QuantiGene® ViewRNA For FFPE Samples
Contents
1. incubate at RT for 3 min Move the slide rack up and down several times to wash the sections Decant the Wash Buffer and add fresh Wash Buffer Incubate the slides in Wash Buffer at RT for 3 min and move the slide rack up and down several times Repeat step 10b 11 Move the slides to a dry paper towel on the bench Add 200 uL of the AP Enhancer Solution and incubate at RT for 5 10 minutes while preparing the Fast Red Substrate 12 Prepare the Fast Red Substrate a Remove the required number of Fast Red tablets from the refrigerator Allow tablets to reach RT b Drop one Fast Red tablet into 5 mL of Naphthol Buffer Vortex to dissolve tablet Note For best results use the prepared Fast Red Substrate within 1 hour QuantiGene ViewRNA FFPE User Manual 17 Section VI In Situ Hybridization Procedure 18 Step Action 13 Apply the Fast Red Substrate a Decant the AP Enhancer Solution and place the slides on a dry paper towel with tissue section on top If using Then do this Thermobrite a Add 200 uL Fast Red Substrate move slides to oven Thermobrite hybridization station close the lid and incubate at 40 C for 30 min in dark b Decant the Fast Red Substrate from slides place slides in a Tissue Tek Slide Rack and submerge the slides in a Tissue Tek Staining Dish containing 200 mL of 1X PBS c Move the slide rack up and down several times t2. Ansty be aan ones ed Salad gaa Sede Ada a E mn 5 Optimization experiment kk kk kk kk kk KK KK KK KI KK KI KI KK KI KK KI KK KK eee 5 Expected ResUlts asyaya Zawa n a a wa d e teeta 5 Section IV Assay Workflow kk kk kk kk kK KK KK KK KK KK KK KK KK KK KK KK kK kk 6 Section V Sample Pretreatment kk kk kk KK KK KK K KIRR KK KK KIR KI K K KI KIRI KK 7 OVEIVIE WS cata a Gea ala hdd Ae ta o LE ata 7 Important Procedural Note kk kk kk kK KK KK KK KK KK KK KK KK KI RIK eee 7 Before YOU Mateo ar RdA E A oe 7 Procede a 8 Section VI In Situ Hybridization Procedure 2 2 2 0 0000 eee 10 OVEIVIE siria sl tii ad ic 10 Important Procedural Notes 0000 20 KK KK KI KK KK KIRI KI KK K KK 10 Before You Start es nis xalk k y ka WAW da W AA AKA AW A i 10 Procedures xw A rab 11 Section VII Troubleshooting u kk kk kk kk KK KEK KK KK KK KK kK KIRI kk 19 Troubleshooting No or Weak Signals KK KK KK KK KK KK KK KK 19 High Background on the Tissue Section 0 0 0 ee 19 High Background on Slide 61 eee 20 Hematoxylin Staining is too Strong 1 00 kk KK KK KK eee 20 Section VIII Rat Kidney FFPE Tissue Example KK KK cee eee eee 21 QuantiGene ViewRNA FFPE User Manual iii Table of Contents v QuantiGene ViewRNA FFPE User Manual Section l Introduction Section l Introduction About This Manual The QuantiGene ViewRNA Reagent Sys
3. Stopping point Dry slides and store at RT QuantiGene ViewRNA FFPE User Manual Section V Sample Pretreatment To pretreat samples continuea Step Action 4 Treat sample with 1X Pretreatment Solution a Fill a 1000 mL beaker with 800 mL of 1X Pretreatment Solution and bring the solution to boil 100 104 C by heating it on a hot plate As soon as the solution reaches boiling cover the beaker and turn down the heat to maintain 100 C while preventing evaporation IMPORTANT The pretreatment solution should completely cover tissues on the slides IMPORTANT To prevent boil over make sure the pretreatment solution is not vigorously boiling while submerging the slides b Submerge the slide rack loaded with slides into the boiling pretreatment solution cover the beaker and incubate for the time as determined by using the optimization procedure in Section Ill Recommendations for Experimental Design and Assay Optimization on page 5 c At the end of time point remove the slide rack with slides in it and submerge the entire rack with slides into a Tissue Tek Staining Dish containing ddH O d If you are performing the optimization assay remove the slides at the appropriate time points and insert the slides in a rack submerged in Tissue Tek Staining Dish containing ddH O Wait until you collect all of the slides with various time points for pretreatment before proceeding e Rinse the slides once more b
4. not to introduce any bubbles in between the section and the coverslip c Move slides to the 40 C Thermobrite close the lid and incubate for 3 hours Tissue culture incubator a Place slides on the aluminum slide rack b Add 200 uL Working Probe Set to each tissue section addition of cover slip is not necessary c Put slides in a 40 C humidified incubator for 3 hours QuantiGene ViewRNA FFPE User Manual Section VI In Situ Hybridization Procedure Step Action 3 Wash the samples 4 times for Thermobrite with Wash Buffer If using Then do this Thermobrite oven a Remove slides from the 40 C Thermobrite and submerge the slides in a Tissue Tek Staining Dish without rack inside containing 200 mL of Wash Buffer The coverslips should fall off the section b As soon as the coverslips fall off transfer the slides into a Tissue Tek Slide Rack submerged in another Tissue Tek Staining Dish containing 200 mL of Wash Buffer and incubate at RT for 2 min Move the slide rack up and down several times to wash the sections c Decant the Wash Buffer and add fresh Wash Buffer Incubate the slides in Wash Buffer at RT for 2 min and move the slide rack up and down several times d Repeat step 3c Tissue culture incubator a Remove slides from the 40 C incubator decant the Hyb A solution from slides and transfer slides to a Tissue Tek Slide Rack submerged i
