PrimeFlow™ RNA Assay User Manual and Protocol
Contents
1. 9 affymetrix PrimeFlow RNA Assay Protocol Page 2 of 20 eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only General Notes Assay Specifications Sample Type Species Plex level Assay format Single cell suspensions including human whole blood and PBMC mouse dissociated tissues and cell lines See Appendix A5 for a complete list of validated cell types Mammalian Up to 3 RNA targets simultaneously 1 5 mL microcentrifuge tube Instrumentation for RNA detection Flow cytometer equipped with e blue 488 nm and red 633 640 nm lasers e filter sets for FITC bandpass 530 30 APC bandpass 660 20 and APC eFluor 780 APC Cyanine7 bandpass 780 60 Assay Guidelines 1 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 lysis 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 Valid2. Note If needed please see Best Protocols Staining Cell Surface Antigens for 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 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 Add 1 mL of Flow Cytometry Staining Buffer to each sample invert to mix and spin down at 600xg for 5 minutes then 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 Fixation Buffer 1A and 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 Ad
3. Rotor Eppendorf model A 4 44 PRESSE Adaptor for 15 mL conical tube Eppendorf model 5804 755 006 Adaptor for 1 5 mL microcentrifuge tube Eppendorf model 5804 750 004 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 amp 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 T Example shown d V Vacuum bottle Argos Technologies model EV432 Aw Aspirator Argos Technologies model EV514 d 5 ViewRNA Temperature Validation Kit NIST traceable thermometer with temperature probe in 1 5 mL E i microcentrifuge tube shown on left Affymetrix cat QV0523 Not for further distribution without written consent 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com 9 affymetrix PrimeFlow RNA Assay Protocol Page 12 of 20 eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only A3 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 fluorochrome often spills over into the other det
4. GAPDH adherent cells limited testing Not for further distribution without written consent 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com PrimeFlow RNA Assay Protocol Page 19 of 20 9 affymetrix eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only A6 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 and or weak signal The incubator should be validated before use following these instructions Materials required Incubator capable of maintaining temperature at 40 1 C Affymetrix cat QS0704 or QS0712 ViewRNA Temperature Validation Kit Affymetrix cat QV0523 1 5 mL microcentrifuge tube Metal heat block for 1 5 mL microcentrifuge tube VWR cat 13259 002 Parafilm Experimental Procedure 1 Prepare the incubator a Turn on the incubator b Set the temperature to 40 C c Place the metal heat block into the incubator near the center of the middle shelf of the incubator d Allow the incubator and heat block to equilibrate overnight Assemble the temperature validation unit a Insert the battery to activate the digital thermometer b Usea pointed object e g a ballpoint pen to drill a hole into the center of the lid of a 1
5. Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com 9 affymetrix PrimeFlow RNA Assay Protocol Page 5 of 20 eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only Experimental Procedure Day 1 Antibody staining fixation and permeabilization 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 amp 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 1x10 cells mL 1 2 Pre warm Wash Buffer to room temperature Aliquot 1 5x10 cells in Flow Cytometry Staining Buffer or 100 uL of whole blood into the 1 5 mL tubes provided in the kit Note When using whole blood it is not necessary to lyse the red blood before beginning the assay However if lysis is desired the use of 10X RBC Lysis Buffer cat 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 conjugated antibodies at their optimal concentration for 30 minutes at 2 8 C
6. 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 being used in the experiment As with single color controls there should be an FMO control for every fluorochrome being 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 granulocytes from monocytes is recommended Please see Appendix A4 for examples of sample set up and experimental design Not for further distribution without written consent 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 Fax 858 642 2046 e www ebioscience com info ebioscience com
