QuantiGene FlowRNA User Manual
Contents
1. a flow cytometer M Suspension cells with fixed RNA QuantiGene FlowRNA Reagent System Contents and Storage Conditions The components of the QuantiGene FlowRNA Assay Kit and their recommended storage conditions are listed below Refer to the package insert for quantities of individual components supplied Kit components have a shelf life of six months from the date of receipt Each QuantiGene FlowRNA Assay Kit is supplied in three separate parts based on storage temperature Box 1 Shipped on dry ice and store at 20 C Component Description Storage 100x Label Probe Mix Fluorescent dye labeled oligonucleotides in aqueous buffered 20 C solution containing Alexa 647 LP1 uses APC channel Alexa 488 LPA uses FITC channel and Alexa 750 LP6 uses APC Cy7 channel Box 2 Shipped on blue ice and store at 2 8 C Component Description Storage Target Probe Diluent Aqueous solution containing formamide and detergent 2 8 C PreAmp Mix DNA PreAmp1 PreAmp4 and PreAmp6 in aqueous solution containing 2 8 C formamide and detergent Amp Mix DNA Amp1 Amp4 and Amp6 in aqueous solution containing formamide 2 8 C and detergent Label Probe Diluent Aqueous solution containing detergent 2 8 C Wash Buffer Aqueous buffered solution 2 8 C Storage Buffer Aqueous buffered solution 2 8 C 1 5 mL microfuge tube Clear copolymer tube with a flat cap RNase free for low cell2. affymetrix eBioscience User Manual QuantiGene FlowRNA Assay in situ gene expression assay for flow cytometry p pe A P N 19221 Rev A ii QuantiGene Flow User Manual For research use only Not for use in diagnostic procedures Trademarks E Affymetrix and AX are registered trademarks of Affymetrix Inc QuantiGene is a registered trademark exclusively licensed to Affymetrix 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 FlowRNA Assay in Publications When describing a procedure for publication using this product please refer to it as the QuantiGene FlowRNA Assay from Affymetrix Disclaimer Affymetrix Inc reserves the r
3. Probe Set contains on average 20 oligonucleotide pairs Next signal amplification is achieved via a series of sequential hybridization steps The PreAmplifier molecules hybridize to their respective pair of bound Probe Set oligonucleotides then multiple Amplifier molecules hybridize to their respective PreAmplifier Finally Label Probe oligonucleotides conjugated to fluorescent dye hybridize to their corresponding Amplifier molecules A fully assembled signal amplification tree has 400 Label Probe binding sites When all target specific oligos in the Probe Set bind to the target mRNA transcript an 8 000 fold amplification is achieved Affymetrix currently offers three different amplification tree structures that allow simultaneous measurement of up to three different RNA targets for multicolor flow cytometric analysis Once the cells have been processed by the QuantiGene FlowRNA Assay the data can be collected and analyzed on a flow cytometer Sample Preparation Target Hybridization Signal Amplification ZZ Gene specific PreAmplifier Fluorescence labeled probes Zz Label Extenders LE Add labeled probes to cells Gene specific Blocking Probes BL r Amplifier Label proteins with Incubate cells with Hybridize with antibody optional gene specific probe PreAmplifier and sets Type 1 4 or 6 Amplifier DNA Fix and permeabilize cells Type 1 4 or 6 in suspension Process cells using
4. binding Room DO NOT SUBSTITUTE Temp Box 3 Fixation and Permeabilization Kit Shipped at room temperature and store at 2 8 C Component Description Storage Solution A Aqueous buffered solution 2 8 C Solution B Aqueous buffered solution 2 8 C Solution C Aqueous buffered solution containing detergent 2 8 C Solution D Aqueous buffered solution 2 8 C QuantiGene FlowRNA Probe Sets In addition to the QuantiGene FlowRNA Assay Kit Probe Set s specific to your RNA target s of interest must be purchased separately Refer to the Product Insert for more information Our current probe set catalog can be found at http www ebioscience com application flowrna htm Component Description Storage QuantiGene FlowRNA Probe sets RNA specific oligonucleotides designed against a target of 20 C interest and are compatible with TYPE 1 4 or 6 Signal Amplifiers 4 QuantiGene FlowRNA Assay User Manual WARNING All chemicals should be considered potentially hazardous We recommend that this product and its components be handled by those trained in laboratory techniques and be used according to the principles of good laboratory practice Required Equipment and Materials Not Supplied Required Equipment Material Source Part Number 1696 Paraformaldehyde PFA Electron Microscopy Sciences 15710 aqueous solution in 10 mL amber tube Methanol HPLC grade 99 996 pure Fisher S
