PrimeFlow™ RNA Assay User Manual and Protocol
Contents
1. OSZ4V VNYW g W ZU LID OSZAV VNYW g W ZULID 0SZ4V 4 u zueu_p OSZAV VNYW g awAzueI5 G G o o 5 O 5 N J N lt 00 lt 3 U 3 m wo o Set Set m m lt A 5 S 5 j 2 5 O SST o10N 4 eX9 V VNYW 79 1 887 o10N 4 eX9 V VNMU 79 1 A I I o o o 9 9 U S 3 o o D 2 5 T 5 o o T c o 2 e E U1 U1 o o 5 1 019 6 0 34 842 019 10 34 842 17 Appendix 5 Validated cells and recommended positive control genes Human peripheral blood whole blood or PBMC fresh and cryopreserved RPL13A B2M Mouse splenocytes tissue fresh and cryopreserved ACTB RPL13A Mouse thymocytes tissue ACTB RPL13A Mouse bone marrow ACTB Human monocytic lymphoma U937 RPL13A B2M Human T cell lymphoma Jurkat RPL13A B2M Human cervical carcinoma HeLa RPL13A GAPDH Human lung carcinoma PC9 RPL13A GAPDH adherent cells limited testing Appendix 6 Temperature validation procedure for incubator Temperature control is critical for the success of the PrimeFlow RNA assay Improper hybridization temperature will result in high background or weak signal or both The incubator should be validated before use following these instructions This temperature validation procedure is appropriate for both tube and plate based proto2. markings on the 1 5 mL tubes provided in the kit to assist Note PrimeFlow RNA PreAmp Mix PrimeFlovv RNA Amp Mix and diluted Label Probes should be pipetted directly into the 100 uL of residual volume and samples should be mixed well before incubating Do not pipette these solutions onto the walls of the tubes 28 Pre warm samples and PrimeFlow RNA Wash Buffer to room temperature 29 Pre warm PrimeFlow RNA PreAmp Mix PrimeFlow RNA Amp Mix and PrimeFlow RNA Label Probe Diluent to 40 C 30 Thaw PrimeFlow RNA Label Probes 100X on ice in the dark Note This can be done during the Amp Mix incubation Step 34 31 Add 100 uL of PrimeFlow RNA PreAmp Mix directly into the cell suspension for each sample and briefly vortex to mix Incubate for 1 5 hours at 40 C 32 Add 1 mL of PrimeFlow RNA Wash Buffer to each sample invert to mix and spin down at 800 x g for 5 minutes Aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently 33 Repeat Step 32 two times for a total of three washes 34 Add 100 uL of PrimeFlow RNA Amp Mix directly into the cell suspension for each sample and briefly vortex to mix Incubate for 1 5 hours at 40 C 35 Add 1 mL of PrimeFlow RNA Wash Buffer to each sample invert to mix and spin down at 800 x g for 5 minutes Aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently 36 Repeat Step 35 37 Dilute PrimeFlow RNA
3. 4 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 the ViewRNA Temperature Validation Kit Affymetrix cat no QV0523 3 Insufficient washing 2 3 Ensure the PrimeFlovv RNA Wash Buffer is used at room temperature Follow instructions for washing steps in the protocol Ensure uniform cell resuspension by gentle vortexing 4 Cytometer not set up properly Reduce voltage settings on your cytometer Note that this assay may require lower settings than typical antibody staining 5 Over fixation Follow the protocol for proper fixation time 6 Excessive Target Probe used Dilute Target Probes at a 1 20 dilution Appendix 2 Instrument and equipment setup guide This guide illustrates the setup of typical equipment and their specifications for PrimeFlow RNA Assay Consult your equipment manufacturers to make sure the equipment meets the specifications The major equipment include incubator svvinging bucket centrifuge flow cytometer aspiration system for vvashing and temperature validation kit Specifications 1 Incubator with heat block inside 1 Validated to maintain 40 1 C Equipment 2 Metal heat block for 1 5 mL microcentrifuge tube Example shown Incubator Affymetrix
4. Fluor 647 high sensitivity that is best for low or unknown expression levels Type 4 Alexa Fluor 488 intermediate to low sensitivity that is best for medium to high expression levels Type 6 Alexa Fluor 750 intermediate to low sensitivity that is best for medium to high expression levels data for Type 6 Alexa Fluor 750 probe sets should be collected in the APC eFluor 780 channel with a 780 60 bandpass filter or equivalent The autofluorescence of cells is increased following this protocol when compared to live cells or fixed cells This primarily impacts the FITC PE eFluor 450 and eFluor 506 channels Other channels up to approximately 650 nm may also be affected depending on the cell type laser line and instrument settings Please take this into consideration when setting voltages and when designing multicolor panels Please contact eBioscience Technical Support tech ebioscience com for more information Protect samples from light after they have been stained with fluorochrome conjugated antibodies and labeled for RNA We recommend performing the assay in two days as follows Day 1 Antibody staining Fixation and permeabilization Target Probe hybridization Day 2 Signal amplification Flow cytometric analysis Fluorochrome compatibility I 4 2 4 Organic fluorochromes are compatible with this assay such as FITC eFluor 450 eFluor 660 and Alexa Fluor 700 BV dyes are
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6. PrimeFlow RNA Storage Buffer RNase Inhibitor 2 100X Store at 20 C RNase Inhibitor 1 1 000X Positive Control Target Probe Sets 20X one for each RNA detection channel PrimeFlow RNA Label Probes 100X Materials required but not included Flow Cytometry Staining Buffer cat no 00 4222 Fluorochrome conjugated antibodies as needed Fixable Viability Dye as needed 12 x 75 mm polystyrene tubes e g Corning cat no 352008 PrimeFlow Compensation Kit cat no 88 17001 OPTIONAL IC Fixation Buffer cat no 00 8222 OPTIONAL 96 well v bottom polystyrene plate cat no 44 17005 see Appendix A7 for modified protocol Instruments and equipment Refer to Appendix A2 of this user manual Experiment duration Day 1 6 8 hours Day 2 6 hours Antibody staining Signal amplification Fixation and permeabilization Flow cytometric analysis Target probe hybridization Experimental procedure Note This procedure is written based on the use of the 1 5 ml tubes provided in the kit throughout the assay The use of these tubes is important during the hybridization and signal amplification steps to control residual volumes However staining with antibodies and or fixable viability dyes as well as fixation and permeabilization Steps 1 16 may be done in bulk and in any tube desired If these procedures are done in bulk use volumes such that cells do not exceed 1 x 10 cells mL To
