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1. Working Probe Set Solution per Slide Reagent Volume Probe Set Diluent QT prewarmed to 40 C 380 uL QuantiGene ViewRNA TYPE 1 Probe Set 10 uL QuantiGene ViewRNA TYPE 6 Probe Set 10 uL Total volume 400 uL B Remove each slide and flick it to remove 1X PBS Tap the slide on its edge then wipe the backside on a laboratory wipe Place the slides flat face up on the aluminum slide rack and immediately add 400 uL Working Probe Set Solution to each tissue section Place the aluminum slide rack in the humidified incubator and incubate at 40 C for 2 hr Place the slides flat face up on the aluminum slide rack and immediately add 400 uL of the Decant the 1X PBS refill with 200 mL of fresh 1X PBS and rinse by moving slide rack up and Transfer the 496 formaldehyde solution to a 200 mL capacity container keep for later use Using the table below as a guide prepare the Working Probe Set Solutions by diluting the Chapter 3 QuantiGene ViewRNA ISH Tissue Assay Procedure 17 Step Action Step 9 Wash Slides A Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer B After incubation decant the Working Probe Set Solution from the slides and insert them 10 min into the slide rack C Incubate the slides in Wash Buffer at RT for 2 min with frequent agitation D Decant the Wash Buffer refill with 200 mL fresh Wash Buffer and incubate the slides at RT for 2 min with frequent agitation Repeat t
2. Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer After incubation decant the Working Label Probe 1 AP Solution from the slides and insert them into the slide rack Incubate the slides in Wash Buffer at RT for 3 min with frequent agitation Decant the Wash Buffer refill with 200 mL fresh Wash Buffer and incubate the slides at RT for 3 min with frequent agitation Repeat this step one more time for a total of 3 washes Step 23 Apply Fast Red Substrate 1 hr Remove each slide and flick it to remove the Wash Buffer Tap the slide on its edge then wipe the backside on a laboratory wipe Place slides flat face up on the lab bench Immediately add 400 uL of the AP Enhancer Solution to each tissue section pipet directly from bottle and incubate at RT for 5 min while preparing the Fast Red Substrate Prepare the Fast Red Substrate in a 15 ml conical tube add 5 ml of Naphthol Buffer and one Fast Red Tablet Vortex at high speed to completely dissolve the tablet Decant the AP Enhancer Solution by flicking Tap the slide on its edge then wipe the backside on a laboratory wipe Place the slide on the aluminum slide rack and immediately add 400 uL of Fast Red Substrate onto each tissue section Place the aluminum slide rack in the humidified incubator and incubate at 40 C for 30 min Insert an empty slide rack into a clear staining dish containing 200 mL of 1X PBS After incubation decan
3. iv QuantiGene ViewRNA ISH Tissue Assay User Manual Appendix A Assay Optimization Procedures 000 kk kk KK eee eens 29 About the Optimization and Typical Results 2 2 kk kk kk KK RR KK RR KK KK eee 29 Optimization Procedure Overview s 4 xis Sea dala eee eee 29 Optimization Experimental Design Layout kk kk kk kK KK KK KK K KI KIR eee ee 29 Important Procedural Notes and Guidelines 0 0 0 0 000 RR KIRR KI KIR KK 30 Essential Keys for a Successful Assay Jk kk kk kk kk kK KK K KK KK KK ee 30 Sample Preparation and Target Probe Hybridization kk kK KK KK K K KK K K 30 Procedure tegiga EC TETTE LTD 30 Appendix B Assay Optimization Lookup Table 2225922 KK KK KK RR RR KK RR RES 35 Appendix C Templates for Drawing the Hydrophobic Barrier 2 37 Introduction About This Manual This manual provides complete instructions for performing the QuantiGene ViewRNA ISH Tissue Assay for visualization of 2 target RNAs in formalin fixed paraffin embedded FFPE samples prepared in accordance with the guidelines provided This manual provides assay procedures that utilize a dry oven and humidified incubator Related User Documents See the following documents for assay instructions that utilize additional sample types and equipment Table 1 1 Related Manuals for Other Sample Types and Equipment Document Format Sample Type Equipment QuantiGene ViewRNA ISH Tissue 2 Plex FFPE
4. Pour 200 mL of 9596 ethanol into a clear staining dish Lift the slide rack from the Histo Clear and submerge into the clear staining dish containing 95 ethanol and incubate for 1 min with frequent agitation Decant the 9596 ethanol refill with 200 mL of fresh 9596 ethanol and incubate for 1 min with frequent agitation Remove the slides from the slide rack and place them face up on a paper towel to air dry for 5 min at RT Discard the 9596 ethanol If using xylene A Pour 200 mL of xylene in a fume hood into green clearing agent dish B Load the slides into the slide rack C Transfer the slide rack to the green clearing dish containing 200 mL xylene D Incubate in a fume hood at RT for 10 min with frequent agitation E Liftthe slide rack from the xylene and submerge into the clear staining dish containing 200 mL 1X PBS and incubate for 1 min with frequent agitation F Decant the 1X PBS refill with 200 mL fresh 1X PBS and incubate for 1 min with frequent agitation G Remove the slides from the slide rack and place them face up on a paper towel to air dry for 20 min at RT Discard the 1X PBS Step 5 Draw Hydrophobic A Dab the hydrophobic pen on a paper towel several times before use to ensure proper flow Barrier of the hydrophobic solution B Tocreate a hydrophobic barrier place the slide over the template image below tissue 1hr sections should fall inside blue rectangle and lightly trace the thick blue rectangle
5. 1X PBS 95 Ethanol Wash Buffer and Storage Buffer Rinse staining dishes in between steps with ddH O Typical processing times included in the assay procedure assume that preparation for the following step is being done during the incubation periods Essential Keys for a Successful Assay Prepare samples following Tissue Preparation Guidelines on page 9 Organize the preparation of the assay before you start o Verify that all materials and equipment are available u Be mindful of the incubation times temperatures there are small tolerances o Double check all reagent calculations concentration of reagents is critical Employ good washing techniques Frequently this washing is performed too gently Adequate washing is important for consistent low backgrounds Verify and validate temperatures for all equipment using the QuantiGene View Temperature Validation Kit DO NOT let tissues dry out where indicated in the procedure D Incorporate controls both positive and negative so that ambiguous results can be interpreted See Experimental Design Guidelines on page 9 Refer to the Quick Reference Guide to quickly get an overview of the assay workflow Once you become familiar with the procedures you can rely on this quick guide and a Reagent Preparation Guide for running the assay 14 QuantiGene ViewRNA ISH Tissue Assay User Manual Part 1 Sample Preparation and Target Probe Hybridization Part 1 Procedure Step Action Ste
6. Prewarm 40 mL of 1X PBS and Probe Set Diluent QT to 40 1 C l Thaw Probe Set s Place on ice until use Step 3 Fix Slides A Ina fume hood pour 200 mL of 10 formaldehyde into clear staining dish B Insert slides into an empty slide rack and submerge into a clear staining dish containing 1 hr 5 min 1096 formaldehyde Incubate for 1 hour at room temperature RT in a fume hood C Remove the slide rack from the 1096 formaldehyde and submerge it into a clear staining dish containing 200 mL of 1X PBS Incubate for 1 min with frequent agitation D Decantthe 1X PBS refill with 200 mL of fresh 1X PBS and incubate for 1 min with frequent agitation E Remove each slide and flick it to remove the 1X PBS Tap the slide on its edge then wipe the backside on a laboratory wipe Place the slides face up on a paper towel to air dry Make sure the slides are completely dry before going to the next step F Set dry oven to 80 1 C 32 QuantiGene ViewRNA ISH Tissue Assay User Manual Step Action 30 min Step 4 Deparaffinization If using Histo Clear A C G Pour 200 mL of Histo Clear into a green clearing agent dish Insert the slides into an empty slide rack and bake the slides in the dry oven at 80 C for 3 min The paraffin should melt as soon as the slides are in the dry oven After baking insert the slide rack into dish containing Histo Clear and incubate at RT for 10 min with frequent agitation