5. for 1 5 mL microfuge tube Example shown Incubator Affymetrix QS0704 Metal heat block VWR 13259 002 Rotor with adaptor for 15 mL conical tube Adaptor for 1 5 mL microfuge tube Swinging bucket with adaptors for 15 mL conical tubes and 1 5 mL microfuge tubes With refrigeration to 4 C Examples shown Centrifuge Eppendorf 5810R Rotor Eppendorf A 4 44 Adaptor for 15 mL conical tube Eppendorf 5804 755 006 Adaptor for 1 5 mL microfuge tube Eppendorf 5804 750 004 NOTE Fixed angle centrifuge is NOT recommended Flow cytometer Two lasers blue 488 nm and red 633 nm or similar Detection optics optimized for FITC APC and APC Cy7 fluorophores Example shown BD LSRFortessa Aspiration system for washing e pw D Aspiration system Aspiration rate adjusted to 0 5 mL sec Can use in house vacuum line or vacuum pump Example shown Vacuum bottle Argos Technologies EV432 Aspirator Argos Technologies EV514 QuantiGene ViewRNA Temperature Validation Kit UN a NIST traceable thermometer with temperature probe in 1 5 mL microfuge tube shown on left a Affymetrix cat QV0523 14 QuantiGene FlowRNA Assay User Manual A3 Cytometer Setup The QuantiGene FlowRNA Assay Kit utilizes up to three fluorescent channels for detection of RNA on a flow cytometer To ensure optimal detection of RNA it is important that the cytometer is set up properly In a m
6. hole of the provided 1 5 mL tube containing 0 2 mL of deionized water E Wrap parafilm around the top of the 1 5 mL tube and probe to form a seal Avoid an excessive amount of parafilm around the sides of the tube otherwise it may not fit properly into the heat block F Turn on the digital thermometer device Step Action Step 3 Measure and adjust incubator temperature A Insert the 1 5 mL tube with probe and deionized water into the heat block that has been pre warmed in the incubator in Step 1 B Close the door making sure there is sufficient slack in the wiring C Wait 15 min for temperature to equilibrate D Record the temperature E If necessary adjust the dry incubator temperature settings so that the digital thermometer reads 40 C After adjustment allow the incubator and heat block to equilibrate Then recheck the temperature F Repeat Step E as needed IMPORTANT We recommend calibrating the incubator at least once a month to ensure accuracy Step 4 Assess incubator temperature uniformity Repeat step 3 to measure the temperature at multiple positions in the incubator to determine temperature uniformity IMPORTANT The temperature for all positions should be 40 1 C Step 5 Assess ramp up time A Open the incubator door for 1 min then close the door Measure the time needed for the temperature to return to 40 C and monitor the temperature profile during re
7. 7 Dilute Label Probe Mix 1 100 with Label Probe Diluent Store in the 40 C incubator and protect from light until ready to use Reagent 1 Tube 10 Tubes 24 Tubes 1 Label Probe 1 2 uL 12 uL 28 8 uL 2 Label Probe Diluent 118 8 uL 1188 uL 2851 2 uL Total 120 uL 1200 uL 2880 uL Step 8 Remove tubes from incubator and add 1 mL of Wash Buffer to each tube Invert tubes to mix Centrifuge the tubes at 800 x g at RT for 5 min to pellet the cells then aspirate the supernatant leaving a residual volume of 100 uL Briefly vortex tubes to resuspend the cell pellets Step 9 Repeat wash Step 10 Add 100 uL of Working Label Probe Solution prepared in Step 7 to each tube and briefly vortex tube to mix Place tubes in the pre warmed heat block inside the incubator Incubate at 40 C for 1 hour Step 11 Turn on the flow cytometer to allow the lasers to warm up Step 12 Remove tubes from incubator and add 1 mL of Wash Buffer to each tube Invert tubes to mix Centrifuge the tubes at 800 x g at RT for 5 min to pellet the cells then aspirate leaving a residual volume of 100 uL Briefly vortex tubes to resuspend the cell pellets Step 13 Repeat wash Step 14 Add 1 mL of Storage Buffer to each tube and invert tubes to mix Centrifuge the tubes at 800 x g at RT for 5 min to pellet the cells then aspirate supernatant leaving 100 uL residual volume Briefly vortex tubes to resuspend the cell pellets Protect samples from light NOTE Optional stop point store samp
8. ay User Manual Step Action Step 4 Repeat wash by adding 1 mL of Wash Buffer Optional add 1 uL mL of RNasin if used as stop point and invert tubes to mix Centrifuge the tubes at 800 x g at RT for 5 min to pellet the cells then aspirate leaving a residual volume of 100 uL Briefly vortex tubes to resuspend the cell pellets NOTE Optional stop point store samples overnight at 4 C in the dark Ill Signal Amplification and Detection Step Step 1 Perform the following tasks a Pre warm PreAmp Mix Amp Mix and Label Probe Diluent to 40 C b Equilibrate Wash Buffer Storage Buffer and the samples to RT Step 2 Add 100 uL of pre warmed PreAmp Mix with green cap to each tube and briefly vortex tubes to mix Place tubes in the pre warmed heat block inside the incubator Incubate at 40 C for 1 5 hours Step 3 Remove tubes from incubator and add 1 mL of Wash Buffer to each tube Invert tubes to mix Centrifuge the tubes at 800 x g at RT for 5 min to pellet the cells then aspirate leaving a residual volume of 100 uL Briefly vortex tubes to resuspend the cell pellets Step 4 Repeat wash Step 5 Add 100 uL of pre warmed Amp Mix to each tube and briefly vortex tubes to mix Place tubes in the pre warmed heat block inside the incubator Incubate at 40 C for 1 5 hours Step 6 During Amp Mix incubation thaw Label Probe Mix on ice Step