7. cat nos QS0704 or QS0712 Metal heat block VWR cat no 13259 002 2 Refrigerated swinging bucket centrifuge Svvinging bucket centrifuge with adaptors for 15 mL conical tubes and 1 5 mL microcentrifuge tubes Optional with refrigeration to 4 C Examples shown Centrifuge Eppendorf model 5810R Rotor Eppendorf model A 4 44 Adaptor for 15 mL conical tube Eppendorf model 5804 755 006 Adaptor for 1 5 mL microcentrifuge tube Eppendorf model 5804 750 004 Rotor with adaptor for Adaptor for 1 5 mL 15 mL conical tube microfuge tube 3 Flow cytometer 1 Two lasers blue 488 nm and red 633 nm or similar 2 Detection optics optimized for FITC APC and APC eFluor 780 APC Cyanine7 Example shown BD LSRFortessa 4 Aspiration system for washing Aspiration rate adjusted to 0 5 mL sec Can use in house vacuum line or vacuum pump Example shown Vacuum bottle Argos Technologies model EV432 Aspirator Argos Technologies model EV514 NIST traceable thermometer with temperature probe in 1 5 mL microcentrifuge tube shown on left Affymetrix cat no QV0523 5 ViewRNA Temperature Validation Kit Appendix 3 Cytometer setup The PrimeFlow RNA assay 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 multicolor assay signal from a given
8. for every fluorochrome used in the experiment FMO controls facilitate assessment of background on gated events and allow fine tuning of compensation for optimal performance Negative controls such as samples with the target probe omitted or samples labeled with a target probe not expressed in the cells of interest e g DapB a bacterial gene are highly recommended Negative control samples comprised of or containing cells known to be negative for the gene of interest e g unstimulated are also recommended to confirm specificity of the target probes If using whole blood there is no need to pre lyse red blood cells however due to changes in forward scatter and side scatter properties of granulocytes the use of antibodies to distinguish leukocyte subpopulations is recommended Please see Appendix A4 for examples of sample set up and experimental design This protocol is for use with 1 5 mL microcentrifuge tubes See Appendix 7 for the 96 well plate protocol Materials included Store at room temperature PrimeFlow RNA tubes 1 5 mL microcentrifuge tubes Store at 2 8 C PrimeFlow RNA Fixation Buffer 1A PrimeFlow RNA Fixation Buffer 1B PrimeFlow RNA Permeabilization Buffer 10X PrimeFlow RNA Fixation Buffer 2 8X PrimeFlow RNA Wash Buffer PrimeFlow RNA Target Probe Diluent PrimeFlow RNA PreAmp Mix PrimeFlow RNA Amp Mix PrimeFlow RNA Label Probe Diluent
9. histogram plot for every fluorescent channel used and a two parameter plot for every pairwise combination of fluorescent channels used in the experiment Place the unstained cell sample on the cytometer and begin to collect events a Adjust 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 Make a note of these settings b Looking at the histogram plots ensure that the signals are on scale and adjust PMT voltages if necessary c Stop collecting events and remove sample from the cytometer Place a single color sample on the cytometer and begin to collect events Note UltraComp eBeads may require different forward and side scatter voltage settings to be visualized Adjust these settings as needed and make a note of these settings a Looking at the histogram plot for the fluorochrome collected ensure that events are on scale for the fluorescent channel collected and adjust PMT voltages if necessary b Ensure that the signal is at least 0 5 log brighter in the channel of interest than in all other channels Adjust other PMT voltages as necessary c Stop collecting events and remove sample from the cytometer d Repeat for each of the single color samples Contirm that the voltage settings are appropriate for the experimental samples a Place an experimental sample on the cytometer and begin collecting events Note If
10. oy Y 88t o10N 4 ex V 88t o10N 4 ex V 88t o10N 4 Ex ly 88t 10 ex V VNdul eyd e INL VNuw eud e INL VNuu eyd e INL VNuw eydye INL U m o U m o o o lt lt o o o o N p le nuuns 5 1 019 510 34 842 019 510 14 842 15 L9 VNYW quizo g awAzues5 6802 VNYW 29 1 usun duues 0 02 02 d 10SI EPZ DL aqoid ou d 10SI EPZ L 2802 qoid ou usun 0 1 02 ou d 20s 61 Bi VNYW quuzD g uu zue1D esa VNMU 9 1 usun OWN OSY 10 81 ZN g awAzues5 6802 VNYW Z9 LUDSUn OWS 032 JON 4 EX V z L9 VNYW 40120 _ 6802 VNYW 9 1 ulnsun OI Zeuluek gt 3d 91 5 VNYW 40120 g 2 m VNYW 9 4 usun OWN 019 4 n 49 44 Sl 9 4 VNYW quizo g uu zue1D 6802 _ usun OWS 887 JO vl OI VNYW 4120 g 2 802 VNYW 9 I N 5 5 10 02 21 adAjos e756 4 ou adAjosi PZ DL 4802 aqojd ou LUS 0 402 qoid ou d 20s 21 VNYW quuzD g 2 e8dD VNYW 9 J wgs OWN OSY 10 Ll 19 1 g uu zue1D esq VNdu Z9 5 ONT 032 JON 4 Boy 01 49 1 VNYW 0120 e8dD VNYW 9 4 5 ON ulue D 3d 6 L9 VNYW 0120 g awAzuel5 VNYW 9 1 Us OW 019 40N 49 4d 8 49 1 VNYW quizo g W zuLID e8dD m wgs OWS 887 JON 4 E
11. settings S dg a WS are 15777 for the experimental ea Se nu 0 samples a Adjust forward and side scatter 7 7 57 E 7 7 in 907 EE ep Il2a Do not change voltages for any of the fluorescent PMT channels b Place an experimental sample on the in SSC cytometer and begin collecting events e c Looking at each of the tvvo parameter 5 S e plots confirm that the compensation is 9 I Zil set correctly by lookino at the medians z d Stop collecting events and remove A A sample from the cytometer 6 After setting compensation you are ready APC APC to acquire your samples Q1 APC Median 258 Q1 APC Median 0 519 7 Use FMO controls after acquisition and 2577715077 SERGE during analysis to optimize compensation Figure A3 2 Single color samples for APC top row or APC eFluor settinos 780 bottom row Uncompensated left or compensated right data are shown Examples of expected results Appendix 4 90S49 Ou Nil VNYW PNI eydje INL esq VNYW BINL usun 5 02 9 22 90S42 414 d 10SI LOD aqojd ou d 10SI LOD esa qo d ou uunsun 021 02 Ou adAjos 77 Nil VNYW DNA eydje INL esq VNYW BINL usun OW 909 Jong 2 905 Ou VNYwW Nil eudie dN1 esq VNYW BINL usun ONJ OSY OZ 90549 Ou ewwep Nil eydje
12. supernatant and resuspend cells in the residual volume by vortexing 48 If a plate adapter is used for sample acquisition resuspend cells in an appropriate volume of PrimeFlow RNA Fixation Buffer or Flow Cytometry Staining Buffer and analyze on a flow cytometer Otherwise transfer samples to 12 x 75 mm polystyrene tubes resuspend cells in an appropriate volume of Storage Buffer or Flow Cytometry Staining Buffer and analyze on a flow cytometer Note Samples may be stored before analysis If samples have been stained with antibodies conjugated to tandem dyes we recommend storing the samples in IC Fixation Buffer by mixing 100 uL of cells with 100 uL of IC Fixation Buffer Store samples in the dark at 2 8 C for up to three days Quick guide PrimeFlow RNA Assay in 96 well plates Day 1 1 Surface stain cells with antibody and or fixable viability dye 2 Wash once with Flow Cytometry Staining Buffer Spin cells at 500 x g for 4 minutes 3 Fix cells in PrimeFlow RNA Fixation Buffer 1 for 30 minutes at 2 8 C 4 Wash twice with 1X PrimeFlow RNA Permeabilization Buffer with RNase Inhibitors Spin cells at 1 000 x g for 4 minutes 5 Intracellularly stain cells with antibody for 30 minutes at 2 8 C 6 Wash once with 1X PrimeFlow RNA Permeabilization Buffer with RNase Inhibitors Spin cells at 1 000 x g for 4 minutes 7 Fix cells in 1X PrimeFlow RNA Fixation Buffer 2 for 60 minutes at room temperature 8 Wash twice with PrimeFlow RNA W