5. pretreatment condition and probed with the same housekeeping gene for example Ubc Once an optimal condition for in situ hybridization is determined you can apply this pretreatment condition to tissue sections in combination with the target probe of your interest The following table shows the optimization experiment set up Protease Pretreatment Boiling Time min Incubation Time min 0 5 10 20 0 Slide 1 10 Slide 2 Slide 5 Slide 8 20 Slide 3 Slide 6 Slide 9 40 Slide 4 Slide 7 Slide 10 Expected Results For section treated with O min pretreatment boiling 0 min protease incubation Slide 1 expect to see no signal For sections treated with 5 10 or 20 min pretreatment boiling and 10 20 or 40 min protease incubation Slide 2 10 expect to see at least one slide showing strong signal with good morphology Once a condition has been optimized any Probe Set can be used with similarly treated samples See Section VIII Rat Kidney FFPE Tissue Example on page 21 for an example of an expected result QuantiGene ViewRNA FFPE User Manual 5 Section IV Assay Workflow Section IV Assay Workflow See Step Task Page 1 FFPE sample preparation approximately 1 5 h a Baking slides at 60 C 7 b Fixing with 10 formaldehyde 8 Note Possible stopping point dry slides and store at RT 2 Sample de paraffinization approximately 15 min 8 3 Sample pre
6. than 5 min in 4 formaldehyde after protease digestion Placing the slide on the 40 C Thermobrite before adding Hybridization Buffer Enhancer Solution or FastRed Solution and causing the Probe to dissociate from the target Add the Hybridization Buffer Enhancer Solution or FastRed Solution to slides before moving them to the 40 C Thermobrite High Background Troubleshooting high background on the tissue section on the Tissue Section Probable Cause Recommended Actions Insufficient washing Increase the length of wash by 5 min for each wash step Slide drys up during Working Probe Set hybridization step Make sure to place a cover glass over the tissue sample when using Thermobrite hybridization station at the target probe hybridization step Make sure to wet the strips inside the Thermobrite before starting hybridization Make sure the Thermobrite is placed on a leveled bench QuantiGene ViewRNA FFPE User Manual 19 Section VII Troubleshooting Fast Red substrate drys up during incubation Make sure enough Fast Red substrate is added to each section Make sure to close the Thermobrite lid during digestion Make sure to wet the strips inside the Thermobrite before starting Fast Red reaction High Background Troubleshooting high background on glass slide on Slide Probable Cause Recommended Actions Over protease digestion of the sample
7. to the 40 C Thermobrite close the lid to incubate for 25 min Tissue a Place slides on an aluminum slide rack and add culture 200 uL of Working PreAmp 1 to each slide incubator b Put the slide rack into the 40 C humidified tissue culture incubator for 25 min 6 Wash the samples 3 times with Wash Buffer If using Then do this Thermobrite a Remove slides from the 40 C Thermobrite decant oven the Hyb B solution from slides and submerge the slides in a Tissue Tek Staining Dish containing 200 mL of Wash Buffer Incubate at RT for 2 minutes Move the slide rack up and down several times to wash the sections b Decant the Wash Buffer and add fresh Wash Buffer Incubate the slides in Wash Buffer at RT for 2 min and move the slide rack up and down several times c Repeat step 6b Tissue a Remove slides from the 40 C incubator decant the culture Hyb B solution from slides and transfer slides to a incubator Tissue Tek Slide Rack submerged in a Tissue Tek Staining Dish containing 200 mL of Wash Buffer and incubate at RT for 2 min Move the slide rack up and down several times to wash the sections b Decant the Wash Buffer and add fresh Wash Buffer Incubate the slides in Wash Buffer at RT for 2 min and move the slide rack up and down several times c Repeat step 6b QuantiGene ViewRNA FFPE User Manual Section VI In Situ Hybridization Procedure Step Action 7 Hybridize Am