7. 2 to 4 times with the Hydrophobic Barrier Pen to ensure a solid seal Allow for barrier to dry at RT for 20 30 min Appendix A Assay Optimization Procedures 33 Step Action Step 6 Tissue Pretreatment 25 min H Bring 500 mL of 1X Pretreatment Solution to boil 100 C in a 1 L beaker tightly covered with aluminum foil on a hot plate When boiling is reached use a water proof probe thermometer to measure and maintain the boiling temperature at 95 100 C B Set slide 1 aside on the lab bench Load slides 9 and 10 into the slide rack Using a pair of forceps submerge the slide rack into the boiling 1X Pretreatment Solution Cover the glass beaker with aluminum foil and incubate for 10 min at 95 100 C At the end of 10 min use forceps to add slides 5 6 7 and 8 into the boiling 1X Pretreatment Solution Re cover the glass beaker with aluminum foil and incubate for 5 min At the end of 5 min use forceps to add slides 2 3 and 4 into the boiling 1X Pretreatment Solution Re cover the glass beaker with aluminum foil and incubate for 5 min Using a pair of forceps remove the slide rack loaded with slides and submerge it into a clear staining dish containing 200 mL ddH O Incubate for 1 min with frequent agitation Decant the ddH O and refill the clear staining dish with fresh ddH O Incubate for 1 min with frequent agitation Transfer the slide rack to a clear staining dish containing 1X PBS
8. IMPORTANT From this point forward do not let the tissue sections dry out 34 QuantiGene ViewRNA ISH Tissue Assay User Manual Step Action 50 min Step 7 Protease Digestion and Fixation Set the humidified incubator to 40 1 C and verify bottom tray is filled with ddH O Using the table below as a guide prepare the Working Protease Solution by diluting the Protease QF 1 100 in prewarmed 1X PBS Scale reagents according to the number of assays to be run Include one slide volume overage Working Protease Solution per Slide Reagent Volume Protease QF 4uL 1X PBS prewarmed to 40 C 396 uL Total volume 400 uL n z 2 F O o m Leave slide 1 on the lab bench as it is excluded from this step Remove slides 4 and 8 and flick off excess 1X PBS Tap the slides on their edges then wipe the backside on a laboratory wipe Leave remaining slides in 1X PBS until appropriate incubation time Place the slides face up on an aluminum slide rack and add 400 uL of the Working Protease Solution onto the tissue section Carefully move the aluminum slide rack in the humidified incubator and incubate for 20 min at 40 C After 19 min remove slides 3 6 7 and 10 from the clear staining dish and flick off excess 1X PBS Tap the slides on their edges then wipe the backside on a laboratory wipe Place the slides face up on an aluminum slide rack and add 400 uL of the Working P
9. Probe Set Hybridization day 1 Part 2 Signal Amplification and Detection day 2 We do not recommend stopping the procedure at any other point in the assay The two conditions to be optimized tissue pretreatment boiling time and protease digestion time are included in Part 1 Sample Preparation An example of typical results for both brightfield and fluorescence detection can be found in the QuantiGene ViewRNA ISH Tissue Assay Supplemental Reference Guide Optimization Procedure Overview You will need to prepare ten 5 1 um thick FFPE tissue sections from a block or blocks which were prepared in the same way fixation time section thickness and tissue type as the FFPE tissue of your interest Each slide will be treated with a different set of conditions as described in Table A 1 With the exception of Slide 7 hybridize every slide with medium expression housekeeping genes for example ACTB and GAPD These control targets should have consistent homogenous expression in your samples Once an optimal assay condition is determined for your sample type apply those conditions to your targets of interest Optimization Experimental Design Layout Table A 1 Optimization Experiment Setup Protease Incubation Pretreatment Boiling Time min Time min 0 5 10 20 0 Slide 1 with probe 10 Slide 2 Slide 5 Slide 9 with Probe with probe with probe 20 Slide 3 Slide 6 Slide 10 with probe with probe with probe Slide 7 wit
10. Scientific Tissue Tek Vertical 24 Slide Rack American Master LWSRA24 Tech Scientific Aluminum slide rack 3 VWR 100493380 Double distilled water ddH 0 MLS major laboratory supplier 95 Ethanol VWR 89015 512 10X PBS pH 7 2 7 4 Bio Rad 161 0780 Laboratories or Invitrogen 700134 032 Gill s Hematoxylin American Master HXGHE1LT Tech Scientific Histo Clear or National Diagnostics HS 200 xylene Sigma 247642 37 Formaldehyde Fisher Scientific F79 1 27 30 Ammonium Hydroxide VWR JT9726 5 6 QuantiGene ViewRNA ISH Tissue Assay User Manual Table 2 QuantiGene ViewRNA Tissue Assay Materials and Equipment Continued maintaining 80 C Material Source Part Number Hydrophobic Barrier Pen Affymetrix or QVCO0500 Vector Laboratories H4000 Ultramount or DAKO 1964 Advantage Mounting Media Innovex NB300 Cover Glass 24 mm x 55 mm VWR or 48382 138 Affymetrix QVC0501 Optional DAPI Invitrogen D3571 Equipment Dry incubator or oven utilizing horizontal airflow and capable of Affymetrix QS0704 QS0700 QS0701 120V or QS0714 QS0710 QS0711 220V Temperature humidity meter Fisher Scientific 11 661 19 Tissue culture incubator 40 C without CO and greater than 8596 Napco 51201082 humidity VWR 9150860 QuantiGene View Temperature Validation Kit Affymetrix QV0523 Water proof remote probe thermometers validated for 95 100 C VWR 46610 024 1000 mL Glass Beaker MLS Pipettes P20 P200 P
11. Slides 5 min n Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer After incubation decant the Fast Blue Substrate from the slides and insert them into the slide rack Incubate the slides in Wash Buffer at RT for 2 min with frequent agitation Decant the Wash Buffer refill with 200 mL fresh Wash Buffer and incubate the slides at RT for 2 min with frequent agitation 20 QuantiGene ViewRNA ISH Tissue Assay User Manual Step Action Step 21 Label Probe 1 AP Hybridization 20 min Briefly vortex and spin down Label Probe 1 AP before using Using the table below as a guide prepare Working Label Probe 1 AP Solution by diluting 1 1000 in prewarmed Label Probe Diluent QF and briefly vortexing to mix Scale reagents according to the number of assays to be run Include one slide volume overage Working Label Probe 1 AP Solution Per Slide Reagent Volume Label Probe Diluent QF prewarmed to 40 C 399 6 uL Label Probe 1 AP 0 4 uL Total volume 400 uL Remove each slide and flick it to remove the Wash Buffer Tap the slide on its edge then wipe the backside on a laboratory wipe Place slides flat face up on the aluminum slide rack and immediately add 400 uL of Working Label Probe 1 AP solution directly to each tissue section Place the aluminum slide rack in the humidified incubator and incubate at 40 C for 15 min Step 22 Wash Slides 15 min
12. Tissue Sections ThermoBrite denaturation Assay User Manual hybridization system QuantiGene ViewRNA ISH Tissue 2 Plex OCT Embedded ThermoBrite denaturation Assay User Manual Frozen Tissue Sections hybridization system QuantiGene ViewRNA ISH Tissue 2 Plex OCT Embedded Dry Oven and Humidified Incubator Assay User Manual Frozen Tissue Sections See the QuantiGene ViewRNA ISH Tissue Assay Supplemental Reference Guide for the following information u Guidelines for data interpretation a Typical results for assay optimization Troubleshooting images examples of backgrounds Assay specificity Guidance for fluorescence and brightfield imaging Procedure for remounting slides Procedure for RNase treatment of samples Procedure for DNase treatment of samples Assay Overview In situ hybridization ISH techniques are used to visualize DNA or RNAs within cells in tissue However the in situ analysis of RNA has always been limited by low sensitivity and difficult probe synthesis The QuantiGene ViewRNA ISH Tissue Assay based on branched DNA signal amplification technology has the sensitivity and robustness to measure two different genes at single copy sensitivity The assay design is illustrated and explained in Figure 1 1 2 QuantiGene ViewRNA ISH Tissue Assay User Manual How it Works Figure 1 1 QuantiGene ViewRNA ISH Tissue Assay Workflow Target specific Probe Sets PreAmplifier Mix Sample Target Signal amplifi
13. in Assay Optimization Procedures on page 29 to identify the optimal pretreatment boiling time and protease digestion time to un mask the mRNA Applying the optimal condition will not only provide a favorable environment for the QuantiGene ViewRNA Probe Set to bind to the target mRNA but will also have an impact on the final chromogenic staining quality and tissue morphology Once identified the same optimal condition can be used for different Probe Sets Tissues fixed in 4 PFA may not have the same optimization conditions as tissues fixed in 10 NBF Therefore if working with mixed set of samples some prepared using 4 PFA and other prepared using 10 NBF separate optimization assays should be run based on the fixative used If you are limited on samples for optimization use the Assay Optimization Lookup Table on page 35 for a general guideline If you do not obtain the desired results we recommend performing the assay optimization procedure Probe Set Considerations QuantiGene ViewRNA Probe Sets TYPE specific are designed to a specified region of a target RNA TYPE 1 Probe Set s will be visualized using a Fast Red substrate alkaline phosphatase breaks down the substrate to form a dark red precipitate wherever the target RNA molecule is localized This vivid red color is easily visible to the eyes in brightfield TYPE 6 Probe Set s will be visualized using a Fast Blue substrate alkaline phosphatase breaks down the substrate to form a dark b