13. viability dye Wash once with Flow Cytometry Staining Buffer Spin cells at 500 x g for 5 minutes Fix cells in PrimeFlow RNA Fixation Buffer1 for 30 minutes at 2 8 C Wash twice with 1X PrimeFlow RNA Permeabilization Buffer with RNase Inhibitors Spin cells at 800 x g for 5 minutes Intracellularly stain cells with antibody for 30 minutes at 2 8 C Wash once with 1X PrimeFlow RNA Permeabilization Buffer with RNase Inhibitors Spin cells at 800 x g for 5 minutes Fix cells in 1X PrimeFlow RNA Fixation Buffer 2 for 60 minutes at room temperature Wash twice with PrimeFlow RNA Wash Buffer Spin cells at 800 x g for 5 minutes 9 Perform Target Probe hybridization for 2 hours at 40 C Invert to mix after 1 hour 10 Wash once with PrimeFlow RNA Wash Buffer Spin cells at 800 x g for 5 minutes 11 Wash once with PrimeFlow RNA Wash Buffer with RNase Inhibitor 1 Spin cells at 800 x g for 5 minutes ES Store samples overnight Day 2 EZ 14 15 16 Perform PreAmp hybridization for 1 5 hours at 40 C Wash three times with PrimeFlovv RNA Wash Buffer Spin cells at 800 x g for 5 minutes Perform Amp hybridization for 1 5 hours at 40 C Wash twice with PrimeFlow RNA Wash Buffer Spin cells at 800 x g for 5 minutes 17 Perform Label Probe hybridization for 1 hour at 40 C 18 19 20 Wash tvvice with PrimeFlow RNA Wash Buffer Spin cells at 800 x g for 5 minutes Wash once with PrimeFlow R
14. 8 Repeat Step 17 Note It is critical that the residual volume is as close to 100 uL as possible Use the markings on the 1 5 mL tubes provided in the kit to assist 19 OPTIONAL Cells may be stored in PrimeFlow Wash Buffer with RNase Inhibitor 1 overnight in the dark at 2 8 C To do so add RNase Inhibitor 1 1 000X to PrimeFlow RNA Wash Buffer at a 1 1 000 dilution and use in Step 18 Note You will need 1 mL of this buffer per sample Prepare this buffer in bulk to accommodate all samples This buffer should be prepared fresh Dispose of any unused buffer Day 1 Target probe hybridization Note It is critical that the residual volume after all washes be as close to 100 uL as possible Use the markings on the 1 5 mL tubes provided in the kit to assist Note Diluted Target Probes should be pipetted directly into the 100 uL of residual volume and samples should be mixed well before incubating Do not pipette solutions onto the walls of the tubes 20 Thaw Target Probes including Positive Control Target Probe Sets 20X at room temperature 21 Pre warm PrimeFlow RNA Target Probe Diluent to 40 C 22 Dilute Positive Control Target Probe Sets 20X 1 20 in PrimeFlow RNA Target Probe Diluent Mix thoroughly by pipetting up and down Note You will need 100 uL of diluted Target Probes for each sample If you are adding more than one Target Probe per sample adjust the volume of the PrimeFlow RNA Target Probe Diluent accordingly so that
15. D affymetrix eBioscience PrimeFlovv RNA Assay User Manual and Protocol eBioscience GeneChip USB PrimeFlovv RNA Assay User Manual and Protocol For Research Use Only Introduction PrimeFlow RNA assay is an in situ hybridization assay that combines the power of branched DNA technology with the single cell resolution of flow cytometry This assay enables the simultaneous detection of up to three RNA targets in combination with immunophenotyping for cell surface and intracellular proteins using tluorochrome conjugated antibodies to allow further discrimination of specific cell subpopulations In the PrimeFlow RNA assay branched DNA technology is used to amplify the detection of a RNA transcript rather than the target RNA itself In the first hybridization step of the assay a gene specific oligonucleotide Target Probe set that contains 20 40 probe palrs binds to the target RNA sequence An individual probe pair is designed to bind adjacent to each other in order for signal amplification to take place Signal amplification is then achieved through a series of sequential hybridization steps The PreAmplifier molecules confer an additional level of specificity because they will hybridize to the Target Probes only when both halves of a respective probe pair have bound to their target sequence Multiple Amplifier molecules subsequently hybridize to their respective PreAmplifier Finally Label Probe oligonucleotides conjugated to a fluor
16. Fixation Permeabilization table and refer to the column for After IC Fixation and Perm Wash to determine if the antibody clone will recognize a fixed epitope Experimental design guidelines 1 To ensure proper assay performance use Positive Control Probe Sets in every experiment For your convenience these probe sets are included with PrimeFlow RNA Assay RPL13A for human leukocytes and beta actin ACTB for mouse tissues See Appendix A5 for specific cell types and other recommended positive control genes For proper compensation we recommend using PrimeFlow Compensation Kit cat no 88 1 001 Please refer to the product data sheet for instructions on use Alternatively cells stained with positive control probe sets may be used for single color compensation samples Do not use fluorochrome conjugated antibodies to set compensation for RNA signal If using a combination of cells and beads as compensation controls be sure to use the appropriate negative population for settino compensation e for beads use the negative bead population for cells use unlabeled cells that have undergone the PrimeFlow RNA assay Please contact eBioscience Technical Support tech ebioscience com for more information Fluorescence Minus One FMO controls are highly recommended The FMO control is a sample that contains all but one of the fluorochromes used in the experiment As with single color controls there should be an FMO control
17. Flow RNA Fixation Buffer 2 8X with 875 uL of PrimeFlow RNA Wash Buffer per sample Mix gently by inverting Note You will need 1 mL of this buffer per sample Prepare this buffer in bulk to accommodate all samples For example for 10 samples combine 1 25 mL of PrimeFlow RNA Fixation Buffer 2 8X with 8 75 mL of PrimeFlow Wash Buffer Avoid vortexing or vigorously shaking this buffer This buffer should be prepared fresh Dispose of any unused buffer 14 Add 1 mL of 1X PrimeFlow RNA Fixation Buffer 2 to each sample and invert to mix and then incubate for 60 minutes in the dark at room temperature Note t is important to fix the samples at room temperature Do not perform this step on ice 15 OPTIONAL Cells may be stored in 1X PrimeFlow RNA Fixation Buffer 2 overnight in the dark at 2 8 C instead of incubating for 60 minutes at room temperature i e skip Step 14 16 Spin down at 800 x g for 5 minutes then aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently Note If staining fixation and permeabilization were performed in bulk the cells should be transferred into the 1 5 mL tubes provided in the kit during the following wash steps and before storage overnight 17 Add 1 mL of PrimeFlow RNA Wash Buffer to each sample invert to mix and spin down at 800 x g for 5 minutes then aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently 1