8. treatment approximately 5 20 min 9 Note Possible stopping point store slides in 1X PBS at RT 4 Protease digestion approximately 5 40 min a Protease treatment 11 b Post fixation in 4 formaldehyde 11 5 Target probe hybridization approximately 3 h a Incubate with target probe 12 b Wash 3 times 13 Note Possible stopping point store slides in 6X SSC at RT 6 PreAmp hybridization approximately 25 min a Incubate with PreAmp 14 b Wash 3 ti as imes 14 7 Amp hybridization approximately 15 min a Incubate with Amp 15 b Wash 3 times 15 8 LP AP hybridization approximately 15 min a Incubate with LP AP 16 b Wash 3 times 17 9 FastRed substrate incubation approximately 30 min a Incubate with FastRed substrate 18 b Rinse off substrate and fix in 4 formaldehyde 18 10 Counterstain with Gills Hematoxylin 18 11 Mount samples and observe under brightfield microscope 18 6 QuantiGene ViewRNA FFPE User Manual Section V Sample Pretreatment Section V Sample Pretreatment Overview Here we provide recommendations for preparation of samples prior to in situ hybrid ization For a successful in situ hybridization prepare the FFPE samples as follows Fixin 10 neutral buffer formalin for 16 24 hours as soon as the tissues are extracted Y Section FFPE blocks into 4 6 um thickness and attach to positively charged slides Superfrost Plus Fisher Scientific P N 12 550 15 Make sure FFPE tissu
9. 0 in 1X PBS and mixing well Place at 40 C until ready to use b Pre warm the section by putting the slides on Thermobrite set at 40 C and add 200 uL 1X PBS to each section make sure the buffer does not dry up on the sections c Decant the 1X PBS from the slides one at a time followed by placing the slide back onto the Thermobrite and immediately add 200 uL of Working Protease Solution onto the tissue section Use a pipette tip to spread the protease and make sure the entire tissue section is covered d Close the lid and incubate for the amount of time determined in the optimization experiment see Section III Recommendations for Experimental Design and Assay Optimization on page 5 e Remove the slides from Thermobrite decant the Protease Solution and place slides in a Tissue Tek Slide Rack f If you are performing the optimization assay remove the slides from the Thermobrite decant the protease solution and insert the slides in a Tissue Tek Slide Rack submerged in Tissue Tek Staining Dish containing 200 mL 1X PBS Wait until you collect all of the slides before proceeding g Submerge the slides into a Tissue Tek Staining Dish containing 200 mL 1X PBS h Decant the 1X PBS and replace with fresh 1X PBS Move the slide rack up and down several times i Move the slides using a slide rack into a Tissue Tek Staining Dish containing 4 formaldehyde and incubate for 5 min at RT j Fill another clean Tissue Tek Sta
10. Decant the Wash Buffer and add fresh Wash Buffer Incubate the slides in Wash Buffer at RT for 2 min and move the slide rack up and down several times Repeat step 8b Tissue culture incubator Remove slides from the 40 C incubator decant the Hyb B solution from slides and transfer slides to a Tissue Tek Slide Rack submerged in a Tissue Tek Staining Dish containing 200 mL of Wash Buffer and incubate at RT for 2 min Move the slide rack up and down several times to wash the sections Decant the Wash Buffer and add fresh Wash Buffer Incubate the slides in Wash Buffer at RT for 2 min and move the slide rack up and down several times Repeat step 8b QuantiGene ViewRNA FFPE User Manual 15 Section VI In Situ Hybridization Procedure Step Action 9 Hybridize Label Probe a Prepare Working LP AP by first diluting LP AP 1 10 in prewarmed Hyb C and mix well followed by making a 1 100 dilution yielding a 1 1000 final dilution b Place at 40 C until use Scale reagents according to the number of assays to be run Include 10 overage Use the table below as a guide Component 1 slide 10 slides Hyb C prewarmed 197 8 uL 1978 uL LP AP 1 10 diluted 2 2 uL 22 uL Total Volume 220 uL 2200 uL c Remove the slides from the staining dish and decant the Wash Buffer from the slides Make sure the section is wet and the hydrophobic rectangle is dry Always u
11. Optimize the protease digestion time as indicated in the Section Ill Recommendations for Experimental Design and Assay Optimization on page 5 Insufficient washing Increase the length of wash by 5 min for each wash step Slide drys up during Working Probe Set hybridization step Make sure to place a cover glass over the tissue sample when using Thermobrite hybridization station at the target probe hybridization step Make sure to wet the strips inside the Thermobrite before starting hybridization Make sure the Thermobrite is placed on a leveled bench Fast Red substrate drys up during incubation Make sure enough Fast Red substrate is added to each section Make sure to close the Thermobrite lid during digestion Make sure to wet the strips inside the Thermobrite before starting Fast Red reaction Hematoxylin Troubleshooting Hematoxylin staining Staining is too Strong 20 Probable Cause Recommended Actions Slides are incubated in Gill s Hematoxylin solution for too long Wash slides several times in ddH20 to reduce blue stain Reduce the time the slides are in Gill s Hematoxylin solution QuantiGene ViewRNA FFPE User Manual Section VIII Rat Kidney FFPE Tissue Example Example ssue Rat Kidney FFPE Ti Section VIII Pretreatment boiling time min 5 10 20 Hematoxylin nuclei FastRed labeled GAPD mRNA more hematoxylin staining the be