7. vns Nar eude3ni edo ums omxesviomiextv 8 gseqd us tomesafuseosiomze 1 c a RER EREECHEN wuN Tue Loes g ie s A ee ue opone awer ag v o 7 pL a ws EECHER pte use Less apue sera exer z OZ OLS OS OS t 09 082 0Z 019 uondu sepeqni 90g14on je OSrpjon je OG Jony exe v JeuIUeAD Fd 0L9 J0njJe 3d 88y Jon exay Jose cor Jose 9 19581 88p DNS8dA Sue 4eejonuouous poojq jejeudued ueuny jeuuou pejejnuugs ui eydje 4N pue ewweb yy Jo uoronpui je 400 0 jaued 10 02 9 e 104 dn Jas Jejuauiuedx3 VF V 9 81 Not for further distribution without written consent O 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com PrimeFlow RNA Assay Protocol Page 15 of 20 49 affymetrix lOscience eB PrimeFlow RNA Assay User Manual and Protocol Research Use Only pejeoipui se eujojKo Bupuodsa1109 y 10 832 Jayya jo Buiuiejs Apogijue moys sexe ay au sexe x ay uo UMOYS Be YNY Jo uojoasjeg SsisAJeue 104 pesn alam ajeb eyoouduuM y ui sje vN YU Jo jueuieunseaui ay 104 AJ aArjoedsai mor uiojjoq 10 doj seqoud 196181 0G auonjJ exe y euiueD yy ueunH 9 ed 10 ggr amp onjJ exejv eudje 4NL ueuinH p adA_ uy pejeqe ueuj aiau SIO 6L 4 8b 789 OGr GuonjJe ewueb N 4 ueung guy pue 6r 4 sc 389 LeulueAD F_ eudje JN
8. 5 mL microcentrifuge tube c Add 0 2 mL of deionized water to the tube and then close the lid d 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 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 Measure and adjust the temperature of the incubator a Place the 1 5 mL tube containing the probe into the prewarmed heat block from Step 1 b Close the door making sure there is sufficient slack in the wiring c Wait 15 minutes for temperature to equilibrate d 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 e Repeat the step above to adjust temperature until the incubator is 40 1 C Note We recommend calibrating the incubator at least once a month to ensure accuracy Not for further distribution without written consent 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com PrimeFlow RNA Assay Protocol Page 20 of 20 9 affymetrix eBioscience PrimeFlow RNA As
9. 9 affymetrix PrimeFlow RNA Assay Protocol Page 4 of 20 eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only 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 PrimeFlow RNA Storage Buffer RNase Inhibitor 2 100X Store at 20 C RNase Inhibitor 1 1000X 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 00 4222 Fluorochrome conjugated antibodies as needed Fixable Viability Dye as needed 12x75 mm polystyrene tubes e g BD Falcon cat 352008 PrimeFlow Compensation Kit cat 88 17001 IC Fixation Buffer cat 00 8222 Instruments and equipment Refer to Appendix A2 of this user manual Experiment duration Day 1 6 8 hours Antibody staining Fixation amp permeabilization Target Probe hybridization Day 2 6 hours Signal amplification Flow cytometric analysis Not for further distribution without written consent 2000 eBioscience
10. Add 100 uL of PreAmp Mix directly into the cell suspension for each sample briefly vortex to mix and then incubate for 1 5 hours at 40 C Add 1 mL of Wash Buffer to each sample invert to mix and spin down at 800xg for 5 minutes Aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently Repeat wash with Wash Buffer two more times for a total of three washes Add 100 uL of Amp Mix directly into the cell suspension for each sample briefly vortex to mix and then incubate for 1 5 hours at 40 C Add 1 mL of Wash Buffer to each sample invert to mix and spin down at 800xg for 5 minutes Aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently Repeat wash with Wash Buffer Dilute Label Probes 1 100 in 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 diluted Label Probes directly into the cell suspension for each sample briefly vortex to mix and then incubate for 1 hour at 40 C Not for further distribution without written consent 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 Fax 858 642 2046 e www ebioscience com info ebioscience com 9 affymetrix PrimeFlow RNA Assay Protocol Page 8 of 20 eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only Quick Guid
11. Amp Mix and Label Probes are used in the specified order 1 Incubator must be able to hold temperature at 40 1 C 2 Use ametal 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 during initial incubator setup 5 Measure temperature using the ViewRNA Temperature Validation Kit Affymetrix cat QV0523 Do not substitute Wash Buffer with Storage Buffer or any other buffer Ensure Target Probe Diluent is used with Target Probes and Label Probe Diluent is used with Label Probes Not for further distribution without written consent 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com 9 affymetrix eBioscience PrimeFlow RNA Assay Protocol Page 10 of 20 PrimeFlow RNA Assay User Manual and Protocol Research Use Only C High Background 7 Incorrect dilution of Target Probes or Label Probes 8 Gene expressed at very low level 9 Incorrect or insufficient RNase Inhibitor used 1 Compensation not set up correctly 2 Incorrect incubator temperature 3 Insufficient washing 4 Cytometer not set up properly 5 Over fixation 6 Excessive Target Probe used 1 Ensure Target Probes are diluted 1 20 with Target Probe Diluent 2 Ensure Label