9. cientific A452 4 QuantiGene ViewRNA Temperature Affymetrix QV0523 Validation Kit Flow cytometer with the following MLS e g BD Biosciences Beckman Many components Two lasers blue 488 nm and red 633 nm or similar Detection channels optimized for FITC APC and APC Cy7 fluorophores Coulter e g Calibur from BD Biosciences FC500 from Beckman Coulter Refrigerated low speed centrifuge with swinging bucket for 15 mL tubes with inserts for 1 5 mL microfuge tubes MLS e g Eppendorf Eppendorf 5804 with A 4 44 Rotor swing bucket Incubator validated to maintain 40 1 C Affymetrix QS0704 120V QS0712 220V Metal tube rack heat block for 1 5 mL microfuge tubes placed inside incubator MLS e g VWR Fisher Scientific VWR 13259 002 Vacuum aspirator adjustable to 0 5 mL sec MLS e g Fisher Scientific VWR Fisher Scientific 07200564 Vortexer MLS e g VWR Fisher Scientific VWR 58816 121 Optional 15 mL polypropylene tubes MLS e g Fisher Scientific BD Fisher Scientific 14 959 70C Optional RNasin 1 Multiple Lab Supplier Experimental Guidelines Promega 1 Start with cell culture in good physiological conditions N2611 Cells should be in active growth phase to preserve RNA integrity and minimize cell lysis in processing If using freshly isolated primary cells make sure the cells are healthy aft
10. covery B Repeat the Step 5A two more times IMPORTANT Do not use the incubator for the assay if it takes more than 5 minutes to return to 40 C or if it overshoots by more than 2 C during recovery 23 24 QuantiGene FlowRNA Assay User Manual
11. d for RNA levels using the QuantiGene FlowRNA Assay Due to the nature of the RNA labeling protocol additional considerations for staining are warranted The following two options are recommended for staining with antibodies 1 Cells may be stained with fluorochrome conjugated antibodies before the Fixation and Permeabilization Procedure only if the fluorochromes are resistant to exposure to methanol Methanol resistant and methanol sensitive fluorochromes are listed in the table below Although some fluorochromes are resistant to methanol there may still be some loss in signal intensity therefore performance of a specific reagent in this assay should be determined empirically 2 If the antibody is not conjugated to a methanol resistant fluorochrome it may be possible to utilize an indirect staining protocol Cells may be stained with an unconjugated antibody before the Fixation and Permeabilization Procedure A secondary reagent fluorochrome conjugated anti Ig secondary antibody can then be used to detect the primary antibody Staining with the secondary reagent must be done after Signal Amplification and Detection It is important to note that only one unconjugated antibody from a given host may be used unless the isotypes of the unconjugated antibodies used are different In this case isotype specific secondaries must be used a pan anti mouse IgG secondary antibody will bind to all mouse IgG antibodies present on the cells Methanol Res
12. er purification 2 Selection of probe type and fluorescent channel for optimal RNA detection Type 1 650 probe set produces the most sensitive detection when compared to Type 4 488 and Type 6 750 probe sets Type 1 650 channel is about two to five folds more sensitive than the other two channels We recommend using the following probe type excitation line for the low medium and high expression genes respectively In addition background fluorescence in the Type 4 488 channel is higher compared to Type 1 650 and Type 6 750 channels Gene Expression Level Probe Type Cytometer Ex Em Laser line Low Type 1 633 650 668 Medium to High Type 4 488 495 519 Medium to High Type 6 633 749 775 3 QuantiGene FlowRNA Assay Controls Below are recommended assay controls that we recommend for first time users to perform Control Gene Catalog Number for Probe Set Positive Control B2M high expression in Human PBMC Type 1 VA1 10611 Type 4 VA4 13460 Type 6 VA6 11782 Mouse Type 1 VB1 11142 Type 4 VB4 14643 Type 6 VB6 12836 GAPDH medium expression Human in PBMC Type 1 VA1 10119 Type 4 VA4 10641 Type 6 VA6 10337 Mouse Type 1 VB1 10150 Type 4 VB4 10414 Type 6 VB6 10574 Negative assay control No Target Probe Not Applicable Bacterial DapB gene Type 1 VF1 11712 Type 4 VF4 10408 Type 6 VF6 10407 6 QuantiGeneG FlowRNA Assay User Manual Assay Procedure Th