12. QuantiGene ViewRNA For FFPE Samples User Manual Panomics Panomics Inc QuantiGene ViewRNA For FFPE Samples User Manual Copyright O 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 now a part of Affymetrix 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 Em
13. ail 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 I Introd cton ecer ss ZE kak W Ere al a alan a uza sa 1 About This Manual As s ats sunne kd Paneth Ged Ho W Flank ee eee lad AAS 1 QuantiGene ViewRNA Assay BaSicS 000 0c KK KK KK KK ees 1 HOw It WOrkSia2 seus hate ated ata Aran 1 Section Il Required Materials kk kk kk kK kK KK KK KK KI ete eee 2 QuantiGene ViewRNA FFPE Sample Optimization Kit Optional 2 QuantiGene ViewRNA Assay Kit for FFPE SampleS o o ocoo ooo o 2 QuantiGene ViewRNA Chromogenic Signal Amplification Kit 2 QuantiGene ViewRNA Assay Probe Sets KK KK KK KK KK RIK KK 3 Optional QuantiGene ViewRNA FFPE Positive Control Kit Rat Kidney 3 Required Materials Not Provided 020 eee eee KK KK 3 Section lll Recommendations for Experimental Design and Assay Optimization 5 OVErRIOW cai hae
14. ccessory Product QuantiGene ViewRNA TYPE 1 Probe Set Description Storage 20 C RNA specific oligonucleotides for use with PreAmp1 Amp1 LP AP Visit www panomics com for the most up to date listing of available QuantiGene ViewRNA Probe Sets If we do not yet offer a QuantiGene ViewRNA Probe Set for your RNA of interest please submit the accession number include version or provide gi number or RNA sequence with your order and we will design and synthesize it for you in approximately 2 weeks The QuantiGene ViewRNA FFPE Positive Control Kit is designed for use with QuantiGene ViewRNA Assay Kit for FFPE Samples and QuantiGene ViewRNA Chromogenic Signal Amplification Kit The kit includes 8 4 6 um thick rat kidney FFPE sections attached to positively charged slides as well as TYPE 1 Probe Set for rat Ubc Refer to the Product Insert for the optimized conditions and recommendations for use Other materials required to perform the QuantiGene ViewRNA assay for FFPE Samples that are not included in the Assay Kit for FFPE Samples or the Chromogenic Signal Amplification Kit are listed here When specified do not use alternate materials or suppliers Table 1 All Sample Types Required Material Supplier Part Number Deionized Water ddH 0 MLSa 95 Ethanol VWR 89015 512 10X PBS pH 7 2 7 4 Bio Rad Laboratories or 161 0780 Invitrogen 70013 032 Gill s Hematoxylin American Master Tech HXGHE1LT Scient
15. e sections on each slide are smaller than 22 mm x 22 mm in size If larger than 22 mm x 22 mm in size you will require twice as much prepared working reagents per slide reducing the total number of assays in the kit Important All reagent volumes shown are based on hydrophobic barrier size of 22 mm x 25 mm Procedural Note area Before You Start Step Action 1 Set one Thermobrite to 60 C or a dry incubator to 60 C 2 Bake the slides in Thermobrite with lid open at 60 C for 30 minutes Alternatively bake slides in a 60 C dry oven for 30 min This will increase the tissue attachment to the slide 3 Prepare one Coplin jar with 10 formaldehyde in 1X PBS add 10 mL of 37 formaldehyde to 27 mL 1X PBS 4 Prepare 350 mL of 1X PBS 5 Prepare a second Coplin jar with 1X PBS 6 Prepare 1X Pretreatment Solution by diluting 8 mL of 100X Pretreatment Solution to 792 mL ddH O 7 Dispense 200 mL of 95 EtOH into a clean Tissue Tek Staining Dish Ensure 400 mL 95 Ethanol and ddH 0 are available 9 Add 200 mL HistoClear to Tissue Tek Clearing Agent Dish green QuantiGene ViewRNA FFPE User Manual 7 Section V Sample Pretreatment Procedure To pretreat samples Step Action 1 Incubate the sections with 10 formaldehyde in a fume hood for 1 hour at room temperature c Submerge the slide sections into the Coplin Jar containing 10 formaldehyde solution and incubate for 1 h
16. f individual components supplied Kits have a shelf life of 6 months from the date of delivery The components of the QuantiGene ViewRNA Assay Kit for FFPE samples and their recommended storage conditions are listed below QuantiGene ViewRNA Assay Kits for FFPE samples are available in two sizes QV0050 and QV0051 sufficient for 24 or 96 assays respectively Each kit is configured for processing a minimum of 6 slides per experiment Refer to the product insert for quantities of individual components supplied Kits have a shelf life of 6 months from date of delivery Component Description Storage 100X Pretreatment Aqueous buffered solution 2 8 C Solution Protease 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 containing detergent 2 8 C Hyb 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 a IMPORTANT Do not freeze The components of the QuantiGene ViewRNA Chromogenic Signal Amplification Kit and their recommended storage conditions are listed below This kit is available in 2 sizes QVO200 and QV0201 sufficient for 24 or 96 assays respectively Each kit is configured f