14. laboratory wipe Place slides flat face up on the aluminum slide rack 20 min and immediately add 400 uL of Amplifier Mix QT directly to each tissue section C Place the aluminum slide rack in the humidified incubator and incubate at 40 C for 15 min Step 16 Wash Slides A Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer B After incubation decant the Amplifier Mix QT from the slides and insert them into the slide 10 min rack C Incubate the slides in Wash Buffer at RT for 2 min with frequent agitation D Decant the Wash Buffer refill with 200 mL fresh Wash Buffer and incubate the slides at RT for 2 min with frequent agitation Repeat this step one more time for a total of 3 washes Chapter 3 QuantiGene ViewRNA ISH Tissue Assay Procedure 19 Step Action Step 17 Label Probe 6 AP Hybridization 20 min Briefly vortex and spin down Label Probe 6 AP before using Using the table below as a guide prepare Working Label Probe 6 AP Solution by diluting 1 1000 in prewarmed Label Probe Diluent QF and briefly vortexing to mix Scale reagents according to the number of assays to be run Include one slide volume overage Working Label Probe 6 AP Solution Per Slide Reagent Volume Label Probe Diluent QF prewarmed to 40 C 399 6 uL Label Probe 6 AP 0 4 uL Total volume 400 uL C Remove each slide and flick it to remove the Wash Buffer Tap the slide on its
15. min Transfer the 496 formaldehyde solution to a 200 mL capacity container keep for later use Proceed to Step 8 Target Probe Set Hybridization on page 16 to continue the assay procedure Assay Optimization Lookup Table The table below contains a list of the tissues that we prepared according to the guidelines outlined in this manual using 10 NBF Tissue Preparation Guidelines on page 9 and optimized using the recommended pretreatment assay optimization procedure You can use this table as a guideline to minimize the number of conditions if you do not have sufficient slides to perform the recommended pretreatment optimization procedure These guidelines are specific to tissues prepared using 10 NBF and may not be applicable to tissues prepared using 4 PFA You must also include a negative control slide without Probe Set to ensure no background is visible and well defined cell morphology is achieved If your tissue type is not listed in the Table B 1 and you have only a few slides available for optimization Table B 2 lists recommended boiling and protease incubation times Table B 1 Assay Optimization Lookup Table Tissue Information Optimal Conditions Range for Tolerance Conditions Species Type Boiling at 95 100 C Protease at Boiling Protease min 40 1 C min min Human Brain 20 10 10 10 10 20 Breast 20 15 25 15 30 20 25 20 Colon 5 20 5 10 Kidney 20 10 L
16. 1000 MLS Fume hood for dispensing formaldehyde and ammonium hydroxide MLS Isotemp Hot Plates Fisher Scientific 11 300 49SHP 120V 11 302 49SHP 230V Table top microtube centrifuge MLS Water Bath capable of maintaining 40 1 C MLS Microscope and imaging equipment Required for fluorescence detection See QuantiGene ViewRNA ISH Tissue Assay Imaging Options on page 7 Chapter 1 Introduction 7 Microscopy and Imaging Equipment Guidelines for QuantiGene ViewRNA ISH Tissue Assay A unique benefit of the Affymetrix QuantiGene ViewRNA ISH Tissue Assay is that the stains used to label RNA can be visualized using both brightfield and fluorescence microscopy The stain colors are described in Table 1 5 Table 1 5 QuantiGene ViewRNA ISH Tissue Assay Stains Stain Detection Molecule Staining Reagent Stain Color Fluorescence RNA 1 using TYPE 1 probe Fast Red Red dot Red dot RNA 2 using TYPE 6 probe Fast Blue Dark blue dot Far red dot Nuclear stain Hemotoxylin DAPI Light blue Dark blue Table 2 QuantiGene ViewRNA ISH Tissue Assay Imaging Options Olympus BX series a Zeiss Axio Lab Scope Imager Or equivalent Viewing and Microscope Type Recommended Microscope Required Optics Recommended Filter Digital System Capturing Options Brightfield Standard brightfield Leica DM series Requires 20 and 40x Requires neutral density viewi
17. 1X Pretreatment Solution to boil 100 C in a 1 L beaker tightly covered with aluminum foil on a hot plate When boiling is reached use a water proof probe thermometer to measure and maintain the boiling temperature at 95 100 C n Load the slides into the slide rack Using a pair of forceps submerge the slide rack into the boiling 1X Pretreatment Solution Cover the glass beaker with aluminum foil and incubate at 95 100 C for the optimal time as determined in Assay Optimization Procedures on page 29 Using a pair of forceps remove the slide rack and submerge it into a clear staining dish containing 200 mL ddH O Incubate for 1 min with frequent agitation Decant the ddH O and refill the clear staining dish with fresh ddH O Incubate for 1 min with frequent agitation Transfer the slide rack to a clear staining dish containing 1X PBS IMPORTANT From this point forward do not let the tissue sections dry out 16 QuantiGene ViewRNA ISH Tissue Assay User Manual Step Action Step 7 Protease Digestion and Fixation 30 50 min depending on optimized time A Set the humidified incubator to 40 1 C and verify bottom tray is filled with ddH O Using the table below as a guide prepare the Working Protease Solution by diluting the Protease QF 1 100 in prewarmed 1X PBS Scale reagents according to the number of assays to be run Include one slide volume overage Working Protease
18. KG Affymetrix User Manual QuantiGene ViewRNA ISH Tissue Assay Format 2 Plex Sample FFPE Tissue Sections Equipment Dry Oven and Humidified Incubator P N 18959 Rev A 120414 For research use only Not for use in diagnostic procedures Trademarks 2 Affymetrix XC and QuantiGene are trademarks of Affymetrix Inc All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Citing QuantiGene ViewRNA in Publications When describing a procedure for publication using this product please refer to it as the QuantiGene ViewRNA ISH Tissue assay Disclaimer Affymetrix Inc reserves the right to change its products and services at any time to incorporate technological de
19. Solution per Slide Reagent Volume Protease QF 4 uL 1X PBS prewarmed to 40 C 396 uL Total volume 400 uL C Remove each slide and flick it to remove excess 1X PBS Tap the slide on its edge then wipe the backside on a laboratory wipe Working Protease Solution onto the tissue section Place the slide rack into the humidified incubator and incubate at 40 C for the optimal time as determined in the Assay Optimization Procedures on page 29 Pour 200 mL of 1X PBS into a clear staining dish and insert an empty rack into it After incubation decant the Working Protease Solution from the slides insert them into the slide rack and rinse by moving up and down for 1 min down for 1 min Transfer the slide rack into the clear staining dish containing 496 formaldehyde and incubate under a fume hood for 5 min at RT Decant the clear staining dish containing 1X PBS and refill with 200 mL of fresh 1X PBS K Transfer the slide rack from the 496 formaldehyde solution to the clear staining dish containing 1X PBS and incubate for 1 min with frequent agitation Decant the 1X PBS refill with 200 mL of fresh 1X PBS and rinse by moving slide rack up and down for 1 min Step 8 Target Probe Set Hybridization 2 hr 10 min QuantiGene ViewRNA Probe Set s 1 40 in prewarmed Probe Set Diluent QT and briefly vortex Scale reagents according to the number of assays to be run Include one slide volume overage
20. ansfer slide rack to clear staining dish containing Wash Buffer and incubate for 2 min with frequent agitation 5 min B Decant Wash Buffer refill with 200 mL fresh Wash Buffer and incubate for 2 min with frequent agitation Step 13 PreAmp A Set the humidified incubator to 40 1 C and verify the bottom tray is filled with ddH O Hybridization B Swirl PreAmplifier Mix QT bottle briefly to mix the solution a C Remove each slide and flick it to remove the Wash Buffer Tap the slide on its edge then 30 min wipe the backside on a laboratory wipe Place slides flat face up on the aluminum slide rack and immediately add 400 uL of PreAmplifier Mix QT directly to each tissue section D Place the aluminum slide rack in the humidified incubator and incubate at 40 C for 25 min Step 14 Wash Slides A Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer B After incubation decant the PreAmplifier Mix QT from the slides and insert them into the 10 min slide rack C Incubate the slides in Wash Buffer at RT for 2 min with frequent agitation D Decant the Wash Buffer refill with 200 mL fresh Wash Buffer and incubate the slides at RT for 2 min with frequent agitation Repeat this step one more time for a total of 3 washes Step 15 Amp A Swirl Amplifier Mix QT bottle briefly to mix the solution Hybridization B Remove each slide and flick it to remove the Wash Buffer Tap the slide on its edge then wipe the backside on a