18. Label Probes 100X 1 100 in PrimeFlow RNA Label Probe Diluent Note You will need 100 uL of diluted Label Probes for each sample Prepare diluted Label Probes in bulk to accommodate all samples 38 Add 100 uL of diluted Label Probes directly into the cell suspension for each sample and briefly vortex to mix and then incubate for 1 hour at 40 C 39 Add 1 mL of PrimeFlow RNA Wash Buffer to each sample invert to mix and spin down at 800 x g for 5 minutes Aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently 40 Repeat Step 39 41 42 Quick guide Add 1 mL of PrimeFlow RNA Storage Buffer or Flow Cytometry Stainino Buffer to each sample invert to mix and spin down at 800 x g for 5 minutes Aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently Transfer samples to 12 x 75 mm polystyrene tubes resuspend in an appropriate volume of PrimeFlovv RNA Storage Buffer or Flow Cytometry Staining Buffer and analyze samples on a flow cytometer Note Samples may be stored before analys s If samples have been stained vvith ant bodles conjugated to tandem dyes we recommend storing the samples in IC Fixation Buffer by mixing 100 ul of cells with 100 uL of IC Fixation Buffer Store samples in the dark at 2 8 C for up to three days PrimeFlow RNA Assay in 1 5 mL tubes Day 1 E WN Ul Surface stain cells with antibody and or fixable
19. NA Storage Buffer Spin cells at 800 x g for 5 minutes Analyze samples on a flow cytometer Appendix 1 Troubleshooting A Poor Cell Recovery Low 1 Cell Count on Cytometer Cell lysis due to cells in poor physiological condition during processing 1 If using frozen cells thaw cells carefully following proper cell culture procedures and rest cells for 30 minutes at 37 C before starting the assay Make sure cells are cryopreserved while still in log phase growth 2 Check cell viability usino trypan blue or other viability dye before beginning the assay 2 Improper centrifuge settings 1 Confirm settings are for RCF x g not RPM 2 Set centrifuge to the speed specified in the protocol 3 Improper tubes used Only use the 1 5 mL tubes provided with the kit Tubes from other vendors may result in significant cell loss 4 High aspiration rate by pipetting or Ensure vacuum setting for aspirator is low Aspirate vacuum aspirator when removing no faster than 0 5 mL sec Place a 10 uL pipette tip supernatant on the tip of the aspirator to help reduce aspiration rate While aspirating follow the meniscus down to the 100 uL mark on the side of the tube 5 Rough handling Avoid excessive vortexing to prevent cell damage Vortex in short pulses 6 Incorrect cell count by flow Verify the flow rate of cytometer using beads of known cytometer concentration such as the Flow Cytometry Absolute Count Sta
20. Probes l Ensure Target Probes are diluted 1 20 with PrimeFlow RNA Target Probe Diluent Ensure PrimeFlow RNA Label Probes 100X are diluted 1 100 with PrimeFlow RNA Label Probe Diluent 8 Gene expressed at very low level 9 Incorrect or insufficient RNase Inhibitor used Check biological model and confirm gene is expressed in sample by an alternative method such as QuantiGene 2 0 Assay for lysate Keep in mind that protein expression might not correlate with RNA expression in some cases Ensure that 1X PrimeFlow RNA Permeabilization Buffer contains both RNase Inhibitor 1 1 000X at a final dilution of 1 1 000 and RNase Inhibitor 2 100X at a final dilution of 1 100 Ensure that the PrimeFlow RNA Wash Buffer for overnight sample storage in Step 19 or Step 25 or both contains RNase Inhibitor 1 at a final dilution of 1 1 000 C High Background 1 Compensation not set up correctly See Appendix for how to set compensation Use the PrimeFlow Compensation Kit sold separately or samples labeled with one each of the included Positive Control Target Probe Sets 20X to set compensation Do not use fluorochrome conjugated antibodies to set compensation for RNA detection 2 Incorrect incubator temperature See Appendix for how to validate an incubator Incubator must be able to hold temperature at 40 1 C 3 Use a metal heat block for hybridization
21. X PrimeFlow RNA Fixation Buffer 2 overnight in the dark at 2 8 C instead of incubating for 60 minutes at room temperature i e skip Step 15 Spin down at 1 000 x g for 4 minutes then discard supernatant and resuspend cells in the residual volume by vortexing Add 200 uL of PrimeFlow RNA Wash Buffer to each well pipet to mix and spin down at 1 000 x g for 4 minutes then discard supernatant and resuspend cells in the residual volume by vortexing Repeat Step 18 OPTIONAL Cells may be stored in PrimeFlow RNA Wash Buffer with RNase Inhibitor 1 overnight in the dark at 2 8 C with lid on To do so add RNase Inhibitor 1 1 000X to PrimeFlow RNA Wash Buffer at a 1 1 000 dilution and use in Step 19 Note You will need 100 uL of this buffer per well Prepare this buffer in bulk to accommodate all samples This buffer should be prepared fresh Dispose of any unused buffer Day 1 Target probe hybridization Note It is critical that the residual volume after all washes does not exceed 10 ul Note Diluted Target Probes should be pipetted directly into the cell suspension and samples should be mixed well before incubating The total volume for Target Probe hybridization should be 200 pL per well 21 22 23 24 Thaw Target Probes including Positive Control Probe Sets 20X at room temperature Pre warm PrimeFlovv RNA Target Probe Diluent to 40 C Dilute Target Probes 1 20 in PrimeFlow RNA Target Probe Diluent Mix thoroughl
22. ace the 1 5 mL tube containing the probe from Step l into the prewarmed heat block from Step 1 2 Close the door making sure there is sufficient slack in the wiring Wait 15 minutes tor temperature to equilibrate 4 Record the temperature If necessary adjust the temperature settings so that the digital thermometer reads 40 C After adjustment allow the incubator and heat block to equilibrate Then recheck the temperature 5 Repeat the step above to adjust temperature until the incubator is 40 1 C UJ Note We recommend calibrating the incubator at least once a month to ensure accuracy Step IV Assess incubator temperature uniformity 1 Repeat Step Ill to measure the temperature at multiple positions in the incubator to determine temperature uniformity Note The temperature for all positions should be 40 1 C Step V Assess temperature ramp up time 1 Remove the 1 5 mL tube containing the probe from the prewarmed heat block from Step 11 and allow it to return to room temperature 2 Open the incubator door for 1 minute then place the 1 5 mL tube containing the probe from Step V1 into the heat block and close the door Measure the time needed for the temperature to return to 40 C and monitor the temperature profile during recovery 3 Repeat Steps V1 2 two more times Note Do not use the Incubator for the assay if it takes more than 10 minutes to return to 40 C or if it overshoots by more than 2 C during recov