17. ific HistoClear National Diagnostics HS 202 QuantiGene ViewRNA FFPE User Manual 3 Section II Required Materials Table 1 All Sample Types continued 37 Formaldehyde gt Fisher Scientific F79 1 27 30 Ammonium Hydroxide VWR JT9726 5 20X SSC used only for optional stop Ambion AM9763 point in the procedure ImmEdge Hydrophobic Barrier Pen Vector Laboratories H4000 UltraMount DAKO 1964 Tissue Tek Staining Dish clear color 3 American Master Tech LWT4457EA required Scientific Tissue Tek Clearing Agent Dish green American Master Tech LWT4456EA color 1 required Scientific Tissue Tek Vertical 24 Slide Rack 1 American Master Tech LWSRA24 required Scientific Coplin Jar two 40 50 mL size MLS 1000 mL Glass Beaker MLS Cover Glass 22 mm x 30 mm not Fisher Scientific 12 530A required if using optional tissue culture incubator ThermoBrite Hybridization Chamber holds up to 12 slides Abbot Molecular 07J91 010 110V 0791 020 240V Humidifying Strips Abbot Molecular 30 144115 Isotemp Hot Plate Fisher Scientific 11 300 49SHP 120V 11 302 49SHP 230V 40 1 C Temperature humidity meter optional if Fisher Scientific 11 661 19 using tissue culture incubator Optional Tissue culture incubator 40 C Napco 51201082 ye Ae without CO and 80 90 humidity for VWR 9150860 processing 12 or more slides per experimen
18. ining Dish with 1X PBS while waiting for the incubation k Rinse the slide by moving the slide rack from the 4 formaldehyde solution to 1X PBS solution Keep the 4 formaldehyde solution for later use Move the slide rack up and down several times to make sure the formaldehyde is rinsed off QuantiGene ViewRNA FFPE User Manual 11 Section VI In Situ Hybridization Procedure 12 Step Action 2 Hybridize the Probe Set Note If you are using the Sample Optimization Kit skip the preparation of Working Probe Set and instead use species specific prewarmed Hyb A a Prepare Working Probe Set by diluting the Probe Set 1 50 in pre warmed Hyb A and mixing well b Place at 40 C until use Scale reagents according to the number of assays to be run Include 10 overage Use the table below as a guide Component 1 slide 10 slides Hyb A prewarmed 215 6 uL 2156 uL QuantiGene ViewRNA TYPE 1 4 4 uL 44 uL Probe Set Total Volume 220 uL 2200 uL c Continue the hybridization according to the method of incubation If using Then do this Thermobrite oven a Before moving tissue sections to the Thermobrite add 200 uL Working Probe Set to each tissue section IMPORTANT The hydrophobic barrier must be drawn at 22 mm W x 25 mm L to accommodate this volume once the coverslip is put in place b Gently add a 30 x 22 mm coverslip on top of each section Make sure
19. n a Tissue Tek Staining Dish containing 200 mL of Wash Buffer and incubate at RT for 2 min Move the slide rack up and down several times to wash the sections b Decant the Wash Buffer and add fresh Wash Buffer Incubate the slides in Wash Buffer at RT for 2 min and move the slide rack up and down several times c Repeat step 3b 4 Optional stopping point Prepare 6X SSC Submerge your slides in a slide rack into a Tissue Tek Staining Dish containing 200 mL 6X SSC Slides can be left overnight at RT QuantiGene ViewRNA FFPE User Manual 13 Section VI In Situ Hybridization Procedure 14 Step Action 5 Hybridize Pre Amplifier a Prepare Working PreAmp1 by diluting PreAmp1 1 100 in pre warmed Hyb B and mixing well b Place at 40 C until use Scale reagents according to the number of assays to be run Include 10 overage Use the table below as a guide Component 1 slide 10 slides Hyb B prewarmed 197 8 uL 2178 uL PreAmp1 2 2 uL 22 uL Total Volume 220 uL 2200 uL c Remove the slides from the staining dish and decant the Wash Buffer from the slides Make sure the section is wet and the hydrophobic rectangle is dry Always use the corner of a laboratory tissue to wipe the hydrophobic barrier dry If using Then do this Thermobrite a Before moving the slides to the Thermobrite add oven 200 uL of Working PreAmp1 to each slide b Move the slides