12. Probes are diluted 1 100 with Label Probe Diluent 1 Check biological model and confirm gene is expressed in sample by an alternative method such as QuantiGene 2 0 Assay for lysate 2 Keep in mind that protein expression might not correlate with RNA expression in some cases 3 Ensure that 1X Permeabilization Buffer contains both RNase inhibitor 1 at a final dilution of 1 1000 and RNase inhibitor 2 at a final dilution of 1 100 Ensure that the Wash buffer in Step 19 and or Step 25 for overnight sample storage contains RNase inhibitor 1 at a final dilution of 1 1000 1 See Appendix A3 for how to set compensation Use the PrimeFlow Compensation kit sold separately or samples labeled with one each of the included Positive Control Probe sets to set compensation 3 Do not use fluorochrome conjugated antibodies to set compensation for RNA detection 1 Incubator must be able to hold temperature at 40 1 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 assay as temperature may drift during initial incubator setup 5 Measure temperature using the ViewRNA Temperature Validation Kit Affymetrix cat QV0523 1 Ensure the Wash Buffer is used at room temperature 2 Follow instructions for washing steps in the protocol 3 Ensure uniform cell resuspension by gentle vo
13. gauen e8d9 w io US OWS 092 Joni Ee OL Jon wazo f soon wawy uns OWS JeutueRO dd 6 1954 vwNMuduzo augue LW ums ONJOI9 je 3d 8 95 wauza asus vado gt ums ONJssrjendexv Z ea o o dT ass 9o afus osp onde 9 en eg aaa osz jen exa S gt Lc T c ue o ums 190 eBuseuuefo dd Y so Burs Oro ene d E SE EECH uns EA 0S 0Sv 09 084 09 084 uonduosep eqni OSp1on je 09410n j exe v 7 euiue 3d 0L9 Joni Je 3d 89r Jon exe v Jose cor Jose ee9 Jost 88p sey oouejds esnouu paje nuys ui 9 14 pue g auwAzuele jo uogonpul je 400 oj Jeued 10j 02 G e 104 dn Jas Jejueuiuedx3 Z F V 9 81 Not for further distribution without written consent O 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com PrimeFlow RNA Assay Protocol Page 17 of 20 49 affymetrix lOscience eB PrimeFlow RNA Assay User Manual and Protocol Research Use Only pejeoipui se uiejoud Bupuodsa1109 y 10 800 4euye jo Buiuiejs poquue Moys sexe A ay eJluM sexe x euj uo UMOYS 218 YNY Jo uoosjeg YN YU Jojuauisinseaui ay 10 JaAjedsai MOJ u10jjoq 40 doj seqoug 19818 064 amp uon 4 exe v Y ewAZUeID esnojy 9 ed 10 ggr QUON S exe v 79 14 SNOW p ed yum pajaqe u y BOM Sie 869S 8r 189 or amp uonjJe 9 1X SSNON YUY pue 8688 GZ 389 euiueK2 3d g su zuelo es
14. may be stored in 1X Fixation Buffer 2 overnight in the dark at 2 8 C instead of incubating for 60 minutes at room temperature Step 14 Spin down at 800xg 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 Add 1 mL of Wash Buffer to each sample invert to mix and spin down at 800 xg for 5 minutes then aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently Repeat wash with Wash Buffer Note lt 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 OPTIONAL Cells may be stored in Wash Buffer with RNase Inhibitor 1 overnight in the dark at 2 8 C To do so add RNase Inhibitor 1 to Wash Buffer at a 1 1000 dilution for the last wash 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 No
15. of samples and instructions for use Alternatively cell samples labeled with individual Positive Control Probe Sets may be used for single color Not for further distribution without written consent O 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com PrimeFlow RNA Assay Protocol Page 13 of 20 9 affymetrix eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only compensation controls Do not use fluorochrome conjugated antibodies to compensate for RNA signal a If an autocompensation feature is available for the cytometer being used follow the manufacturer s instructions for use b lf an autocompensation feature is not available or to set compensation manually i Place the unstained sample on the cytometer and begin to collect events ii 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 Iii For each two parameter plot create statistics windows according to your instrument s acquisition software and include the x and y median for all parameters IV Stop collecting events and remove sample from the cytometer c 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 negative events a small amount of unstained cells may b