13. ge tubes at 800 x g at 4 C for 5 min and aspirate the supernatant leaving a residual volume of 100 uL Briefly vortex tubes to resuspend cell pellet Step 14 To each tube add 1 mL of freshly prepared 496 PFA solution in Solution C prepared in Step 10a Invert the tubes to mix and incubate at RT for 30 min Invert tubes once during incubation Step 15 Centrifuge tubes at 800 x g at RT for 5 min and aspirate the supernatant leaving a residual volume of 100 uL Briefly vortex tubes to resuspend the cell pellets Step 16 To each tube add 1 mL of Solution D Invert tubes to mix and centrifuge tubes at 800 x g at RT for 5 min then aspirate the supernatant leaving a residual volume of 100 pL Briefly vortex tubes to resuspend the cell pellets Step 17 Repeat wash with Solution D and leave a residual volume of 100 jL Il Target Probe Hybridization Step Action Step 1 Dilute the target probes 1 20 with Target Probe Diluent Add 100 uL of diluted target probes to each tube from Step 17 Step 2 Briefly vortex tubes to mix Place tubes in the pre warmed heat block inside the incubator Incubate at 40 C for 2 hours Invert tubes to mix after 1 hour of incubation Step 3 To each tube add 1 mL of Wash Buffer and invert tubes to mix Centrifuge the tubes at 800 x g at RT for 5 min to pellet the cells then aspirate leaving a residual volume of 100 pL Briefly vortex tubes to resuspend the cell pellets 8 QuantiGene FlowRNA Ass
14. h RNA expression in some cases High Background Incorrect incubator temperature Incubator must be able to hold temperature at 40 1 C Minimize traffic to incubator Temperature tolerance is 1 2 C Ensure incubator has been stable at 40 C for at least 12 hours before starting assay as temperature may drift during initial incubator setup Measure temperature using QG ViewRNA Temperature Validation Kit from Affymetrix cat QV0523 Insufficient washing Ensure the Wash Buffer is used at room temperature Follow instructions of washing steps in the protocol Ensure uniform cell resuspension by vortexing Cytometer not set up properly Reduce voltage settings of your cytometer Note that this assay may require lower settings than typical antibody staining Over fixation Follow the protocol for proper fixation time Excessive Target Probe used Dilute Target Probe at a 1 20 dilution A2 Lab Setup Guide This guide illustrates the setup of typical equipment and their specifications for the QuantiGene FIowRNA Assay Consult your equipment manufacturers to make sure the equipment meets the specifications The major equipment include incubator swinging bucket centrifuge flow cytometer aspiration system for washing and temperature validation kit 13 Equipment Specifications Incubator with heat block inside Metal heat block a Validated to maintain 40x1 C Metal block
15. h plot such that the cells fall within the lower left quadrant see Figure 1 Figure 1 Unstained sample APC eFluor 780 3 For each two parameter plot create statistics windows tables according to your instrument s acquisition software and include the median for the x and y parameters 4 Stop collecting events and remove sample from the cytometer Place a single color sample on the cytometer and begin to collect events NOTE If your single color sample does not contain any negative events a small amount of unstained cells may be added to each of the single color samples just prior to loading the sample onto the cytometer Alternatively the MFI of the unstained sample run in Step 3B above must be noted for all parameters 1 Looking at the two parameter plots for the fluorochrome being collected adjust the quadrants so that the positive events are fully contained within either the lower right if the parameter is on the x axis Figure 2A left or the upper left 1f the parameter is on the y axis Figure 2B left quadrant 2 From the statistics window note the y medians if the parameter is on the x axis Figure 2A left or the x medians if the parameter is on the y axis Figure 2B left of the two populations 3 Adjust each of the compensation values for the fluorochrome being collected subtract its fluorescence out of each of the other channels being used in the experiment until the medians are the same Figure 2A a
16. ight 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 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 Contents iii About This User Mari al 3 etre RES Rid Hber e e EET CEBIT Y v3 1 Contacting Technical SUPOTE x i a babet du bic Per b Padre dtd 1 About the QuantiGene FlowRNA Assay Kits kk kk kk kk kK kK kK KK KK KK KK RR KK R KK K KK KK 1 sewi li SNN re9 ee a een ieee ane 2 QuantiGene FlowRNA Reagent System Contents and Storage Conditions 3 Required Equipment and Materials Not Supplied o an annaa ee ee 4 Experimental Guidelines asss sk s ca amin a n mana dd ad dh dh d ala RAN NEEN DESEES EF 4 Assay Procedure Fixation and Permeabilization ciel 6 ll Target Probe Hybridization lt cssc se mmm RO yy RR Cs s 7 Ill Signal Amplification and Detection kk kk kk kk kK KK KK KK eI I 9 Appendix AT Troubleshooting e eeraa sx alal aya an ala lala Re ig a ee Paga a RT al dg D P ala e a daji ask 10 A2 Lab setup guide 2 kk kk kk kk kk kk KK KK KK KK KK hr 13 A3 Cytometer Setup 2 da do ape x ob XR ue hee dob Rex ned EE N M Ro eee di Rogo and 14 A4 Incorporati
