL well Working Amp Mixture b Cover plate with plate lid and incubate at 40 1 C for 60 minutes 7 Invert plate over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel for 1 2 seconds Wash 3 times with 150 uL well Wash Buffer 8 IMPORTANT Protect samples from light during this and all subsequent steps Hybridize Label Probes LPs a Invert plate over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel for 1 2 seconds Add 60 uL well Working Label Probe Mixture b Cover plate with plate lid and incubate at 40 1 C for 60 minutes 9 Wash three times with Wash Buffer a Invert plate over appropriate receptacle and gently expel contents then invert plate on a clean dry paper towel for 1 2 seconds Wash 3 times with 150 uL well Wash Buffer b Remove as above Wash Buffer and add 150 uL well 1X PBS At this point the plate is stable at room temperature for several hours 32 QuantiGene ViewRNA Plate Based Assay User Manual Appendix V Alternative Assay Procedure Manual Processing To perform hybridizations Step Action 10 Cou
46. the volume from 50 uL well to 100 pL well IMPORTANT Do not apply the residual volume technique to this procedure To dehydrate cells for storage or for shipping Step Action 1 To dehydrate cells a Aspirate the final 1X PBS wash then dispense 50 uL well 50 ethanol Incubate at room temperature RT for 2 minutes Aspirate 50 ethanol then dispense 50 uL well 70 ethanol Incubate at RT for 2 minutes Aspirate 70 ethanol then dispense 50 uL well 100 ethanol Aspirate 100 ethanol then dispense 50 uL well fresh 100 ethanol Cover the plate with its lid and seal with parafilm S dB Los ge Store at 20 C Cells can be stored for several weeks Proceed to step 3 when you are ready to run the rest of the assay i Proceed to step 2 if you plan to ship the plate IMPORTANT Glass bottom plates may crack when stored at 20 C In this case store at 4 C for up to 48 hours 2 To prepare for shipping a Aspirate the ethanol b Sealthe plate with an adhesive seal c Ship the plate overnight at 4 C d Immediately upon receipt of the plate add 10096 ethanol and store at 20 C 3 When ready to run the rest of the assay rehydrate the cells a Aspirate 10096 ethanol and dispense 50 uL well 70 ethanol Incubate at RT for 2 minutes Aspirate 70 ethanol and dispense 50 uL well 5096 ethanol Incubate at RT for 2 minutes Aspirate 5096 ethanol and dispense 50 pL well 1X PBS Incubate at RT for 10
47. tions can be left in wells at room temperature for 30 minutes before washing steps PreHyb Buffer must be kept at 40 C at all times during the procedure to prevent precipitation To prepare reagents Step Action 1 Thaw warm the following reagents Prewarm PreHyb Hyb A Hyb B and Hyb C Buffers to 40 C for 30 minutes Thaw Probe Sets PreAmp or PreAmps Amp or Amps and LP or LPs and place on ice IMPORTANT PreHyb Buffer must remain at 40 C during the entire procedure to prevent precipitation 2 Prepare the appropriate volume of the following reagents 1X PBS 8 Formaldehyde Solution in 1X PBS prepare fresh each time WARNING Formaldehyde is a poison and irritant Avoid contact with skin and mucous membranes QuantiGene ViewRNA Plate Based Assay User Manual Section IV Assay Procedures for Automated or Batch Processing Residual Volume Method To prepare reagents continued Step Action 3 Prepare Wash Buffer For example to a 1 000 mL graduated cylinder add the following in this order 800 mL nuclease free water 3mL Wash Buffer Comp 1 5mL Wash Buffer Comp 2 Adjust volume to 1 liter with nuclease free water Scale preparation according to the number of plates to be processed 4 For steps 5 9 prepare an appropriate volume of working reagents Plan for 30 uL well for 96 well and
48. tive with numeric aperture equal to or greater than 0 75 Set exposure time to obtain optimal signal to background ratio Normally DAPI stain requires 10 50X less exposure time than for target RNA 1 and 2 Use the following table to determine the multi bandpass fluorescence microscope filter sets to visualize signals For the best viewing signal set the gray scale or intensity level as follows min 2X above background outside of cell max 4X above background outside of cell Adjust as necessary Peak Peak Nucleic Excitation Emission Acid Fluorophore nm nm Purpose Target RNA 1 Fluorescein 495 519 For detection of 1 copy cell Target RNA 2 Rhodamine 546 573 For detection of greater than 1000 copies cell Nuclear DNA DAPI 358 461 For focusing and cell identification 34 QuantiGene ViewRNA Plate Based Assay User Manual
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