16. ueung guv 8800 19 162 019 amp 40njje 3d egqo ueuinH nuv YM peuiejs AuenjjeoeJjui 849 S N essy YNY Mo Jeuiug eu Bursn pezAjeue u y Gyu sinoy 104 6265 00 389 Suojigiyul yodsuen urejoud snid j654202 uogejnuugs 11980 eu uy pejejnuuis Jo ya pejejnuusun 848 s ja9 1esjonuouou poojq JBjeudued ueuinu J eEUUON L py e4nDr4 0543v VNYU ewweb HA Di Je VN3U euuweb HA 05L3IV WH ewweb HA 019 10130 3d 809 988 JV VNYu eude JN L9uiue 5 3d edie INL 019 10nj409 3d 802 eujueA9 3d Pude JN 019 10n49 3d 802 paje nuns paje nupysun Not for further distribution without written consent 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com e info ebioscience com PrimeFlow RNA Assay Protocol Page 16 of 20 9 affymetrix lOSCIence eB PrimeFlow RNA Assay User Manual and Protocol Research Use Only Lom w quzo geufzueliD egqo vNutu jem unsun aiduesioio9 S 0Z_ E O AA A AA E VNMuUquzo geufzueio egg9 wg om umsun OlOSryJonde ot AAA AAA ee OWS OZ Joni exelv LI 4954 vNuuguzO ee Tag ien unsu IE TESECES NES O OA ee OW 019 10n149 3d SL 49 VNHUuquzo gaw zuelio egg umsun OWS 887 On 4 exelv y 290 wanna ques geuwuer Se wiel Uns E El pogosrecopII osque hed UN eget UNS i PRISAS SONAS El gewizues egg9 weieng uns T ONOS onde LL
17. PrimeFlow RNA Assay Protocol Page 1 of 20 9 affymetrix eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only Introduction The PrimeFlow RNA Assay is an in situ hybridization assay that combines the power of the Affymetrix ViewRNA branched DNA technology with the single cell resolution of flow cytometry This assay enables simultaneous detection of up to three RNA targets in combination with immunophenotyping for cell surface and intracellular proteins using fluorochrome 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 pairs 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 sides of a respective probe pair have bound Multiple Amplifier molecules then hybridize to their respective PreAmplifier Finally Label Probe oligonucleotides conjugated to a fluorescent dye hybridize to their corresponding Am
18. ation Kit Affymetrix cat QV0523 following the instructions in Appendix A6 of this user manual This assay allows detection of up to three 3 RNA targets in a single sample Type 1 Alexa Fluor 647 high sensitivity best for low or unknown expression levels Type 4 Alexa Fluor 488 intermediate to low sensitivity best for medium to high expression levels Type 6 Alexa Fluor 750 intermediate to low sensitivity 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 amp 450 and eFluor 506 channels but other channels up to approximately 650 nm may also be affected and depends 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 techOebioscience 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 amp permeabilization Target Probe hybridization Day 2 Signal amplification Flow cytometric analysis Not for further
19. bitor 1 by adding RNase Inhibitor 1 to Wash Buffer at a 1 1000 dilution 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 Add 1 mL of Wash Buffer with RNase Inhibitor 1 to each sample invert to mix and spin down at 800xg for 5 minutes Aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently 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 lt 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 PreAmp Mix Amp Mix and diluted Label Probes should be pipetted directly into the 100 LL of residual volume and samples should be mixed well before incubating Do not pipette these solutions onto the walls of the tubes 28 29 30 31 32 33 34 35 36 37 38 Pre warm samples and Wash Buffer to room temperature Pre warm PreAmp Mix Amp Mix and Label Probe Diluent to 40 C Thaw Label Probes on ice in the dark Note This can be done during the Amp Mix incubation Step 34
20. d 1 mL of prepared Fixation Buffer 1 to each sample invert to mix and then incubate for 30 minutes at 2 8 C Spin down at 800xg for 5 minutes then discard supernatant and resuspend cells in the residual volume Prepare 1X Permeabilization Buffer with RNase Inhibitors by diluting Permeabilization Buffer 10X to 1X with RNase free water and then add RNase Inhibitor 1 at a 1 1000 dilution and RNase Inhibitor 2 at a 1 100 dilution Mix gently by inverting Keep at 2 8 C Note You will need 3 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 1X Permeabilization Buffer with RNase Inhibitors to each sample invert to mix and spin down at 800xg for 5 minutes then discard supernatant and resuspend cells in the residual volume 10 Repeat wash with 1X Permeabilization Buffer with RNase Inhibitors Not for further distribution without written consent O 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com 9 affymetrix PrimeFlow RNA Assay Protocol Page 6 of 20 eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only 11 Intracellularly stain cells with fluorochrome conjugated antibodies at their optimal concentration for 30 minutes at 2 8 C Note lf int