23. also reported to work Most protein based fluorochromes are compatible with this assay including PE PE eFluor 610 PE Cyanine5 PE Cyanine5 5 PE Cyan ne7 APC and APCeFluor 780 PerCP PerCP Cyanine5 5 and PerCP eFluor 710 should not be used We recommend using PE Cyanine5 or PE Cyanine5 5 instead Qdot nanocrystal and eVolve conjugated antibodies are not compatible with this assay Intracellular antibody staining compatibility 1 E The fixation and permeabilization buffers in this kit are compatible with most eBioscience antibodies used for intracellular staining Some changes in performance are expected when compared to performance in the Intracellular Fixation and Permeabilization Buffer Set cat no 88 8824 or Foxp3 Transcription Factor Staining Buffer Set cat no 00 5523 Antibody performance should be determined empirically using the PrimeFlow Fixation Permeabilization Buffer Set cat no 88 17000 The fixation and permeabilization buffers in this kit are compatible with most eBioscience phospho specific antibodies however phospho specific antibodies that will work only in the IC Fixation Methanol protocol are not compatible Please see the Phospho Flow Cytometry Antibody Clone Buffer Selection Guide or the datasheet for the individual antibody for more information Staining for some surface markers may be done after fixation and permeabilization Please see the Antibody Clone Performance Following
24. ash Buffer Spin cells at 1 000 x g for 4 minutes 9 Perform Target Probe hybridization for 2 hours at 40 C 10 Wash twice with PrimeFlow RNA Wash Buffer Spin cells at 1 000 x g for 4 minutes 11 Wash once with PrimeFlow RNA Wash Buffer with RNase Inhibitor 1 Spin cells at 1 000 x g for 4 minutes 12 Store samples overnioht Day 2 13 Perform PreAmp hybridization for 1 5 hours at 40 C 14 Wash twice with PrimeFlow RNA Wash Buffer Spin cells at 1 000 x g for 4 minutes 15 Perform Amp hybridization for 1 5 hours at 40 C 16 Wash twice with PrimeFlow RNA Wash Buffer Spin cells at 1 000 x g for 4 minutes 17 Perform Label Probe hybridization for 1 hour at 40 C 18 Wash twice with PrimeFlow RNA Wash Buffer Spin cells at 1 000 x g for 4 minutes 19 Wash once with PrimeFlow RNA Storage Buffer Spin cells at 1 000 x g for 4 minutes 20 Analyze samples on a flow cytometer 23 Notes 24 D affymetrix eBioscience Customers in countries where direct sales are not available may contact their local eBioscience distributor listed at www ebioscience com distributors or email us at tech ebioscience com Technical support USA 1 888 810 6168 toll free 1 858 642 2058 Europe 43 1 796 4040 120 Affymetrix Inc 3420 Central Expressway Santa Clara CA 95051 GOGH www ebioscience com FC05776 2 PrimeFlow User Manual 0915 2015 Affymetrix Inc All rights reserved
25. ation Buffer 1 to each sample and pipet to mix Incubate for 30 minutes at 2 8 C 8 Spin down at 1 000 x g for 4 minutes then discard supernatant and resuspend cells in the residual volume by vortexing 9 Prepare 1X PrimeFlow RNA Permeabilization Buffer with RNase Inhibitors by diluting PrimeFlow RNA Permeabilization Buffer 10X to 1X with RNase free water Then add RNase Inhibitor 1 1 000X at a 1 1 000 dilution and RNase Inhibitor 2 100X at a 1 100 dilution Mix gently by inverting Keep at 2 8 C Note You will need 700 uL of this buffer per sample Prepare this buffer in bulk to accommodate all samples Avoid vortexing or vigorously shaking this buffer This buffer should be prepared fresh 10 Add 200 uL of 1X PrimeFlow RNA Permeabilization Buffer with RNase Inhibitors to each Sample pipet to mix and spin down at 1 000 x g for 4 minutes then discard supernatant and resuspend cells in the residual volume by vortexing 20 11 12 E 14 Ka 16 17 18 19 20 Repeat Step 10 Note Whole blood samples being prepared in bulk should be transferred into a 96 vvell plate before proceeding to Step 12 Intracellularly stain cells with tluorochrome conjugated antibodies at their optimal concentration in the 1X PrimeFlow RNA Permeabilization Buffer with RNase Inhibitors for 30 minutes at 2 8 C Note f intracellular staining is not desired skip this step and proceed to Step 14 Note Stainin
26. cols in this user manual Materials required Incubator capable of maintaining temperature at 4041 C Affymetrix cat nos QS0704 or Q50712 ViewRNA Temperature Validation Kit Affymetrix cat no QV0523 1 5 mL microcentrifuge tube included in the PrimeFlow RNA Assay kit Metal heat block for 1 5 mL microcentrifuge tube VWR cat no 13259 002 Parafilm wrap Calibration procedure Step l Prepare the incubator Turn on the incubator Set the temperature to 40 C Place the metal heat block into the incubator near the center of the middle shelf of the incubator Allow the incubator and heat block to equilibrate overnight WN Step II Assemble the temperature validation unit Insert the battery to activate the digital thermometer 2 Use a pointed object eg a ballpoint pen to drill a hole into the center of the lid of a 1 5 mL microcentrifuge tube 3 Add 0 2 mL of deionized water to the tube and then close the lid 4 Insert the Type K beaded probe into the digital thermometer and place the other end of the probe through the pre drilled hole of the 1 5 mL tube and into the water 5 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 6 Turn on the digital thermometer Step 11 Measure and adjust the temperature of the incubator 1 Pl
27. ery Appendix 7 Protocol using 96 well plates This protocol is validated based on the use of a polystyrene 96 vvell v bottom plate cat no 44 17005 polystyrene u bottom plates may also be used To discard supernatant from the vvells the plate may be inverted using a single motion with adequate force and then gently blotted on a paper towel Alternatively aspiration may be used being careful to not disrupt the pellet The residual volume inside each well should not exceed 10 uL Note Flat bottom plates are not recommended for use with this protocol Experimental procedure Day 1 Antibody staining fixation and permeabilization 1 Pre vvarm PrimeFlow RNA Wash Buffer to room temperature This buffer will first be used In Step 14 2 Aliquot 1 5 x 106 cells in 100 uL of Flow Cytometry Staining Buffer per well Note To use whole blood it is necessary to pre lyse the red blood cells before beginning the assay using the 10X RBC Lysis Buffer cat no 00 4300 Please see the product datasheet or Best Protocols Red Blood Cell Lysis Protocol Protocol A for instructions for use Alternatively perform Steps 2 11 in bulk refer to the 1 5 mL tube protocol above Steps 2 10 to maintain the optimal sample to reagent ratio 3 Surface stain cells with fluorochrome conjugated antibodies at their optimal concentration tor 30 minutes at 2 8 C Note If needed please see Best Protocols Staining Cell Surface Antigens for Flow Cytome
28. es then discard supernatant and resuspend cells in the residual volume by vortexing Repeat Step 36 Add 100 uL of PrimeFlow RNA Wash Buffer to each well Then add 100 uL of PrimeFlow RNA Amp Mix directly into the cell suspension and pipet to mix Incubate plate with the lid on for 1 5 hours at 40 C Spin down at 1 000 x g for 4 minutes then discard supernatant and resuspend cells in the residual volume by vortexing Add 200 uL of PrimeFlow RNA Wash Buffer and spin down at 1 000 x g for 4 minutes then discard supernatant and resuspend cells in the residual volume by vortexing Repeat Step 40 Dilute PrimeFlow RNA Label Probes 100X 1 100 in PrimeFlow RNA Label Probe Diluent Note You will need 100 ul of diluted Label Probes for each sample Prepare diluted Label Probes in bulk to accommodate all samples Add 100 uL of PrimeFlow RNA Wash Buffer to each well Then add 100 uL of diluted PrimeFlow RNA Label Probes directly into the cell suspension and pipet to mix Incubate plate with the lid on for 1 hour at 40 C Spin down at 1 000 x g for 4 minutes Discard supernatant and resuspend cells in the residual volume by vortexing Add 200 uL of PrimeFlow RNA Wash Buffer and spin down at 1 000 x g for 4 minutes then discard supernatant and resuspend cells in the residual volume by vortexing Repeat Step 45 Add 200 uL of PrimeFlow RNA Storage Buffer or Flow Cytometry Staining Buffer and spin down at 1 000 x g for 4 minutes then discard