17. is protocol contains the following sections It can be performed in two days with optional stop points Day 1 I Fixation and Permeabilization II Target Probe Hybridization Day 2 III Signal Amplification and Detection IMPORTANT This protocol is optimized for the swinging bucket centrifuge We do not recommend using a fixed angle centrifuge as severe cell loss may result Fixation and Permeabilization NOTE The fixation and permeabilization can be performed in bulk if desired However polypropylene conical tubes e g Fisher Scientific cat 14 959 70C must be used DO NOT use polystyrene tubes such as FACS tube e g BD Falcon cat 352052 which would lead to low signal Step Action Step 1 Optional Antibody staining see Appendix for details Step 2 Perform the following tasks to prepare enough reagents for 24 samples a Pre warm Target Probe Diluent to 40 C b Equilibrate Solutions A B C and D to room temperature RT c Prepare a 4 paraformaldehyde PFA in Solution A NOTE PFA solution must be freshly prepared before use Add 30 mL of Solution A to a 50 mL conical tube and keep at RT in the dark Open an amber tube containing 10 mL of 1696 PFA stock solution and add the solution to the conical tube containing Solution A Mix well and keep at RT in the dark d Ensure incubator and heat block are calibrated to 40 C using a traceable digital thermometer see QuantiGene ViewRNA Tem
18. istant and Methanol Sensitive Fluorochromes Methanol Resistant Fluorochromes Methanol Sensitive Fluorochromes FITC PE Alexa Fluor 488 PE Cy5 Alexa Fluor 700 PE Cy5 5 Alexa Fluor 647 PE Cy7 eFluor 450 PerCP Pacific Blue PerCP Cy5 5 Pacific Orange PerCP eFluor 710 Brilliant Violet 421 APC Brilliant Violet 6057M APC Cy7 APC H7 APC Alexa Fluor 750 APC eFluor 780 Qdot reagents eFluor Nanocrystal NC reagents How to validate an antibody for QuantiGene FlowRNA Assay Each antibody needs to be validated with the assay protocol to confirm its compatibility Experimental conditions 1 Antibody staining alone 18 QuantiGene FlowRNA Assay User Manual 2 Antibody staining followed by RNA detection procedure without target probe Analyze and compare two samples for 1 Signal intensity of the protein staining 2 Percentage of positive cells For a qualified antibody 1 Sufficient separation between the positive and negative signals should be maintained after the RNA detection procedure to allow identification of desired populations 2 The percentage of desired cell populations should be comparable 19 A5 Expected Results This is an example of typical results of the QuantiGene FlowRNA Assay Experimental conditions Human PBMC were stimulated with 1 ug mL of bacterial lipopolysaccharides LPS and 2 5 ug mL of R 848 for 4 hours Cells were subjected to anal
19. les at 4 C in the dark Step 15 To analyze a sample pipette to mix Then transfer 50 100 uL sample to a reading tube containing 250 uL of 1X PBS and analyze on a flow cytometer For storage keep samples at 4 C in the dark Samples are stable for 1 week when stored at 4 C 10 QuantiGene FlowRNA Assay User Manual Appendix A1 Troubleshooting Observation Probable Cause Recommended Solution Poor Cell Recovery Low Cell Count on Cytometer Cell lysis due to cells in poor physiological condition during processing If using frozen cells thaw cells carefully following proper cell culture procedures Check cell viability using trypan blue or other viability dyes before assay Improper centrifuge and tube adaptor Use swinging bucket for centrifugation Do not use fixed angled centrifuges Use correct tube adaptor for proper g force Improper centrifuge settings Confirm settings are for RCF xg not RPM Set centrifuge to the speed specified in the protocol Improper tubes used Only use the 1 5 mL microfuge tubes provided with the kit Tubes from other vendors may result in significant cell loss High aspiration rate by pipetting or vacuum aspirator when removing supernatant Ensure vacuum setting for aspirator is low Aspirate no faster than 0 5 mL sec Place a 1 10 pL pipette tip on the tip of the aspirator to help reduce aspiration rate Follow the meniscus while a