21. distribution without written consent O 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com 9 affymetrix PrimeFlow RNA Assay Protocol Page 3 of 20 eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only Fluorochrome compatibility LP 2 3 4 Organic fluorochromes are compatible with this assay such as FITC eFluor amp 450 eFluor 660 and Alexa Fluor amp 700 BV dyes are also reported to work Most protein based fluorochromes are compatible with this assay including PE PE eFluor 610 PE Cyanine5 PE Cyanine5 5 PE Cyanine7 APC and APC eFluor 780 PerCP PerCP Cyanine5 5 and PerCP eFluor amp 710 may 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 The fixation and permeabilization buffers in this kit are compatible with most of eBioscience s antibodies used for intracellular staining Some changes in performance are expected when compared to performance in the Intracellular Fixation 84 Permeabilization Buffer Set cat 88 8824 or Foxp3 Transcription Factor Staining Buffer Set cat 00 5523 The fixation and permeabilization buffers in this kit are compatible with most of eBioscience s phospho specific antibodies however
22. e Day 1 Day 2 39 AO 41 DOMUS oon Add 1 mL of Wash Buffer to each sample invert to mix and spin down at 800xg for 5 minutes Aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently Repeat wash with Wash Buffer Wash with 1 mL of Storage Buffer or Flow Cytometry Staining Buffer then transfer samples to 12x75 mm polystyrene tubes and analyze samples 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 for up to 3 days PrimeFlow RNA Assay Surface stain cells with antibody and or fixable viability dye Wash once with Flow Cytometry Staining Buffer Spin cells at 600xg for 5 minutes Fix cells in Fixation Buffer 1 for 30 minutes at 2 8 C Wash twice with 1X Permeabilization Buffer with RNase Inhibitors Spin cells at 800xg for 5 minutes Intracellularly stain cells with antibody for 30 minutes at 2 8C Wash once with 1X Permeabilization Buffer with RNase Inhibitors Spin cells at 800xg for 5 minutes Fix cells in Fixation Buffer 2 for 60 minutes at room temperature Wash twice with Wash Buffer Spin cells at 800xg for 5 minutes Target Probe hybridization for 2 hours at 40 C Invert to mix after 1 hour Wash once with Wash Buffe
23. e added to the single color samples just before loading the sample onto the cytometer Alternatively an unstained cell sample that has been processed through the PrimeFlow Assay must be used and the median fluorescence intensity of that sample should be noted for all parameters l 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 or the upper left quadrant Figure A3 2 left ii Looking at the statistics note the y or x median of the two populations Adjust the compensation for the fluorochrome being collected subtract its Q4 APC eFluor 780 Median 0 411 04 APC eFluor 760 Median 0 143 fluorescence out of the other channels E mM mE being used in the experiment until the medians of the two populations are the same Figure A3 2 right APC eFluor 780 Figure A3 1 Unstained sample d APC eFluor 780 APC eFluor 780 E 03 L T 1 lil Stop collecting events and remove S S CC sample from the cytometer iv Repeat for each of the single color 8 8 samples d Confirm that the compensation settings are appropriate for the experimental samples APC i Place an experimental sample on the Q4 APC Median 0 894 d cytometer and begin collecting events Figure A3 2 Single color samples for APC top il Looking at
24. e the unstained cell sample on the cytometer and begin to collect events l 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 ii Looking at the histogram plots ensure that the signals are on scale and adjust PMT voltages if necessary Iii Stop collecting events and remove sample from the cytometer c Place a single color sample on the cytometer and begin to collect events l 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 ii 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 Iii Stop collecting events and remove sample from the cytometer IV Repeat for each of the single color samples d Confirm that the voltage settings are appropriate for the experimental samples l Place an experimental sample on the cytometer and begin collecting events ii Looking at each of the histogram plots confirm that all fluorescent signals are on scale and adjust PMT voltages if necessary lii Stop collecting events and remove sample from the cytometer Setting compensation Note Use the PrimeFlow Compensation Kit for single color compensation controls Please see the product data sheet for preparation