21. cation Visualization preparation hybridization and detection TYPE 1 TYPE e gt So TYPE 1 o pm Probe Set 72 e Ov BESE j Amplifier Mix TYPE Visualize TYPE 6 72 f TYPE 1 using brightfield Probe Set j or fluorescence microscope v IP AP oD Incubate Het Probet Fix cells and permeabilize Label Probe amp AP Fast Red Substrate 2 r Fast Blue Substrate a RNA 1 Sequential hybridizations RNA 2 Sample Preparation FFPE or frozen tissue sections are fixed then permeabilized to allow target accessibility Target Hybridization A target specific Probe Set s hybridizes to the target RNA s Subsequent signal amplification is predicated on specific hybridization of adjacent Probe Set oligonucleotides indicated by II in the image above A typical Probe Set will contain 20 oligonucleotides pairs 77 for simplicity only two are shown TYPE 1 Probe Sets will generate red dot patterns while TYPE 6 Probe Sets will generate blue dot patterns Separate but compatible signal amplification systems enable the multiplex assay Signal Amplification Signal Amplification is achieved via a series of sequential hybridization steps The PreAmplifier hybridizes to each pair of bound Probe Set oligonucleotides then multiple Amplifier hybridize to each PreAmplifier Next multiple Label Probe oligonucleotides conjugated to alkaline phosphate hybridize to each Amplifier Each Amp
22. cent mode Diffused Signals Chapter 4 Troubleshooting 25 Table 4 2 Troubleshooting diffused signals Probable Cause Recommended Action Tissue dries up during processing Keep tissue section moist starting from the pretreatment boiling step by a Adding respective reagents immediately after decanting solution from slides a Limiting tissue exposure to air for too long before adding hybridization reagents a Adding all working reagents onto the slides before moving them to the 40 C humidified incubator Insufficient washing in 1X PBS Make sure tissues are washed in 1X PBS twice after protease digestion and twice again after subsequent fixing in 496 formaldehyde Fast Red substrate not freshly prepared Prepare Fast Red substrate immediately before use Slides are not dried before mounting Ensure that slide sections are completely dry before mounting about 20 min Mounting solution contained alcohol Use DAKO Ultramount or Innovex Advantage mounting media to mount your tissue Avoid any mounting solution containing alcohol Poor Cell Morphology Table 4 3 Troubleshooting poor cell morphology Probable Cause Recommended Action Incorrect pretreatment conditions See Optimization Experimental Design Layout on page 29 Sample preparation not fixed properly Make sure that freshly dissected tissues are fixed in 10 NBF or 4 PFA for 16 24 hours Section thic
23. de rack and submerge into a clear staining dish containing 1 hr 5 min 1096 formaldehyde Incubate for 1 hour at room temperature RT in a fume hood C Remove the slide rack from the 1096 formaldehyde and submerge it into a clear staining dish containing 200 mL of 1X PBS Incubate for 1 min with frequent agitation D Decant the 1X PBS refill with 200 mL of fresh 1X PBS and incubate for 1 min with frequent agitation E Remove each slide and flick it to remove the 1X PBS Tap the slide on its edge then wipe the backside on a laboratory wipe Place the slides face up on a paper towel to air dry Make sure the slides are completely dry before going to the next step F Set dry oven to 80 1 C Chapter 3 QuantiGene ViewRNA ISH Tissue Assay Procedure 15 Step Action Step 4 Deparaffinization 30 min If using Histo Clear A B C G Pour 200 mL of Histo Clear into a green clearing agent dish Insert the slides into an empty slide rack and bake the slides in the dry oven at 80 C for 3 min The paraffin should melt as soon as the slides are in the dry oven After baking insert the slide rack into dish containing Histo Clear and incubate at RT for 10 min with frequent agitation Pour 200 mL of 9596 ethanol into a clear staining dish Lift the slide rack from the Histo Clear and submerge into the clear staining dish containing 95 ethanol and incubate for 1 min with frequent agitation Decant the 9596 e
24. edge then wipe the backside on a laboratory wipe Place slides flat face up on the aluminum slide rack and immediately add 400 uL of Working Label Probe 6 AP solution directly to each tissue section D Place the aluminum slide rack in the humidified incubator and incubate at 40 C for 15 min Step 18 Wash Slides A Insert an empty slide rack into a clear staining dish containing 200 mL of Wash Buffer B After incubation decant the Working Label Probe 6 AP Solution from the slides and insert 15 min them into the slide rack C Incubate the slides in Wash Buffer at RT for 3 min with frequent agitation D Decant the Wash Buffer refill with 200 mL fresh Wash Buffer and incubate the slides at RT for 3 min with frequent agitation Repeat this step one more time for a total of 3 washes Step 19 Apply Fast Blue A Prepare the Fast Blue Substrate in a 15 mL conical tube add 5 mL of Blue Buffer Add Substrate 105 uL of Blue Reagent 1 vortex add 105 uL of Blue Reagent 2 vortex and add 105 uL Blue Reagent 3 then briefly vortex Store solution away from direct light until use 40 min B Remove each slide and flick it to remove the Wash Buffer Tap the slide on its edge then wipe the backside on a laboratory wipe Place slides flat face up on an aluminum slide rack C Immediately add 400 pL Fast Blue Substrate and incubate in the dark at RT for 30 min NOTE Fast Blue Substrate precipitate inactivates Label Probe 6 AP Step 20 Wash
25. edural steps in a well ventilated area at room temperature unless otherwise noted Discard all reagents in accordance with local state and federal laws 4 QuantiGene ViewRNA ISH Tissue Assay User Manual Required Materials The QuantiGene ViewRNA ISH Tissue Assay is composed of the following 2 modules each sold separately and available in multiple sizes QuantiGene ViewRNA ISH Tissue Assay Kit QuantiGene ViewRNA TYPE and TYPE 6 Probe Sets QuantiGene ViewRNA ISH Tissue Assay Kit QuantiGene ViewRNA ISH Tissue Assay Kits are available in two sizes QVT0010 and QVTO0011 sufficient for 24 or 96 assays respectively Each kit is configured for processing a minimum of 6 or 12 slides respectively per experiment The components of the QuantiGene ViewRNA ISH Tissue Assay Kit and their recommended storage conditions are listed below Refer to the Package Insert for quantities of individual components supplied Kits are shipped in 2 parts based on storage conditions and have a shelf life of 6 months from date of delivery when stored as recommended Table 1 3 Assay Kit Components and Their Storage Conditions Component Description Storage 100X Pretreatment Solution Aqueous buffered solution 2 8 C Protease QF Enzyme in aqueous buffered solution 2 8 C Probe Set Diluent QT Aqueous solution containing formamide detergent and blocker 2 8 C Label Probe Diluent QF Aqu
26. elines may not produce optimal results FFPE Tissue Block Preparation Upon removal of tissue drop 100 mg of tissue into a minimum of 2 mL of fresh 10 Neutral Buffered Formalin NBF for 16 24 hr at room temperature Cut tissue to a maximum of 3 mm thick section to ensure faster diffusion of NBF into tissue To prevent RNA degradation place tissues on dry ice or liquid nitrogen if it is not possible to fix the tissue immediately u Alternatively tissues can be fixed in the same way with 4 paraformaldehyde PFA for 16 24 hr at room temperature Rinse dehydrate and embed in paraffin block u Store FFPE tissue blocks at room temperature FFPE Tissue Slide Preparation u Section FFPE tissue to the thickness of 5 1 um Mount a single section onto one of the following positively charged glass slides n Leica SurgiPath X tra P N 3800200 o Tru Scientific TruBond360 P N 0360W o Mercedes StarFrost Platinum P N MER 7200 u Store sections in a slide box at 20 C until use for up to 6 months avoid freeze thaw a Short term storage or shipping conditions at 4 C is only recommended up to 2 weeks TMA Slide Preparation u Construct TMA block with the following specifications o Core size 1 0 mm diameter or greater o Maximum TMA area 20 mm x 30 mm u Cut TMA sections to a thickness of 5 1 um Mount a single section onto a positively charged glass slide n Leica SurgiPath X tra P N 3800200 o Tru Scientific TruBond360 P N 0360W o Merced