29. escent dye hybridize to their corresponding Amplifier molecules A fully assembled signal amplification tree has 400 Label Probe binding sites When all target specific oligonucleotide probes in the Target Probe set bind to the target RNA transcript 8 000 16 000 fold amplification is achieved We currently offer three different amplification 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 PrimeFlow RNA assay the data can be collected and analyzed on any standard flow cytometer The schematic below illustrates the detection of two unique targets Sample Target Signal 000 Fluorescent Label gt ZZ Gene specific ann ZZ Target Probes PreAmplifier 00 0 robe Add Label Probes to lt cells w m Amplifier Label proteins vvith Incubate cells with Hybridized with antibody optional gene specific Target Pre Amplifier and Probes Type 1 4 or 6 Amplifier DNA Fix and permeabilize Type 1 4 and 6 cells in suspension v Process cells using a standard flovv cytometer IFN gamma protein eFluor 450 Suspension cells IFN gamma mRNA with RNA fixed Alexa Fluor 750 General notes Assay specifications Sample type Single cell suspensions including human whole blood and peripheral blood mononuclear cells PBMC mouse dissociated tissues and cell line
30. fluorochrome often spills over into the other detection channels due to overlapping emission spectra The process of subtractino the spillover signal is called compensation The procedures below describe how to set proper PMT voltage and compensation for multicolor PrimeFlow RNA analysis Materials required Calibration beads Unstained cells that have undergone the PrimeFlow assay Samples prepared using the PrimeFlow Compensation Kit cat no 88 17001 Unstained UltraComp eBeads UltraComp eBeads labeled with PrimeFlow RNA Alexa Fluor 647 Compensation Control UltraComp eBeads labeled with PrimeFlow RNA Alexa Fluor 488 Compensation Control UltraComp eBeads labeled with PrimeFlow RNA Alexa Fluor 750 Compensation Control Single color antibody stained UltraComp eBeads needed only if protein staining is performed Experimental procedure Step I Ensure proper instrument alignment 1 Follow the instrument manufacturer s recommendations for checking instrument alignment Alternatively calibration beads such as Rainbow Calibration Particles 8 peak beads Spherotech cat no RCP 30 5A or Rainbow Fluorescent Particles 1 peak beads Spherotech cat no RFP 30 5A may be used to check the linearity of the PMT PMT voltage range and alignment of the instrument Step Il Set PMT voltages 1 Create an acquisition template workspace that includes a forward scatter versus side scatter plot a single parameter
31. forward and side scatter voltages were changed between Steps 2a and 113 adjust voltages back to those noted in Step ll2a b Looking at each of the histogram plots confirm that all fluorescent signals are on scale and adjust PMT voltages if necessary c Stop collecting events and remove sample from the cytometer Step III Set compensation Note Use PrimeFlovv Compensat on Kit Tor single color compensat on controls Please see the product data sheet Tor preparation of samples and instructions for use Alternatively cell samples labeled with individual Positive Control Probe sets may be used for single color compensation controls Do not use fluorochrome conjugated antibodies to compensate for RNA signal 1 If using PrimeFlow Compensation Kit for single color controls return forward and side scatter voltages to those noted in Step 1 3 in order to visualize the compensation beads Do not change voltages for any of the fluorescent PMT channels 2 Ifan autocompensation feature is available for the cytometer used follow the manufacturer s instructions for use 3 If an autocompensation feature is not available or to set compensation manually a Place the unstained sample on the cytometer and begin to collect events b Looking at the two parameter plots create quadrant regions on each plot such that the cells fall within the lower left quadrant Figure A3 1 c For each two parameter plot create statistics windows according to yo
32. g for some surface markers may be done after fixation and permeabilization Please see the Antibody Clone Performance Following Fixation Permeabilization table on our website and refer to the column for After IC Fixation and Perm Wash to determine if the antibody clone will recognize a fixed epitope Note Use antibodies that are conjugated to approved fluorochromes only see general notes fluorochrome compatibility section above Add 200 uL of 1X PrimeFlow RNA Permeabilization Buffer with RNase Inhibitors to each sample pipet to mix and spin down at 1 000 x g for 4 minutes then discard supernatant and resuspend cells in the residual volume by vortexing Prepare 1X PrimeFlow RNA Fixation Buffer 2 by combining 25 uL of PrimeFlow RNA Fixation Buffer 2 8X with 175 uL of PrimeFlow RNA Wash Buffer per well Mix gently by inverting Note You will need 200 uL of this buffer per well Prepare this buffer in bulk to accommodate all samples For example for 10 samples combine 250 uL of PrimeFlow RNA Fixation Buffer 2 8X with 1 750 uL of PrimeFlow RNA Wash Buffer Avoid vortexing or vigorously shaking this buffer This buffer should be prepared fresh Dispose of any unused buffer Add 200 uL of 1X PrimeFlow RNA Fixation Buffer 2 to each well and pipet to mix Incubate tor 60 minutes in the dark at room temperature Note t is important to fix the samples at room temperature Do not perform this step on ice OPTIONAL Cells may be stored in 1
33. mance Following Fixation Permeabilization table on our website and refer to the column for After IC Fixation and Perm Wash to determine if the antibody clone will recognize a fixed epitope If you prefer to stain after fixation skip this step and proceed to Step 5 Add 1 mL of Flow Cytometry Staining Buffer to each sample invert to mix and spin down at 500 x g for 5 minutes Discard supernatant and resuspend cells in the residual volume Note When using whole blood skip this step and proceed to Step 5 Prepare Fixation Buffer 1 by mixing equal parts of PrimeFlow RNA Fixation Buffer 1A and PrimeFlow RNA Fixation Buffer 1B Mix gently by inverting Note You will need 1 mL of this buffer per sample Prepare this buffer in bulk to accommodate all samples Avoid vortexing or vigorously shaking this buffer This buffer should be prepared fresh Dispose of any unused buffer Add 1 mL of prepared Fixation Buffer 1 to each sample and invert to mix Incubate for 30 minutes al 2 0 G Spin down at 800 x g for 5 minutes Discard supernatant and resuspend cells in the residual volume Prepare 1X PrimeFlow RNA Permeabilization Buffer with RNase Inhibitors by diluting PrimeFlow RNA Permeabilization Buffer 10X to 1X with RNase free water Then add RNase Inhibitor 1 1 000X at a 1 1 000 dilution and RNase Inhibitor 2 100X at a 1 100 dilution Mix gently by inverting Keep at 2 8 C Note You will need 3 mL of this buffer per sam