20. n top of the tissue without making any bubbles g Gently put a cover glass over the sample and avoid making bubbles Leave the samples at room temperature for several hours for the mounting medium to dry 15 View slides under a brightfield microscope QuantiGene ViewRNA FFPE User Manual Section VII Troubleshooting Section VII Troubleshooting Troubleshooting Troubleshooting no or weak signals No or Weak Signals Probable Cause Recommended Actions Tissue drys up during Working Probe Set hybridization step Make sure to place a cover glass over the tissue sample when using Thermobrite hybridization station at the target probe hybridization step Make sure to wet the strips inside the Thermobrite before starting hybridization Make sure the Thermobrite is placed on a leveled bench Tissue drys up during Protease digestion Make sure enough Protease solution is added to each tissue for protease digestion Make sure to close the Thermobrite lid during digestion Make sure to wet the strips inside the Thermobrite before starting hybridization Fast Red substrate drys up during incubation Make sure enough Fast Red Substrate is added to each section Make sure to close the Thermobrite lid during digestion Make sure to wet the strips inside the Thermobrite before starting Fast Red reaction Tissue over fixed after protease digestion Make sure the tissue sections are not fixed for more
21. o rinse off the Fast Red Substrate d Move the slide rack into the Tissue Tek staining dish containing 4 formaldehyde used earlier in the procedure and incubate for 5 min at RT e Decant the 4 formaldehyde and replace with 1X PBS f Move the slide rack up and down several times to rinse off the formaldehyde Tissue a Place slides on aluminum slide rack and add 200 uL culture Fast Red Substrate to each slide Put tray into the incubator 40 C humidified tissue culture incubator for 30 min in the dark b Remove the Fast Red Substrate from slides place slides in a Tissue Tek Slide Rack and submerge the slides in a Tissue Tek Staining Dish containing 200 mL of 1X PBS c Repeat steps 13c 13f from above 14 Counter stain with Gill s Hematoxylin and add cover glass a Move the slide rack into the Tissue Tek staining dish containing Gill s Hematoxylin and incubate for 5 min at RT or until the nuclei are properly stained If they don t stain repeat staining b Move the slide rack into another Tissue Tek staining dish containing ddH O c Decant the ddH O and fill the staining dish with fresh ddH O d Decant the ddH O and fill the staining dish with 0 01 Ammonium Hydroxide and move the rack up and down several times to create a much more blue color nuclear stain e Remove the slides from the rack and remove the solution on the slide as much as possible Allow slides to dry f Place 1 2 drops of the mounting medium o
22. ontains 10 or more oligonucleotide pairs Step 3 Amplify Signal A Pre Amplifier PreAmp molecule hybridizes to each pair of oligonucleotides then multiple Amplifier Amp molecules hybridize to each PreAmp Finally multiple Label Probe oligonucleotides conjugated to alkaline phosphatase LP AP hybridize to each Amp Following the addition of the Fast Red Substrate alkaline phosphatase breaks down the substrate to form a precipitate punctated dots that indicates the presence of the target RNA molecule Step 4 Image Target mRNA is visualized using standard brightfield microscopy and cell nuclei can be identified by the Hematoxylin counterstain QuantiGene ViewRNA FFPE User Manual Section II Required Materials Section Il Required Materials QuantiGene ViewRNA FFPE Sample Optimization Kit Optional QuantiGene ViewRNA Assay Kit for FFPE Samples QuantiGene ViewRNA Chromogenic Signal Amplification Kit For initial optimization of a new tissue this optional cost effective all inclusive kit enables you to optimize assay conditions for up to 2 specific tissues Once you have optimized the conditions those conditions can be used to evaluate targets of interest using the QuantiGene ViewRNA Assay Kit for FFPE samples Chromogenic Signal Amplification Kit and QuantiGene ViewRNA Probe Sets This kit is available for human QV0100 mouse QV0101 or rat QV0102 samples only Refer to the Product Insert for quantities o
23. or processing a minimum of 6 slides per experiment Refer to the Product Insert for quantity of individual components supplied Kits have a shelf life of 6 months from date of delivery Component Description Storage PreAmplifier 1 PreAmp1 DNA in aqueous buffered solution 20 C Amplifier 1 Amp1 DNA in aqueous buffered solution 20 C AP Enhancer Solution Aqueous buffered solution 2 8 C QuantiGene ViewRNA FFPE User Manual QuantiGene ViewRNA Assay Probe Sets Optional QuantiGene ViewRNA FFPE Positive Control Kit Rat Kidney Required Materials Not Provided Section ll Required Materials Fast Red Tablets Red precipitating substrate for the detection of 2 8 C alkaline phosphatase activity Naphthol Buffer Buffer required for preparation of Fast Red 2 8 C Substrate Label Probe AP LP AP Alkaline Phosphatase conjugated 2 8 C oligonucleotide in aqueous buffered solution In addition to the Assay Kit for FFPE Samples and the Chromogenic Signal Amplification Kit QuantiGene ViewRNA TYPE 1 Probe Sets specific to your targets of interest must be purchased separately Probe Sets are available in multiple sizes and should be stored at 20 C Refer to the Product Insert for more information IMPORTANT QuantiGene ViewRNA TYPE 1 Probe Sets hybridize to the PreAmp1 Amp1 LP AP signal amplification system and can be used to visualize 10 RNA copies cell in FFPE samples A