27. eous solution containing detergent 2 8 C PreAmplifier Mix QT DNA in aqueous solution containing formamide and detergent 2 8 C Amplifier Mix QT DNA in aqueous solution containing formamide and detergent 2 8 C Label Probe 6 AP Alkaline phosphatase conjugated oligonucleotide in aqueous 2 8 C buffered solution Blue Buffer Buffer required for preparation for Blue Substrate 2 8 C Blue Reagent 1 Blue precipitating substrate component 1 for the detection of 2 8 C alkaline phosphatase activity Blue Reagent 2 Blue precipitating substrate component 2 for the detection of 2 8 C alkaline phosphatase activity Blue Reagent 3 Blue precipitating substrate component 3 for the detection of 2 8 C alkaline phosphatase activity AP Enhancer Solution Aqueous buffered solution 2 8 C Fast Red tablets Red precipitating substrate for the detection of alkaline 2 8 C phosphatase activity Naphthol Buffer Buffer required for preparation of Red Substrate 2 8 C Label Probe 1 AP Alkaline phosphatase conjugated oligonucleotide in aqueous 2 8 C buffered saline 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 IMPORTANT Do not freeze Chapter 1 Introduction QuantiGene ViewRNA Probe Sets In addition to the QuantiGene ViewRNA ISH Tissue Assay Kit QuantiGene ViewRNA TYPE 1 and TYPE 6 Probe Sets specific to your targets of in
28. es without fixed tissues to determine if the slides are suitable for the assay Decrease Probe Set concentration by diluting target Probe Set 1 50 instead of 1 40 and hybridize for 3 hr at 40 C Insufficient washing Hydrophobic Barrier Falls Off Move the slide rack up and down with frequent agitation Increase wash incubation time by 1 min per wash BB Table 4 6 Troubleshooting hydrophobic barrier problems Probable Cause Recommended Action Incompatible glass slide Use the glass slides from the following recommended vendors a Leica Surgipath X tra P N 3800200 a Tru Scientific TruBond360 P N 0360W Mercedes StartFrost Platinum P N MER 7200 Prevalidate each new batch of slides by running the entire assay including Probe Set on empty slides without fixed tissues to determine if the slides are suitable for the assay Incorrect hydrophobic pen Use Hydrophobic Barrier Pen Affymetrix QVC0500 or Vector Laboratories H4000 Hydrophobic barrier was not dried completely Allow 20 30 min for hydrophobic barrier to dry completely before proceeding to the next step High Background Chapter 4 Troubleshooting 27 Table 4 7 Troubleshooting high background Probable Cause Recommended Action Tissue dries up during hybridization steps Keep tissue section moist starting from the pretreatment boiling step by Adding respective reagents immediately after decanting so
29. es StarFrost Platinum P N MER 7200 Store TMAs in a slide box at 20 C until use for up to 6 months avoid freeze thaw u Short term storage or shipping conditions at 4 C is only recommended up to 2 weeks Experimental Design Guidelines Assay Controls We recommend running positive and negative control slides based on your sample type in every QuantiGene ViewRNA ISH Tissue Assay This will allow you to qualify interpret your results Negative Control This slide undergoes the entire assay procedure and assesses the assay background The negative control can be one of the following 10 QuantiGene ViewRNA ISH Tissue Assay User Manual u Omitting target Probe Set s Using a Probe Set designed to the sense strand of target a Using a target not present in your sample for example the bacterial gene DapB Positive Control This slide undergoes the entire assay procedure using Probe Set s for targets that have a consistently high to medium high homogenous or cell type specific expression in your sample type This control ensures the assay procedure has been run successfully The following are examples of genes to use Housekeeping genes ACTB GAPD or UBC Housekeeping Pan Panel pool the individual housekeeping genes together Replicates We recommend running all assays in duplicate Recommended Assay Optimization When working with a new tissue type we recommend performing the assay optimization procedure as described
30. g the fluorescent microscope then move slide rack into a clear staining dish containing 200 mL DAPI staining solution Incubate the slides for 1 min Decant DAPI staining solution refill with 200 mL fresh ddH O and move the slide rack up and down several times to rinse off DAPI solution Remove the slides from the slide rack and decant the ddH O by flicking Tap the slide on its edge then wipe the backside on a laboratory wipe Place them face up onto a paper towel to air dry Ensure that slide sections are completely dry before mounting about 20 min Step 25 Add Coverslip Dn and Image 20 min If using DAKO Ultramount mounting medium A B C D E Dab the first 2 3 drops of mounting medium onto a paper towel to remove bubbles Add a minimum of 2 drops of DAKO Ultramount mounting medium to tissue section without making any bubbles Use a pipette tip to draw out any air bubbles in the droplets Slowly place the cover glass onto the specimen slide at an angle Make sure the cover glass comes into contact with the mounting medium first before completely releasing the cover glass to overlap with the glass slide After mounting place the slide on its edge on a laboratory wipe to remove excess mounting medium Image the results under a brightfield and or fluorescence microscope Store the mounted slides at 4 C to avoid bubble formation over time If using Innovex Advantage mounting medium A B C P
31. h no probe 40 Slide 4 Slide 8 with probe with probe 30 QuantiGene ViewRNA ISH Tissue Assay User Manual Important Procedural Notes and Guidelines a Procedure assumes running a maximum of 12 slides at a time Do not mix and match kit components from different kit lots Throughout the procedure dedicate one clear staining dish for fixing in formaldehyde we recommend labeling this dish The other two clear staining dishes can be used interchangeably for 1X PBS 95 Ethanol Wash Buffer and Storage Buffer Rinse staining dishes in between steps with ddH O u Typical processing times included in the assay procedure assume that preparation for the following step is being done during the incubation periods Essential Keys for a Successful Assay Prepare samples following the Tissue Preparation Guidelines on page 9 Organize the preparation of the assay before you start u Verify that all materials and equipment are available o Be mindful of the incubation times temperatures there are small tolerances o Double check all reagent calculations concentration of reagents is critical Employ good washing techniques Frequently this washing is performed too gently Adequate washing is important for consistent low backgrounds Verify and validate temperatures for all equipment a DO NOT let tissues dry out where indicated in the procedure BB Incorporate controls both positive and negative so that ambiguou
32. his step one more time for a total of 3 washes Step 10 Stop Point A J slides in a clear staining dish containing 200 mL of Storage Buffer for up to 24 hours at RT 1 min B The following reagent preparations should be stored at RT for use in Part 2 496 formaldehyde 1X PBS Wash Buffer C All other reagent and solution preparations should be discarded D When you are ready to continue the assay proceed to Step 11 Prepare Additional Buffers and Reagents on page 18 18 QuantiGene ViewRNA ISH Tissue Assay User Manual Part 2 Signal Amplification and Detection Part 2 Procedure Step Action Step 11 Prepare A Prepare 1 L of 0 01 ammonium hydroxide in ddH O Additional Buffers and In a fume hood add 0 33 mL 30 ammonium hydroxide to 999 67 mL ddH O and mix well Reagents B Ensure availability of 200 mL Gill s Hematoxylin Pour into a clear staining dish and store at RT away from light until use 10 min C Ifyou plan on using fluorescence detection prepare 200 mL DAPI The final dilution of DAPI should be 3 0 ug mL in 1X PBS Store in the dark at 4 C until use or place on ice D Prewarm PreAmplifier Mix QT Amplifier Mix QT and Label Probe Diluent QF buffers to 40 C E Place Label Probe 1 AP Label Probe 6 AP and Blue reagents on ice F Bring Fast Red Tablets Naphthol Buffer AP Enhancer Solution and Blue Buffer to RT Step 12 Wash Slides A Remove the slides from Storage Buffer tr