34. mix Note We recommend this stopping point for ease of use and a more manageable workflow However if desired Step 30 may be skipped If skipping this step proceed to Step 31 and continue through to the end of the protocol Day 2 Signal amplification Note It is critical that the residual volume after all washes does not exceed 10 ul Note PrimeFlow RNA PreAmp Mix PrimeFlow RNA Amp Mix and diluted Label Probes should be pipetted directly into the samples and mixed well before incubating The total volume for each hybridization is 200 uL per well During hybridization keep lid on the plate Place plates directly on the incubator shelf Do not stack plates CAR 33 34 E 36 37 29 40 41 42 43 44 45 46 47 Pre warm samples and PrimeFlow RNA Wash Buffer to room temperature Pre warm PrimeFlow RNA PreAmp Mix PrimeFlow RNA Amp Mix and PrimeFlow RNA Label Probe Diluent to 40 C Thaw PrimeFlow RNA Label Probes 100X on ice in the dark Note This can be done during the Amp Mix incubation Step 38 Add 100 uL of PrimeFlovv RNA PreAmp Mix directly into the cell suspension for each sample and pipet to mix and then incubate plate with the lid on for 1 5 hours at 40 C Spin down at 1 000 x g for 4 minutes then discard supernatant and resuspend cells in the residual volume by vortexing Add 200 uL of PrimeFlow RNA Wash Buffer and spin down at 1 000 x g for 4 minut
35. ndard Bangs Laboratories cat no 580 B Weak No Signal 1 Cytometer not set up properly Check that your instrument is properly set up according to the manufacturer s recommendations 2 Incorrect samples used for setting Use samples labeled with one each of the included compensation Positive Control Probe Sets to set compensation Do not use fluorochrome conjugated antibodies to set compensation for RNA detection 3 Incorrect preparation of signal Make sure PreAmp Mix Amp Mix and Label amplification reagents Probes are used in the specified order 4 Incorrect incubator temperature 1 Incubator must be able to hold temperature at 401 C 2 Use a metal heat block for hybridization 3 Minimize traffic to incubator Temperature tolerance is 1 2 C 4 Ensure incubator has been stable at 40 C for at least 12 hours before starting the assay as temperature may drift durino initial incubator setup 5 Measure temperature using the ViewRNA Temperature Validation Kit Affymetrix cat no QV0523 5 Incorrect Wash Buffer Do not substitute PrimeFlow RNA Wash Buffer with Storage Buffer or any other buffer 6 Incorrect diluents used Ensure PrimeFlow RNA Target Probe Diluent is used with Positive Control Target Probe Sets 20X and PrimeFlow RNA Label Probe Diluent is used with PrimeFlow RNA Label Probes 100X Appendix 1 Troubleshooting B Weak No Signal 7 Incorrect dilution of Target Probes or Label
36. perform the assay in 96 vvell plates refer to Appendix A7 of this user manual Day 1 Antibody staining fixation and permeabilization 1 Aliquot 1 5 x 106 cells in Flow Cytometry Staining Buffer or 100 uL of whole blood into the UJ Ul O 10 Pre warm PrimeFlow RNA Wash Buffer to room temperature This buffer will first be used in Step 13 1 5 mL tubes provided in the kit Note When using whole blood it is not necessary to lyse the red blood cells before beginning the assay However if pre lysis is desired the use of 10X RBC Lysis Buffer cat no 00 4300 is recommended Please see the product datasheet or Best Protocols Red Blood Cell Lysis Protocol Protocol A for detailed instructions for use Surface stain cells with fluorochrome coniugated antibodies at their optimal concentration for 30 minutes at 2 8 C Note If needed please see Best Protocols Staining Cell Surface Antigens Tor Flow Cytometry Protocol A Cell Suspensions for detailed instructions for staining Note Use antibodies that are conjugated to approved fluorochromes only see general notes fluorochrome compatibility section above Note Cells may be stained with a Fixable Viability Dye before or after surface staining see Best Protocols Viability Staining Protocol Protocol C for detailed instructions Note Staining for some surface markers may be done after fixation and permeabilization Please see the Antibody Clone Perfor
37. ple Prepare this buffer in bulk to accommodate all samples Avoid vortexing or vigorously shaking this buffer This buffer should be prepared fresh Dispose of any unused buffer Add 1 mL of 1X PrimeFlow RNA Permeabilization Buffer with RNase Inhibitors to each sample invert to mix and spin down at 800 x g for 5 minutes then discard supernatant and resuspend cells in the residual volume Repeat Step 9 11 Intracellularly stain cells with fluorochrome conjugated antibodies at their optimal concentration in the 1X PrimeFlovv RNA Permeabilization Buffer with RNase Inhibitors for 30 minutes at 2 8 C Note If intracellular staining is not desired skip this step and proceed to Step 13 Note Staining for some surface markers may be done after fixation and permeabilization Please see the Antibody Clone Performance Following Fixation Permeabilization table on our website and refer to the column for After IC Fixation and Perm Wash to determine if the antibody clone will recognize a fixed epitope Note Use antibodies that are conjugated to approved fluorochromes only see General notes Fluorochrome compatibility section above 12 Add 1 mL of 1X PrimeFlow RNA Permeabilization Buffer with RNase Inhibitors to each sample invert to mix and spin down at 800 x g for 5 minutes then discard supernatant and resuspend cells in the residual volume by vortexing gently 13 Prepare 1X PrimeFlow RNA Fixation Buffer 2 by combining 125 uL of Prime
38. s See Appendix A5 for a complete list of validated cell types Species Mammalian Plex level Up to three RNA targets simultaneously Assay format 1 5 mL microcentrifuge tube or 96 well v bottom plate See Appendix A7 for the protocol to use 96 well plates Instrumentation for RNA detection Flow cytometer equipped with blue 488 nm and red 633 640 nm lasers filter sets for FITC bandpass 530 30 APC bandpass 660 20 and APC eFluor 780 APC Cyanine7 bandpass 780 60 Assay guidelines L Best results are obtained when starting with healthy cells Always begin with cells that are in good physiological condition Cells should be in active growth phase to preserve RNA integrity and minimize cell ysis during processing If using sorted primary cells make sure the cells are healthy after purification Addition of a Fixable Viability Dye is recommended to ensure analysis is restricted to those cells that were alive at the start of the protocol This assay is highly temperature dependent Please ensure that the incubator holds temperature at 40 1 C A significant reduction in signal will result from temperature deviations greater than 1 C The incubator must be validated using the ViewRNA Temperature Validation Kit Affymetrix cat no QV0523 following the instructions in Appendix A6 of this user manual This assay allows detection of up to three 3 RNA taroets in a single sample Type 1 Alexa