20. nd 2B right 4 Stop collecting events and remove sample from the cytometer 16 QuantiGene FlowRNA Assay User Manual 5 Repeat for each of the single color samples Figure 2A CD8 APC only sample 8 8 f 5 3 Lu Lu w w o o a a lt lt CDs APC CD8 APC Yo Em i Upper Upper Aight gt Y Median nis i Unger Right Y dediar na iLawer Right gt Medan 4394 iLmwer Right gt Median 447 ibesyer Lait Ydiedien 7 85 E ower Lett YMadian 4 04 Figure 28 D4 APC efluor 730 only sample g gi i Bs 5 5 i 2 3 1 iu uo i o H lt lt a T a a o o APC ropar Len X Mpetan 1236 Upper Lett X Medam 314 oper Right Median n a i Upper Aight lt A Median niz i Lower Right KMedign nt i Hawar fight A Mediate nm itewerief Median 46 5 Lowerteft R idedian 38 7 D Confirm that the compensation settings are appropriate for the experimental samples 1 Place an experimental sample on the cytometer and begin collecting events 2 Looking at each of the two parameter plots confirm that the compensation is set correctly by looking at the medians 3 Stop collecting events and remove sample from the cytometer 17 A4 Incorporating Antibody Staining with the QG FlowRNA Assay Fluorochrome conjugated antibodies can be used to stain surface proteins for the purpose of immunophenotyping cells that will be further analyze
21. nes are properly permeabilized and the RNA is fixed within the cell QuantiGene FlowRNA Assay Kit Contains the reagents for running the assay QuantiGene ViewRNA Probe Set Contains gene specific probe sets for specified RNA targets Sample Type Suspension Cells including PBMC bone marrow cells and suspension cell lines Species mammalian Plex level Up to 3 RNA targets Assay format 1 5 mL microfuge tube Detection fluorescent Instrumentation for detection Standard flow cytometer equipped with blue 488 nm and red 633 nm or similar lasers and filter sets for FITC APC and APC Cy7 2 QuantiGene FlowRNA Assay User Manual How it Works The QuantiGene amp FlowRNA Assay is an expansion of Affymetrix s QuantiGene ViewRNA Assay The QuantiGene FlowRNA assay is an in situ hybridization assay enabling simultaneous detection of up to three RNA targets with high sensitivity and single cell resolution for use with a standard flow cytometer This assay can be combined with immunophenotyping using fluorochrome conjugated antibodies to allow further discrimination of specific cell subpopulations The QuantiGene FlowRNA Assay is based on branched DNA technology to amplify the signal of a RNA target The first hybridization of the assay allows the oligonucleotide probe set to bind to the target RNA sequence Subsequent signal amplification is predicated on specific hybridization of adjacent Probe Set pairs A typical
22. ng antibody staining with the QG FlowRNA Assay kk kK KK KIIR 17 AS Expected FesUlts 2 s auk ett o oe ue mils ar deleta RC La CEU eat Re eet e d 19 AG Validated cell lines llis RR Il 20 A7 Temperature Validation Procedure for Incubator kk kk kk KK iiis 21 iv QuantiGene FlowRNA Assay User Manual About This User Manual This manual is for anyone who has purchased a QuantiGene FlowRNA Assay Kit for the following sample types a Cultured mammalian suspension cells a Peripheral Blood Mononuclear Cells PBMC from cryopreserved or freshly collected whole blood and purified by Ficoll Suspension cells sorted by a flow cytometry sorting instrument e g BD FACSAria or purified by antibody coated magnetic beads Contacting Technical Support NOTE For the most current version of user documentation go to our website at www ebioscience com 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 ebioscience com Location Contact Information North America 1 888 810 6168 tech ebioscience com Europe tech ebioscience com About the QuantiGene FlowRNA Assay Kits The QuantiGene FlowRNA RNA Assay System consists of 3 modules each sold separately QuantiGene FlowRNA Fixation and Permeabilization Kit Contains reagents and buffers to ensure that the cell membra
23. or 5 min and aspirate the supernatant leaving a residual volume of 100 uL Briefly vortex tubes to resuspend the cell pellets Step 9 To each tube add 1 mL of ice cold methanol solution Briefly vortex the tubes to mix and incubate on ice for 10 min NOTE Optional stop point For storage transfer cells to 80 C after adding methanol Samples should be stable for 3 months However we do not recommend sample storage in methanol if antibody staining has been performed as in Step 1 Step 10 Perform the following tasks NOTE PFA solution must be freshly prepared before use a Prepare 496 PFA in Solution C Add 30 mL of Solution C to a 50 mL conical tube Keep at RT in the dark Open an amber tube containing 10 mL of 1696 PFA stock solution and add the solution to the conical tube containing Solution C Mix well and keep at RT in the dark b Prepare a 1 1 solution of methanol and 496 PFA in Solution C Add 13 mL of ice cold methanol to a 50 mL conical tube Add 13 mL of 4 PFA in Solution C from Step 10a Keep on ice in dark c Thaw the target probe sets at RT and then place on ice Step 11 Centrifuge tubes at 800 x g at 4 C for 5 min and aspirate the supernatant leaving a residual volume of 100 uL Briefly vortex tubes to resuspend the cell pellets Step 12 To each tube add 1 mL of ice cold methanol 496 PFA in Solution C 1 1 prepared in Step 10b Invert the tubes to mix and incubate on ice for 5 min Step 13 Centrifu