24. our at room temperature d Move the slides to the Coplin jar containing 1X PBS replace with fresh1X PBS e Remove slides from 1X PBS and entirely remove the 1X PBS from the slides by placing them on edge on a laboratory wipe Lightly tap until all liquid is removed and allow to air dry Remove the paraffin from the samples a Place the FFPE slides on 80 C Thermobrite with lid open or in a dry incubator at 80 C for 3 min The paraffin should be melted as soon as the slides are on the heat block b Immediately load the slides into a Tissue Tek Vertical Slide Rack and submerge the slides into a Tissue Tek Clearing Agent Dish green containing 200 mL HistoClear c Incubate the slides in HistoClear in a fume hood at room temperature RT for 10 min with frequent agitation d Remove the slides from the HistoClear solution by lifting the slide rack Immediately transfer the slides into a Tissue Tek Staining Dish with 95 EtOH e Rinse off the residual HistoClear by moving the slide rack up and down several times f Decant the 95 EtOH and rinse the slides with fresh 95 EtOH g Lift the slides out of the 95 EtOH and let the slides air dry on a paper towel for 5 min at RT IMPORTANT Make sure the slide has dried completely before proceeding to step 2h h Create a22 mm x 25 mm hydrophobic rectangle with the tissue section at the center using the ImmEdge Hydrophobic Barrier Pen and allow to air dry Optional
25. plifier a Prepare Working Amp1 by diluting Amp1 1 100 in pre warmed Hyb B and mixing well b Place at 40 C until use Scale reagents according to the number of assays to be run Include 10 overage Use the table below as a guide Component 1 slide 10 slides Hyb B prewarmed 197 8 uL 1978 uL Ampl 2 2 uL 22 uL Total Volume 220 uL 2200 uL c Remove the slides from the staining dish and decant the Wash Buffer from the slides Make sure the section is wet and the hydrophobic rectangle is dry Always use the corner of a laboratory tissue to wipe the hydrophobic barrier dry If using Then do this Thermobrite a Before moving the slides to the Thermobrite add oven 200 uL of Working Amp1 to each slide b Move the slides to the 40 C Thermobrite close the lid to incubate for 15 min Tissue a Place slides on an aluminum slide rack and add culture 200 uL of Working Amp1 to each slide incubator b Put the slide rack into the 40 C humidified tissue culture incubator for 15 min 8 Wash the samples 3 times with Wash Buffer If using Then do this Thermobrite oven a Remove slides from the 40 C Thermobrite decant the Hyb B solution from slides and put the slides into a Tissue Tek rack submerged in Tissue Tek Staining Dish containing 200 mL of Wash Buffer and incubate at RT for 2 min Move the slide rack up and down several times to wash the sections
26. se the corner of a laboratory tissue to wipe the hydrophobic barrier dry If using Then do this Thermobrite a Before moving the slides to the Thermobrite add oven 200 uL of Working LP AP to each slide b Move the slides to the 40 C Thermobrite close the lid to incubate for 15 min Tissue a Place slides on an aluminum slide rack and add culture 200 uL of Working LP AP to each slide incubator b Put the slide rack into the 40 C humidified tissue culture incubator for 15 min 16 QuantiGene ViewRNA FFPE User Manual Section VI In Situ Hybridization Procedure Step Action 10 Wash the samples 3 times with Wash Buffer If using Then do this Thermobrite oven a Remove slides from the 40 C Thermobrite decant the Hyb C solution from slides and put the slides into a Tissue Tek rack submerged in Tissue Tek Staining Dish containing 200 mL of Wash Buffer and incubate at RT for 3 min Move the slide rack up and down several times to wash the sections Decant the Wash Buffer and add fresh Wash Buffer Incubate the slides in Wash Buffer at RT for 3 min and move the slide rack up and down several times Repeat step 10b Tissue culture incubator C Remove slides from the 40 C incubator decant the Hyb C solution from slides and transfer slides to a Tissue Tek Slide Rack submerged in a Tissue Tek Staining Dish containing 200 mL of Wash Buffer and
27. t Optional Aluminum slide rack for use in VWR 100493380 tissue culture incubator Optional Incubator or oven utilizing Panomics QS0700 QS0701 horizontal airflow and capable of 120V or QS0710 maintaining 80 C QS0711 220V Bright Field Microscope MLS Water Bath capable of maintaining MLS a MLS Major Laboratory Supplier b WARNING Formaldehyde is a poison and irritant Avoid contact with skin and mucous membranes c WARNING Highly volatile Use in fume hood QuantiGene ViewRNA FFPE User Manual Section III Recommendations for Experimental Design and Assay Optimization Section III Recommendations for Experimental Design and Assay Optimization Overview In this section we provide recommendations for identifying optimal pretreatment condition for the FFPE tissue in situ hybridization One optimization experiment is required for each tissue type of interest Because the quality of FFPE blocks vary depending on their source optimization is required We recommend preparing the FFPE blocks the same way you prepare the tissue sections you plan to use Optimization You will need to prepare ten 4 6 um thick FFPE tissue sections from a block which experiment was prepared in the same way fixation time section thickness and tissue type as the FFPE tissue of your interest The optimization experiment will enable you to identify the optimal condition for in situ hybridization Each slide will be treated with a different