39. the final volume remains 100 uL per sample 23 Add 100 uL of diluted Target Probets directly into the cell suspension for the appropriate samples and briefly vortex to mix and then incubate for 2 hours at 40 C Invert samples to mix after 1 hour 24 Add 1 mL of PrimeFlow RNA Wash Buffer to each sample invert to mix and spin down at 800 x g for 5 minutes Aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently 25 Prepare PrimeFlow RNA Wash Buffer with RNase Inhibitor 1 by adding RNase Inhibitor 1 1 000X at a 1 1 000 dilution to the PrimeFlow RNA Wash Buffer Mix gently by inverting Note You will need 1 mL of this buffer per sample Prepare this buffer in bulk to accommodate all samples This buffer should be prepared fresh Dispose of any unused buffer 26 Add 1 mL of PrimeFlow RNA Wash Buffer with RNase Inhibitor 1 to each sample invert to mix and spin down at 800 x g for 5 minutes Aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently 27 Store samples overnight in the dark at 2 8 C Note We recommend this stopping point for ease of use and a more manageable workflow However if desired Step 27 may be skipped If skipping this step proceed to Step 28 and continue through to the end of the protocol Day 2 Signal amplification Note It is critical that the residual volume after all washes be as close to 100 uL as possible Use the
40. try Protocol A Cell Suspensions for detailed instructions for staining Note Use antibodies that are conjugated to approved fluorochromes only see General notes Fluorochrome compatibility section above Note Cells may be stained with a Fixable Viability Dye before or after surface staining see Best Protocols Viability Staining Protocol Protocol C for detailed instructions Note Staining for some surface markers may be done after fixation and permeabilization Please see the Antibody Clone Performance Following Fixation Permeabilization table on our website and refer to the column for After IC Fixation and Perm Wash to determine if the antibody clone will recognize a fixed epitope If you prefer to stain after fixation skip this step and proceed to Step 5 Spin down at 500 x g for 4 minutes then discard supernatant Add 200 uL of Flow Cytometry Staining Buffer to each sample pipet to mix and spin down at 500 x g for 4 minutes then discard supernatant and resuspend cells in the residual volume by vortexing 6 Prepare Fixation Butter 1 by mixing equal parts of PrimeFlow RNA Fixation Buffer 1A and PrimeFlow RNA Fixation Buffer 1B Mix gently by inverting Note You will need 200 uL of this buffer per well Prepare this buffer in bulk to accommodate all samples Avoid vortexing or vigorously shaking this buffer This buffer should be prepared fresh Dispose of any unused buffer 7 Add 200 uL of prepared PrimeFlow RNA Fix
41. ue sa et am mes 2 Jase SOP Jase 669 1 se1 88t 2 484 Sil JeajpnuouoW poolq jeaaydu d uewny euuou p le nuil s u pudie NL pue ewwep NI JO UOIDNPU 12 1 p o jaUed io o2 9 e 104 dn 195 14901 Up qe L lt p 1e2Ipui se uryo 2 Buipuods ui1o2 y JO 802 4 uql Jo Hululeys ApoqnuP MoUs Saxe A y IUAA S Xe X y UO UAAOUS We YNY JO uol12 1 dq sis eue JO p sn diam 2oudU y UI sil WNYW Jo Ju uj inse ui v 104 5 MOJ 1100 JO 001 s qolq 1 OSZ 10N 4 ex v euuuueD NI ueuunH 9 d JO 1On 4d ex jy eydje dN1 ueuunH y YUM P l QE U Y BAM S l 6LEZ 8y OU 152 OSY s1On d ewe NI UPEUMH n y pue 6i 2 GZ ou 7162 ulue O 3d 4N1 UpuuntH DU 8800 19 OU 182 019 4on 4 3d 6802 UEWNH NUY YUM paulejs lIe n 2e41u Sa AeSSV YNY wiAAO d uulid Buisn pazAjeue u y pue ub sinou JO GZ67 00 ou 1152 sioliqiuu 11odsue1 ul loid snid ile 202 uonpinus il v uA p le nuui s 10 Dall p le nuiijsun JJaM S l 1e j nuououi poolq jeJaydiuad uewny EWJON UP enbi4 OSZ 10 ex V OSZ 10 ex V OSZ 10 ex V OSZ e4On 4 ex V V Nuu ewwep VNYW euweb Nil euweb Nil VNMul euweb NI Z Z LI DI co Sei 3 3 Hi 3 D o o mul o O O gt
42. ur instrument s acquisition software and include the x and y median for all parameters d Stop collecting events and remove sample from the cytometer 4 Place a single color sample on the cytometer and begin to collect events Note If using a single color cell sample that does not contain any ou Q3 negative events a small amount of unstained cells may be added APC to the single color samples just before loading the sample onto Figure A3 1 Unstained the cytometer Alternatively an unstained cell sample that has been sample processed through the PrimefFlovv assay must be used and the medlan fluorescence Intensity of that sample should be noted for all parameters a Looking at the two parameter plots for the fluorochrome collected adjust the quadrants so that the positive events are fully contained within either the lower right or the upper left quadrant Figure A3 2 left b Lookino at the statistics note the y or x median of the two populations Adjust the compensation for the fluorochrome being collected subtract its fluorescence out of the other channels being used in the experiment until the medians of the two populations Q1 Q2 APC eFluor 780 are the same Figure A3 2 right m Stop collecting events and remove sample from the cytometer R R d Repeat for each of the single color S samples 3 5 5 Confirm that the compensation
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44. y by pipetting up and down Note You will need 100 uL of diluted Target Probes for each sample If you are adding more than one Target Probe per sample adjust the volume of the PrimeFlow RNA Target Probe Diluent accordingly so that the final volume remains 100 uL per sample Add 100 uL of PrimeFlow RNA Wash Buffer to each well Then add 100 uL of diluted Target Probe s directly into the cell suspension for the appropriate samples and pipet to mix Incubate plate with the lid on for 2 hours at 40 C Note Plate sealing is not necessary for hybridization Plates should be placed directly onto the incubator shelf Do not stack plates 21 22 Z 26 27 28 29 Store samples overnight in the dark at 2 8 C with lid on Spin down at 1 000 x g for 4 minutes Discard supernatant and resuspend cells in the residual volume by vortexing Add 200 uL of PrimeFlovv RNA Wash Buffer and spin down at 1 000 x g for 4 minutes Discard supernatant and resuspend cells in the residual volume by vortexing Repeat Step 26 Prepare PrimeFlow RNA Wash Buffer with RNase Inhibitor 1 by adding RNase Inhibitor 1 1 000X to PrimeFlow RNA Wash Buffer at a 1 1 000 dilution Mix gently by inverting Note You will need 100 uL of this buffer per well Prepare this buffer in bulk to accommodate all samples This buffer should be prepared fresh Dispose of any unused buffer Add 100 uL of PrimeFlow RNA Wash Buffer with RNase Inhibitor 1 and pipet to
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