33. ion Guidelines 2 2 0 00 0 kk kk KK KK eee 9 Experimental Design Guidelines Ak kk kk eee 9 Probe Set Considerations kk kk kK kk kk kk KK KK KK teen ee 10 Guidelines for Working with Tissue Microarrays TMAS kK KK RR KK KK 11 QuantiGene ViewRNA ISH Tissue Assay Procedure WW ka kk kk kk 13 About the QuantiGene ViewRNA ISH Tissue Assay Procedure 200000 13 Important Procedural Notes and Guidelines 0 0 0 0 0 00000 13 Essential Keys for a Successful Assay Jk a kk kk kk kk eee 13 Part 1 Sample Preparation and Target Probe Hybridization 0000 14 Part 1 Procedute ze er Dee Fg te puce adidid cei Ode ea s Perea ed 14 Part 2 Signal Amplification and Detection kk kk kk kk kk kK KK KK KK KK eee 18 END N aa cts uostri RUD eee at teh E EM he Fea 18 Troubleshooting sce Aw ce aa ee KNEES Au Qur eRe An WA a aa a 23 Contacting Technical SUDDOFE i i ade Boe Ch hb oe ee Ped oe dea obo p PPR 23 Weak or No Signals ce pepe WE W ESS A danay dude a I SS eee qu 23 Diffused Signals 4445 seer nose oot See hh pH pe 25 Poor Cell Morphology 0 kk kk kk kK KK KK eee eee 25 Tissue Detachment from Slide J kk kk kk kK KK KK KK KK KK KK KK KK KK KK KK KI 25 High Non Specific Binding on Glass Slide JWA kk kk kk KK KK KK KK KR KK RR KIRI KK 26 Hydrophobic Barrier Falls Off i ss reams e 26 High Background ausa sa Ghee bods ege Sd eke n awe Soke Ree oo EEE deed 27
34. iver 20 20 10 20 Lung 10 20 Osteoarthritic tissue 20 20 Pancreas 10 10 10 20 5 10 Prostate 10 20 5 10 20 10 10 10 Salivary gland 10 10 5 10 Skin 5 10 Tonsil 10 20 Thyroid 10 20 Rat Kidney 10 20 10 10 20 20 Liver 10 20 Thyroid 10 20 36 QuantiGene ViewRNA ISH Tissue Assay User Manual Table B 1 Assay Optimization Lookup Table Mouse Bone 20 20 Brain 10 10 Heart 10 40 Kidney 20 20 10 20 Liver 20 20 10 20 Lung 10 20 Retina 10 10 Salmon Heart 10 10 Muscle 10 20 Table B 2 Recommended Boiling and Protease Incubation Times for Limited Optimization 5 10 3 10 10 10 20 5 10 5 20 5 10 10 10 20 20 10 5 10 5 20 10 10 7 10 20 20 10 20 20 0 0 Templates for Drawing the Hydrophobic Barrier NOTE To ensure templates print to the correct size make sure that you select none under the page scaling option in the print dialog box 38 QuantiGene ViewRNA ISH Tissue Assay User Manual
35. kness not optimal Make sure tissues are sectioned at 5 1 uim thick Tissue Detachment from Slide Table 4 4 Troubleshooting tissue detachment from slides Probable Cause Recommended Action Insufficient baking of slides a Verify that 30 min baking step was done It may be necessary to increase baking time to 1 hr Incorrect pretreatment conditions Perform full assay optimization procedure to determine optimal boiling time and protease digestion time Improper fixation reagents or concentrations Make sure the correct concentration of NBF was used to fix the slides in the respective steps Temperature of pretreatment condition too high Make sure the temperature is within the tolerance range of 95 100 C For fatty soft tissue such as breast adjust to 95 C Protease treatment is too long or at too high concentration Reduce protease concentration and or incubation time 26 QuantiGene ViewRNA ISH Tissue Assay User Manual High Non Specific Binding on Glass Slide Table 4 5 Troubleshooting non specific binding to slide Probable Cause Recommended Action Incompatible glass slide Use the glass slides from the following recommended vendors a Leica Surgipath X tra P N 3800200 a Tru Scientific TruBond360 P N 0360W Mercedes StarFrost Platinum P N MER 7200 Prevalidate each new batch of slides by running the entire assay including Probe Set s on empty slid
36. lace a 24 mm x 55 mm cover glass horizontally onto a clean flat surface Dab the first 2 3 drops of mounting media onto a paper towel to remove bubbles Add 2 drops of the Innovex Advantage medium directly onto the middle of the cover glass Use a pipette tip to draw out any air bubbles in the droplets Invert the specimen slide and slowly place it onto the mounting medium at an angle Make sure the tissue comes into contact with the mounting medium first before completely letting go of the glass slide to overlap with the cover glass After mounting flip the slide over and place it on its edge on a laboratory wipe to remove excess mounting medium Allow slides to dry at RT for 15 min Do not bake slides to speed up the drying process To prevent bubble formation seal all 4 edges of the cover glass with nail polish Image the results under brightfield and or fluorescence microscope Store slides at RT 22 QuantiGene ViewRNA ISH Tissue Assay User Manual Troubleshooting Contacting Technical Support For technical support contact the appropriate resource provided below based on your geographical location For an updated list of FAQs and product support literature visit our website at www affymetrix com panomics Table 4 6 Technical Support Contacts Location Contact Information North America 1 877 726 6642 option 1 then option 3 pqbhelp affymetrix com Europe 44 1628 552550 techsupport_europe aff
37. lifier branched structure has about 400 binding sites for the Label Probe and each gene typically contains about 20 Amplifiers or 8 000 binding sites Detection The sequential addition of Fast Blue and Fast Red alkaline phosphatase substrates produce precipitate blue and red dots that indicate the presence of the target RNA molecules Target mRNA is visualized using a standard brightfield or fluorescence microscope Performance Highlights Table 1 2 Performance Highlights Chapter 1 Introduction 3 Specification Description Sample types OCT embedded frozen tissue or formalin fixed paraffin embedded FFPE tissue sections Assay area 20 x 30 mm on standard 25 x 75 mm glass slide a FFPE tissue thickness 5 1 um a Fresh frozen thickness 11 x 2 um FFPE tissue microarray TMA Greater than 1 mm diameter and 5 1 um thickness Sensitivity Single RNA molecule per dot Multiplexing Simultaneous detection of 2 target RNAs Detection Chromogenic and fluorescence Nuclear stain Hematoxylin and or DAPI Instrumentation brightfield and or fluorescence microscope or scanner Safety Warnings and Precautions Formaldehyde is a poison and an irritant Avoid contact with skin and mucous membranes Use in a fume hood Ammonium hydroxide is highly volatile Use in a fume hood Xylene is flammable and an irritant Avoid inhalation and contact with skin Use in a fume hood Perform all proc
38. ltiple tissue types within the same TMA block we recommend running an optimization procedure on each individual FFPE tissue type to identify the most favorable pretreatment boiling and protease condition Based on the optimal condition of the tissue morphology signal strength and residual cores you can judge if there is one optimization condition that will be suitable for all the sample types 12 QuantiGene ViewRNA ISH Tissue Assay User Manual QuantiGene ViewRNA ISH Tissue Assay Procedure About the QuantiGene ViewRNA ISH Tissue Assay Procedure The QuantiGene ViewRNA ISH Tissue Assay procedure is broken up into 2 parts that are performed over 2 days Part 1 Sample Preparation and Target Probe Set Hybridization day 1 Part 2 Signal Amplification and Detection day 2 We do not recommend stopping the procedure at any other point in the assay Important Procedural Notes and Guidelines Procedure assumes running a maximum of 12 slides at a time Do not mix and match kit components from different kit lots Before beginning procedure know the pretreatment boiling time and protease digestion time optimized conditions for your sample type If you do not know these optimized conditions refer to Appendix A Assay Optimization Procedures on page 29 Throughout the procedure dedicate one clear staining dish for fixing in formaldehyde we recommend labeling this dish The other two clear staining dishes can be used interchangeably for
39. lue precipitate wherever the target RNA molecule is localized The dark blue color is visible to the eyes in brightfield however the contrast especially when expression is very low and in the presence of hematoxylin nuclear staining may make it more difficult to visualize under lower magnifications 10X Our recommendation when defining which target s will be of a particular TYPE is to assign the lowest expressing target to TYPE 1 and the higher expressing target to TYPE 6 This recommendation is based on the easy of viewing by eye under brightfield using 10X magnification Probe Sets TYPE specific can be combined to create pan panels or target cocktails For example if you wanted to identify epithelial cells you could combine several cytokeretins To do this you would add for example TYPE 1 Probe Sets for KRT5 KRT7 KRT8 KRT10 KRT18 KRT19 and KRT20 into a single assay We do not recommend combining more than 10 targets for a specific signal amplification system for example TYPE 1 or TYPE 6 in a single assay Another example might be creating a pan housekeeping panel as a positive internal assay control to assess RNA integrity In this case you might add TYPE 6 Probe Sets for UBC ACTB PPIB and GAPD into a single assay well Typically QuantiGene ViewRNA Probe Set designs cover approximately 1 000 bases and contain 20 pairs of oligos to achieve maximal sensitivity The requirement for a pair of oligos to bind side by side in orde