24. or the success of the QuantiGene FlowRNA Assay The incubator should be validated before use Improper hybridization temperature will result in high background and or weak signal Required materials Item Description Incubator Capable of maintaining temperature at 40 1 C e g Affymetrix cat QS0704 or QS0712 QuantiGene View Temperature NIST calibrated digital thermometer with Type K beaded probe Validation Kit Affymetrix cat QV0523 1 5 mL microfuge tube 1 5 mL microfuge tube used for assay included in the QuantiGene FlowRNA Assay Kit Metal heat block for 1 5 mL Metal block to hold the microfuge tube e g Fisher Scientific 11 microfuge tube 718 9Q Parafilm Major Laboratory Supplier 22 QuantiGene FlowRNA Assay User Manual Procedure Step Action Step 1 A Turn on the incubator Prepare the incubator B Set the temperature to 40 C C Put the metal heat block into the incubator near the center of the middle incubator D Allow the incubator and heat block to equilibrate for overnight Step 2 Assemble unit A Insert the battery to activate the digital thermometer B Use a pointed object e g a ballpoint pen to drill a hole at the lid of the 1 5 mL microfuge tube along the circular markings C Add 0 2 mL deionized water to the tube and then close the lid D Insert the Type K beaded probe into the digital thermometer and the other end into the pre drilled
25. perature Validation Kit for details e Pre chill methanol to 20 C f Set the temperature of centrifuge to 4 C Step 3 Hybridization must be performed in the 1 5 mL microfuge tubes provided For each reaction start by transferring 1 3 million cells into each tube Step 4 Centrifuge the tubes in a swinging bucket at 400 x g at 4 C for 5 min to pellet the cells Aspirate the supernatant leaving a residual volume of 100 uL Vortex tubes to resuspend cells evenly Add 1 mL Solution A then invert tubes a few times to mix NOTE Always leave a residual volume of 100 pL when aspirating in subsequent steps to avoid disturbing the cell pellet The lowest mark within the graduations on the microfuge tube is 100 pL Step 5 Centrifuge cells at 400 x g at 4 C for 5 min and aspirate the supernatant leaving a residual volume of 100 uL Step 6 Vortex tubes to resuspend cells evenly To each tube add 1 mL of 4 PFA in Solution A prepared in Step 2c Briefly invert the tubes to mix and incubate at RT for 60 min in the dark inverting the tubes 1 2 times during incubation Step 7 Centrifuge the tubes at 800 x g at 4 C for 5 min and aspirate the supernatant leaving a residual volume of 100 uL Briefly vortex tubes to resuspend the cell pellets Step Action Step 8 To each tube add 1 mL of Solution B and invert the tubes to mix Centrifuge cells at 800 x g at 4 C f
26. spirating down to the 100 uL mark on side of tube Rough handling Avoid excessive vortexing to prevent cell damage Vortex in pulses When pipetting to mix do so gently Clog Ensure cytometer is not clogging due to clumps of aggregated cells Try using gentle vortexing and pipette mixing to break up aggregates or use a 70 uL filter Incorrect cell count by flow cytometer Verify the flow rate of cytometer using beads of known concentration such as the Flow Cytometry Absolute Count Standard Bangs Laboratories cat 580 False Positive Population Compensation not set up correctly See Appendix section on how to set up compensation Low No Signal Cytometer not set up properly Check that your instrument is properly set up according to the manufacturer s recommendations Old 496 PFA solution was used in fixation Prepare fresh 496 PFA solution before use 11 Observation Probable Cause Recommended Solution Polystyrene tube e g FACS reading tube was used in fixation and permeabilization Use polypropylene tube e g Fisher Scientific 14 959 70C for fixation and permeabilization Insufficient number of cells recorded on cytometer Determine the number of total events to collect to ensure at least 100 1000 positive cells are recorded for example Total number of cells to count number of positives desired frequency of positives To co