28. tem is an in situ hybridization method for visualization of target RNA within individual cells This manual provides complete instructions for performing QuantiGene ViewRNA assays using formalin fixed paraffin embedded FFPE samples 4 6 um sections and 16 24 hours fixation QuantiGene The QuantiGene ViewRNA assay for FFPE samples is a novel RNA in situ ViewRNA Assay hybridization product based on patent pending Probe Set design and proprietary Basics signal amplification technology that offers 10 copy RNA sensitivity in individual cells 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 How It Works Z Target mRNA specific Probe Sets zz Zz 2 z as zZ zz PreAmp1 r Ampl 0 LP AP 2 eJ FFPE tissue section Target mRNA Step 1 Prepare Sample Step 2 Hybridize Probe Sets Step 3 Amplify Signal Step 4 Image Step 1 Prepare Sample FFPE tissue samples fixed for 16 24 hours at room temperature RT are sectioned to 4 6 um thickness and attached to a positively charged slide FFPE tissue sections are treated with a Pretreatment Solution followed by Protease digestion to allow target accessibility Step 2 Hybridize Probe Sets A gene specific Probe Set hybridizes to the target mRNA For clarity only single oligonucleotide pairs are shown However a typical Probe Set c
29. tissue sections to dry Drying can result in low or no signal Step Action 1 Set one Thermobrite to 40 C and wet the humidifying strips or use humidifying oven without CO 2 Prepare 1000 mL 1X PBS 3 Prepare 4 formaldehyde in 1X PBS in one Tissue Tek Staining Dish add 27 mL of 37 formaldehyde to 223 mL 1X PBS Pre warm Hyb A Hyb B and Hyb C buffers to 40 C 5 Thaw Probe Set PreAmplifier and Amplifier Place on ice until use Note If using the Optimization Kit the Probe Set is not required 6 Fill one Tissue Tek Staining Dish with 200 mL Gill s Hematoxylin 7 Prepare 3000 mL Wash Buffer by adding components to a 3 L capacity container in the following order and then mixing well 2 5LddH 0 27 mL Wash Comp 1 7 5 mL Wash Comp 2 ddH 0 to 3 L Note Adding the components in the order listed above to prevent precipitates from forming that would occur when adding Wash Comp 1 and Wash Comp 2 directly together QuantiGene ViewRNA FFPE User Manual Section VI In Situ Hybridization Procedure Step Action Prepare 1000 mL of 0 01 ammonium hydroxide in ddH O by adding 0 33 mL of 30 ammonium hydroxide into 999 67 mL ddH O and mix well WARNING 30 ammonium hydroxide is highly volatile Use in a fume hood Procedure Step Action Perform Protease digestion a Prepare Working Protease Solution by diluting the Protease 1 10
30. tter morphology more FastRed the better labeling 10 weak staining _ E stronger staining good morphology worst morphology PEN t Ki e gt Protease time min 20 40 Expected Results from Optimization Experiment 21 QuantiGene ViewRNA FFPE User Manual
31. y decanting the ddH O and refill with fresh ddH 0 Optional lf you want to stop at this point you can transfer the slide rack with slides into a Tissue Tek Staining Dish containing 200 mL 1X PBS at RT overnight QuantiGene ViewRNA FFPE User Manual 9 Section VI In Situ Hybridization Procedure Section VI n Situ Hybridization Procedure Overview The QuantiGene ViewRNA in situ hybridization protocol can be completed in approx Important Procedural Notes Before You Start 10 imately 8 hours including pretreatment procedure The instructions provided here are for processing 1 24 sections 1 section per slide If you process less than 6 slides at one time there will be reagent shortages Use the following procedure to fully remove solutions from slides drain solution from the slide surface by placing the slide on its edge on a laboratory wipe and lightly tap until liquid is removed from the hydrophobic barrier Before addition of Working Reagents to samples verify that hydrophobic barrier is dry If necessary use the corner of a laboratory wipe to wick away remaining solution Always use freshly prepared solutions in the Tissue Tek Staining and Clearing Agent dishes Do not save or reuse solutions unless directed to do so for example with the 4 formaldehyde solution Before opening reagents supplied in the microfuge tubes briefly centrifuge to collect contents at the bottom of the tube Do not allow
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