40. lution from the slides Limiting tissue exposure to air for too long before adding hybridization reagents a Adding all working reagents onto the slides before moving them to the 40 C humidified incubator Incomplete removal of paraffin Use fresh Histo Clear solution Immediately submerge the warm slides into the Histo Clear solution after 80 C baking and move the slide rack up and down with frequent agitation Incorrect pretreatment conditions Repeat pretreatment assay optimization procedure to determine optimal boiling time and protease digestion time Insufficient washing Move the slide rack up and down with frequent agitation Increase wash incubation time by 1 min per wash B Concentration of hybridization reagents was too high Double check the dilution calculation for all working solutions Hybridization temperature not optimal Calibrate the humidified incubator at 40 C using the QuantiGene View Temperature Validation Kit Affymetrix P N QV0523 Label Probe AP concentration too high a Verify that the correct concentrations were used Decrease the recommended concentration for Label Probe AP 28 QuantiGene ViewRNA ISH Tissue Assay User Manual Assay Optimization Procedures About the Optimization and Typical Results The QuantiGene ViewRNA ISH Tissue Assay procedure is broken up into 2 parts that are performed over 2 days Part 1 Sample Preparation and Target
41. mmediately after decanting solution from the slides a Limiting tissue exposure to air for too long before adding hybridization reagents a Adding all working reagents onto the slides before moving them to the 40 C humidified incubator Tissue over fixed after protease digestion Make sure the tissue sections are not fixed for more than 5 min in 4 formaldehyde after protease digestion Reagents applied in wrong sequence Apply target Probe Set s PreAmplifier Mix QT Amplifier Mix QT Label Probe AP and substrates in the correct order 24 QuantiGene ViewRNA ISH Tissue Assay User Manual Table 4 1 Troubleshooting Weak or No Signal Probable Cause Recommended Action Incorrect storage condition Store the components at the storage condition as written on the component label or kit boxes Hybridization temperature not optimal Calibrate the humidified incubator at 40 C using a QuantiGene View Temperature Validation Kit Affymetrix P N QV0523 Probe Set hybridization temperature time and or concentration not optimal Decrease hybridization temperature from 40 to 38 C and increase Probe Set concentration by diluting target Probe Set 1 30 instead of 1 40 and hybridize for 2 hr Label Probe AP concentration too low a Verify that the correct concentrations were used a Increase the recommended concentrations for Label Probe AP If this is necessary it may result in higher backgrou
42. nds Mounting solution contained alcohol Use DAKO Ultramount or Innovex Advantage mounting media to mount your tissue Avoid any mounting solution containing alcohol Fast Red and Fast Blue Substrate solutions not freshly prepared Prepare Fast Red and Fast Blue Substrate solutions immediately before use Gene of interest is not expressing Verify expression using other tissue lysate methods such as QuantiGene QuantiGene Plex assay or Affymetrix array Run the same Probe Set on known samples that have been validated to express the gene of interest RNA in tissue is degraded Verify tissue fixation Ensure tissue was freshly harvested and immediately fixed in 10 NBF or 496 PFA for 16 24 hrs Ensure FFPE blocks and sections were stored correctly In the Fast Blue detection channel use a positive control Probe Set such as a pan housekeeping panel which might include TYPE 6 Probe Sets for ACTB GAPD and UBC to assess RNA integrity In that same assay the Fast Red channel would be used to detect target of interest using a TYPE 1 Probe Set Dark hematoxylin stain reduces visibility for the Blue dots Reduce hematoxylin staining time to 5 sec Tissues with lower cell density require longer hematoxylin incubation than tissues that have higher cell density It may be helpful to titrate incubation times a Increase brightness of lamp during viewing View under 40X objective Image under fluores
43. ng microscope Nikon E series objectives filters and or color filters for white balancing Fluorescence viewing and image capture Microscope with camera and fluorescence options Verify camera does not have infrared blocking filter Leica DM series Nikon E series Olympus BX series Zeiss Axio Lab Scope Imager Or equivalent Requires 20 and 40x objectives Numerical Aperture NA 20 5 For Fast Red Substrate use Cy3 TRITC filter set Excitation 530 20 nm Emission 590 20 nm Dichroic 562 nm For Fast Blue Substrate use custom filter set Excitation 630 20 nm Emission 775 25 nm Dichroic 750 nm For DAPI filter set Excitation 387 11 nm Emission 447 60 nm Automated image capture in brightfield and or fluorescence modes Digital pathology scanner system Aperio ScanScope AT XT CS Recommend scanning use FL version for fluorescence at 40x when Leica SCN400 F a Olympus Nanozoomer RS u Or similar expression is low Compatible to above Recommended vendor Semrock Cy7 B Alexa 750 filter modified with excitation filter FF02 628 40 25 8 QuantiGene ViewRNA ISH Tissue Assay User Manual Assay Guidelines Tissue Preparation Guidelines The following are critical guidelines for preparation of FFPE tissue blocks FFPE tissue slides and TMA slides for use with the QuantiGene ViewRNA ISH Tissue Assay Samples prepared outside of these guid
44. p 1 Bake Slides 35 min A B Use a pencil to label the slides Set dry oven at 60 1 C insert slides into slide rack and bake the slides for 30 min NOTE This increases tissue attachment to the slide Step 2 Prepare Buffers and A Prepare 3 L of 1X PBS Reagents While Slides To a 3 L container add 300 mL of 10X PBS and 2 7 L ddH O Bake B Prepare 1096 formaldehyde in 1X PBS in a fume hood To a 200 mL capacity container add 146 ml 1X PBS and 54 mL of 3796 formaldehyde and mix well C Prepare 4 formaldehyde in 1X PBS in a fume hood To a 200 mL capacity container add 22 mL of 3796 formaldehyde to 178 mL 1X PBS and mix well D Prepare 4 L of Wash Buffer To a4 L capacity container add components in the following order and mix well 3L ddH O a 36 mL Wash Comp 1 10mL Wash Comp 2 ddH O to 4 L E Prepare 500 mL of 1X Pretreatment Solution in a 1 L glass beaker Dilute 5 mL of 100X Pretreatment Solution in 495 mL ddH O F Prepare 200 mL of Storage Buffer To a 200 mL container add 60 mL of Wash Comp 2 to 140 mL ddH O and mix well G Ensure availability of a 200 mL Histo Clear or xylene 400 mL 95 ethanol not required if using xylene 400 mL ddH O H Prewarm 40 mL of 1X PBS and Probe Set Diluent QT to 40 1 C l Thaw Probe Set s Place on ice until use Step 3 Fix Slides A Ina fume hood pour 200 mL of 10 formaldehyde into clear staining dish B Insert slides into an empty sli
45. r to build the signal amplification system is at the core of the assays sensitivity and specificity The use of multiple pairs of oligos in a Probe Set ensures there are many opportunities for binding to the Chapter 2 Assay Guidelines 11 specific targets unmasked regions accessible regions and generating signal at that location If you are working with smaller targets or applications such as splice variants or RNA fusions there are only a few available pairs of oligos in a Probe Set and this will directly impact assay sensitivity That is probes will have fewer opportunities to find unmasked areas of the target and generate signal at that location In these cases increasing the Probe Set concentration used in the assay might increase the sensitivity Guidelines for Working with Tissue Microarrays TMAs Process TMA slides using the same assay procedures but with the following two modifications a Increase the initial baking step time from 30 minutes to 60 120 minutes This additional baking time will increase the tissue attachment to the slide reducing the risk of the small gt 1 mm core sections falling off during assay procedure a Increase the volume slide of the Protease Working Solution to prevent drying out of tissues at the edges of the TMA When designing TMAs to be used in the QuantiGene ViewRNA assay it is important to understand that only one optimized condition can be used when running the assay Therefore if you want mu
46. rotease Solution onto the tissue section Carefully move the aluminum slide rack in the humidified incubator and incubate for 10 min Wait 9 min then remove slides 2 5 and 9 from the clear staining dish and flick off excess 1X PBS Tap the slides on their edges then wipe the backside on a laboratory wipe Place the slides face up on an aluminum slide rack and add 400 uL of Working Protease Solution onto the tissue sections Carefully move the aluminum slide rack in the humidified oven and incubate for 10 min Pour 200 mL of 1X PBS into a clear staining dish and insert an empty rack into it At the end of 10 min 40 min total incubation time decant the Working Protease Solution from the slides insert them into the slide rack and rinse by moving up and down for 1 min Retrieve slide 1 and add to slide rack in PBS There should be 10 slides in the slide rack Decant the 1X PBS refill with 200 mL of fresh 1X PBS and rinse by moving slide rack up and down for 1 min Transfer the slide rack into the clear staining dish containing 496 formaldehyde and incubate under a fume hood for 5 min at RT Decant the clear staining dish containing 1X PBS and refill with 200 mL of fresh 1X PBS Transfer the slide rack from the 496 formaldehyde solution to the clear staining dish containing 1X PBS and incubate for 1 min with frequent agitation Decant the 1X PBS refill with 200 mL of fresh 1X PBS and rinse by moving slide rack up and down for 1