27. t voltages for forward scatter and side scatter so that cells events of interest are on scale Set a threshold on forward scatter to eliminate debris from acquisition 2 Looking at the histogram plots ensure that the signals are on scale and adjust PMT voltages if necessary 3 Stop collecting events and remove sample from the cytometer C Place a single color sample on the cytometer and begin to collect events 1 Looking at the histogram plot for the fluorochrome being collected ensure that events are on scale for the fluorescent channel being collected and adjust PMT voltages if necessary 2 Stop collecting events and remove sample from the cytometer 3 Repeat for each of the single color samples D Confirm that the voltage settings are appropriate for the experimental samples 1 Place an experimental sample on the cytometer and begin collecting events 2 Looking at each of the histogram plots confirm that all fluorescent signals are on scale and adjust PMT voltages if necessary 3 Stop collecting events and remove sample from the cytometer 3 Setting compensation A If an autocompensation feature is available for the cytometer being used follow the manufacturer s instructions for use B If an autocompensation feature is not available or to set compensation manually 15 1 Place the unstained sample on the cytometer and begin to collect events 2 Looking at the two parameter plots create quadrant regions on eac
28. ulti color assay signal from a fluorophore often spills over into the other detection channels due to overlapping emission spectra The process of subtracting the spillover signal is called compensation The procedures below describe how to set proper PMT voltage and compensation for multi color RNA analysis Reagents and samples needed Calibration beads Unstained cells Single color samples Type 1 probe Alexa Fluor amp 647 only labeled cells Type 4 probe Alexa Fluor amp 488 only labeled cells Type 6 probe Alexa Fluor amp 750 only labeled cells Single color antibody labeled cells needed only if protein staining is performed 1 Ensure proper alignment following the instrument manufacturer s recommendations Alternatively calibration beads such as Spherotech Rainbow Calibration Particles 8 peak beads catalog number RCP 30 5A or Rainbow Fluorescent Particles 1 peak beads catalog number RFP 30 5A 1 peak may be used to check the linearity of the PMT PMT voltage range and alignment of the instrument 2 Setting PMT Voltages A Create an acquisition template workspace that includes a forward scatter versus side scatter plot one single parameter histogram plot for every fluorescent channel being used and a two parameter plot for every pairwise combination of fluorescent channels being used in the experiment B Place the unstained cell sample on the cytometer and begin to collect events 1 Adjus
29. unt 1 000 positive cells Frequency Total of of Positives cells to count 10 10 000 5 20 000 1 100 000 Incorrect preparation of signal amplification reagents Make sure PreAmp Amp and Label Probe are used in the specified order Incorrect incubator temperature a Incubator must be able to hold temperature at 40 1 C u Use 1 5 mL heat block for hybridization u Minimize traffic to incubator Temperature tolerance is 1 2 C a Ensure incubator has been stable at 40 C for at least 12 hours before starting assay as temperature may drift during initial incubator setup Measure temperature using QG ViewRNA Temperature Validation Kit from Affymetrix PN QV0523 Incorrect Wash Buffer Do not substitute Wash Buffer with Storage Buffer Incorrect diluents used Ensure Target Probe Diluent is used with Target Probe and Label Probe Diluent used with Label Probe Incorrect dilution of Target Probe Label Probe a Ensure Target Probe was added to Target Probe Diluent at a 1 20 dilution Ensure Label Probe Mix was added to Label Probe Diluent at a 1 100 dilution 12 QuantiGeneG FlowRNA Assay User Manual Observation Probable Cause Recommended Solution Gene expressed at very low level u Check biological model and confirm gene is expressed in sample by running QuantiGene 2 0 Assay for lysate Keep in mind that protein expression might not correlate wit
30. ysis by the QuantiGene FlowRNA Assay Cells were stained with anti human CD14 FITC to identify monocytes and labeled with probes for the RNA expression of IL I beta IL 6 IL 8 and TNF alpha Cells in the monocyte gate were used for analysis CD14 staining is shown on the y axis and RNA labels shown on the x axis Result Control Induced Q2 73 3 CD14 Protan g CD44 Protein IL 1 beta l 40 m o3 Tu 445 10 10 10 Td ic 40 107 10 104 IL 1 beta RNA IL 1 beta RNA 4701 Q2 10 oos 0 570 10 i i IL 6 8 8 1 IL 8 amp amp o o 10 ap v at sef IL 8 RNA 10 10 c 2 TNF alpha amp Ew 8 3 10 10 10 n 10 10 10 TNF alpha RNA TNF alpha RNA 20 QuantiGene FlowRNA Assay User Manual A6 Validated Cell Lines The QuantiGene FlowRNA Assay has been validated with the following cell types Primary cells Human PBMC cryopreserved and freshly purified by Ficoll u Human T cells CD3 u Human B cells CD19 Human monocytes CD14 Mouse bone marrow cells Cells were purified by antibody coated magnetic beads Cultured suspension cell lines NKT human natural killer cell line u Jurkat human lymphocytic cell line U937 human monocytic cell line K562 human myelocytic cell line M1 mouse myeloid cell line 21 A7 Temperature Validation Procedure for Incubator Temperature control is critical f
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