47. s results can be interpreted See Experimental Design Guidelines on page 9 Sample Preparation and Target Probe Hybridization Procedure Step Action Step 1 Bake Slides 35 min A B Use a pencil to label the slides Set dry oven at 60 1 C insert slides into slide rack and bake the slides for 30 min NOTE This increases tissue attachment to the slide Appendix A Assay Optimization Procedures 31 Step Action Step 2 Prepare Buffers and A Prepare 3 L of 1X PBS Reagents While Slides To a 3 L container add 300 mL of 10X PBS and 2 7 L ddH O Bake B Prepare 10 formaldehyde in 1X PBS in a fume hood To a 200 mL capacity container add 146 mL 1X PBS and 54 mL of 37 formaldehyde and mix well C Prepare 4 formaldehyde in 1X PBS in a fume hood To a 200 mL capacity container add 22 mL of 37 formaldehyde to 178 mL 1X PBS and mix well D Prepare 4 L of Wash Buffer To a4 L capacity container add components in the following order and mix well 3L ddH O 36 mL Wash Comp 1 10 mL Wash Comp 2 ddH O to 4L E Prepare 500 mL of 1X Pretreatment Solution in a 1 L glass beaker Dilute 5 mL of 100X Pretreatment Solution in 495 mL ddH O F Prepare 200 mL of Storage Buffer To a 200 mL container add 60 mL of Wash Comp 2 to 140 mL ddH O and mix well G Ensure availability of a 200 mL Histo Clear or xylene 400 mL 95 ethanol not required if using xylene 400 mL ddH O H
48. t the Fast Red Substrate from the slides and insert them into the slide rack Move the slide rack up and down several times for 1 min to rinse off the Fast Red Substrate Retrieve 200 mL of 4 formaldehyde used previously and pour in the clear staining dish labeled for formaldehyde Move the slide rack to the clear staining dish containing 200 mL of 496 formaldehyde and incubate for 5 min under a fume hood Rinse off the residual formaldehyde by transferring the slide rack to a clear staining dish containing fresh 1X PBS Move the slide rack up and down several times for 1 min Chapter 3 QuantiGene ViewRNA ISH Tissue Assay Procedure 21 Step Action Step 24 Counterstain 50 min H Transfer the slide rack to the clear staining dish containing the 200 mL of Gill s Hematoxylin Incubate slides for 1 min at RT After incubation transfer the slide rack to a clear staining dish containing ddH O Move the slide rack up and down several times to rinse off the Gill s Hematoxylin Decant the ddH O refill with 200 mL fresh ddH O and move slide rack up and down several times Repeat this step one more time Decant the ddH O refill with 200 mL 0 01 ammonium hydroxide and incubate for 10 sec Decant 0 01 ammonium hydroxide refill with fresh ddH O and move slide rack up and down several times Unused 0 01 ammonium hydroxide solution can be stored at RT for up to one month Optional If you plan to view slides usin
49. terest must be purchased separately Probe Sets are available in multiple sizes and should be stored at 20 C Refer to the Package Insert for quantities provided and design specificities Table 1 4 ViewRNA Probe Set and Storage Conditions Component Description Storage QuantiGene ViewRNA TYPE 1 RNA specific oligonucleotides to your target of interest 20 C Probe Set and compatible with the TYPE 1 Signal Amplification system comprised of PreAmp Mix QT Amp Mix QT Label Probe 1 AP and Fast Red substrate QuantiGene ViewRNA TYPE 6 RNA specific oligonucleotides to your target of interest 20 C Probe Set and compatible with the TYPE 6 Signal Amplification system comprised of PreAmp Mix QT Amp Mix QT Label Probe 6 AP and Fast Blue Substrate Required Materials and Equipment Not Provided Other materials required to perform the QuantiGene ViewRNA ISH Tissue Assay that are not included in the QuantiGene ViewRNA ISH Tissue Assay Kit are listed here Lu IMPORTANT When specified do not use alternate materials or suppliers Table 2 QuantiGene ViewRNA Tissue Assay Materials and Equipment Material Source Part Number Tissue Tek Staining Dish clear color 3 required Affymetrix or QVC0502 clear staining dish American Master LWT4457EA Tech Scientific Tissue Tek Clearing Agent Dish green color Affymetrix or QVC0503 green clearing agent dish American Master LWT4456EA Tech
50. thanol refill with 200 mL of fresh 9596 ethanol and incubate for 1 min with frequent agitation Remove the slides from the slide rack and place them face up on a paper towel to air dry for 5 min at RT Discard the 9596 ethanol If using xylene A Pour 200 mL of xylene in a fume hood into green clearing agent dish B Load the slides into the slide rack C Transfer the slide rack to the green clearing dish containing 200 mL xylene D Incubate in a fume hood at RT for 10 min with frequent agitation E Liftthe slide rack from the xylene and submerge into the clear staining dish containing 200 mL 1X PBS and incubate for 1 min with frequent agitation F Decant the 1X PBS refill with 200 mL fresh 1X PBS and incubate for 1 min with frequent agitation G Remove the slides from the slide rack and place them face up on a paper towel to air dry for 20 min at RT Discard the 1X PBS Step 5 Draw Hydrophobic A Dab the hydrophobic pen on a paper towel several times before use to ensure proper flow Barrier of the hydrophobic solution B Tocreate a hydrophobic barrier place the slide over the template image below tissue 1 hr sections should fall inside blue rectangle and lightly trace the thick blue rectangle 2 to 4 times with the Hydrophobic Barrier Pen to ensure a solid seal Allow for barrier to dry at RT for 20 30 min Step 6 Tissue Pretreatment 10 25 min depending on optimized time E F Bring 500 mL of
51. velopments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Affymetrix Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Copyright 2012 Affymetrix Inc All rights reserved Contents Chapter 1 Introduction seer eean aa a a iI J IM Ba oie Bp usta bak a Been So ae We wba aad 1 Chapter 2 Chapter 3 Chapter 4 About This Manual x s itn ee karaca leba an Ep dib Re mode pce eee 1 Related User Documents A 2 as aA A k RA800 nda k lr 1 Assay OVeIVIeW anci kk ded ODE Re la AW E ANA AE ee eu e ew lw wit Gee A 1 HOW It WORKS xoxo b n w O dab a bb ob oe Ede dil dul ed e Pei 2 Performanc Highlights er o sa Ay k l tere e W dia da LA AA U Aa an WE 3 Safety Warnings and Precautions a As s4 s n An ba xalan n an tee eee 3 Required Materials AA kk kk kk kk kk KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK KK KI KK KK 4 QuantiGene ViewRNA ISH Tissue Assay Kit lA kk kk kk kk kk KK KI KK KI K K KI K R KI KK KIR RIK KK KI 4 QuantiGene ViewRNA Probe Sets kk kk kk kk kk le 5 Required Materials and Equipment Not Provided kK KK eee eee 5 Microscopy and Imaging Equipment Guidelines for QuantiGene ViewRNA ISH Tissue Assay 7 Assay Guidelines s vos acto ex SC kk KK he KK KK kK KK KK Ye were kK kk 9 Tissue Preparat
52. ymetrix com Asia 81 3 6430 430 techsupport_asia affymetrix com Weak or No Signals Table 4 1 Troubleshooting Weak or No Signal Probable Cause Recommended Action Incorrect pretreatment conditions Repeat pretreatment assay optimization procedure to determine optimal boiling time and protease digestion time Sample preparation over fixation Make sure that freshly dissected tissues are fixed in 10 neutral buffered formalin NBF or 4 paraformaldehyde PFA for 16 24 hr Improper fixation reagents or concentrations Make sure correct concentration of NBF was used to fix the slides in respective steps Tissue dries up during hybridization steps Humidified incubator recommendations Verify the bottom tray is filled with ddH 0 before starting hybridization Make sure the humidified incubator has greater than 85 humidity a Calibrate the humidified incubator to 40 C using QuantiGene View Temperature Validation Kit Affymetrix P N QV0523 Prevent sections from drying out by Preparing enough reagents and use the recommended volumes for each step of the assay a Ensuring that you have a solid seal when drawing your hydrophobic barriers a Adding all working reagents onto the slides before moving them to the 40 C humidified incubator Tissue dries up during processing Keep tissue section moist starting from the pretreatment boiling step by Adding respective reagents i

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