25. each of the two parameter row or APC eFluor 780 bottom row plots confirm that the compensation is Uncompensated left or compensated right set correctly by looking at the medians data are shown iii Stop collecting events and remove sample from the cytometer 4 After setting compensation you are ready to acquire your samples 5 Use FMO controls after acquisition and during analysis to optimize compensation settings Not for further distribution without written consent O 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com PrimeFlow RNA Assay Protocol Page 14 of 20 9 affymetrix lOsCIence eB PrimeFlow RNA Assay User Manual and Protocol Research Use Only A4 Examples of Expected Results 90549 GAs t bul seqoidou eat eco seqoidou unsun Eessen zz asou mee adean sm WNAWeaNL usin O3 0o92 10a eei 6 9054eQA4 euueS na wnawn esoo VNMwe4NI unsin ona zaumeko ad 81 ESE vNMuENd de VA E EVA 904eQA4 muuebN wneu nar mde4NI ego ums omiepey 00549 GAs adios rou seqoriou _ edkosiroSu eego seqoidou ums joxuc eqordcuedRos vi ESE adean NTE uns SES 90538 GAS ewwep Ndi vNHUDNJ SO VNMUEJNLI wgs OW4 Z uue o Jd OL 90949 GAS ewwes Nyi vNew DM eude JNI VNHUEJNI Uns OW 01910 49 3d 6 90939 GAs eumebnar
26. ection 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 multicolor PrimeFlow RNA analysis Materials required Calibration beads Samples prepared using the PrimeFlow Compensation Kit cat 88 17001 42 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 amp 750 Compensation Control Single color antibody stained UltraComp eBeads needed only if protein staining is performed Experimental Procedure 1 Ensure proper alignment following the instrument manufacturer s recommendations Alternatively calibration beads such as Rainbow Calibration Particles 8 peak beads Spherotech cat RCP 30 5A or Rainbow Fluorescent Particles 1 peak beads Spherotech cat RFP 30 5A may be used to check the linearity of the PMT PMT voltage range and alignment of the instrument Setting PMT Voltages a Create an acquisition template workspace that includes a forward scatter versus side scatter plot a 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 Plac
27. noyy iuv 1800 19 389 019 amp uon Je Jd e8g2 esnoyW guy yum peuiejs ue njje2eJjui e19M SI A essy YNY wMo Jeuiug ay Bursn p z jeue usuy juBu s ep z 104 L820 9 pue LEO 05L3V VNYU g ew zueso 9 189 spayung epeJc jeuonoun4 8200 pue egqo esnon nuy uim pejejnuuns 10 yel pejejnuunsun aram sejK2ouejds asnon z py e4nBi4 0913V VNYU g ewAZzURID 05L3V VNYU g euzueic 019 40M 4 3d 809 019 1030 34 809 Leuek gt 3d Y eu zuejo 88v JW HA LINW 8873V YNYWU LIN 88 3V VNYW LIDIA 0Sy10M139 uriejoJg 79 4 019 10n 30 3d 809 0 y10n 39 uiejoJd 194 019 10n 30 3d 809 paje nuns paje nuysun Not for further distribution without written consent O 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com e info ebioscience com PrimeFlow RNA Assay Protocol Page 18 of 20 9 affymetrix eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only A5 Validated Cells and Recommended Positive Control Genes Cell Recommended Positive Control Gene Human peripheral blood whole blood or PBMC fresh and HPL13A B2M cryopreserved Mouse splenocytes tissue fresh and cryopreserved ACTB RPL13A Mouse thymocytes tissue ACTB RPL13A Mouse bone marrow ACTB Human monocytic lymphoma U937 HPL13A B2M Human T cell lymphoma Jurkat HPL13A B2M Human cervical carcinoma HeLa RPL13A GAPDH Human lung carcinoma PC9 RPL13A
28. 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 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 De To ensure proper assay performance use Positive Control Probe Sets in every experiment For your convenience these probe sets are included with the PrimeFlow RNA Assay RPL13A for human leukocytes and B actin ACTB for mouse tissues See Appendix A5 for specific cell types and other recommended positive control genes For proper compensation we recommend using the PrimeFlow Compensation Kit cat 88 17001 42 Please refer to the product data sheet on the product page for instructions for use Alternatively 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 setting compensation for beads use the negative bead population for cells use
29. plifier 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 eBioscience currently offers 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 Detection Preparation Hybridization Amplification Es zz Gene specific ooo Fluorescent Label Ree JY Target Probes PreAmplifier 00 0 Probe Add Label Probes to cells p Amplifier M pensi I Label proteins with Incubate cells with Hybridized with antibody optional gene specific Target Pre Amplifier and Probes Type 1 4 or Amplifier DNA Fix and permeabilize 6 Type 1 4 and 6 cells in suspension process cells using a standard flow cytometer Gene 1 Gene 2 IFN gamma eFluor 450 Suspension cells with RNA fixed IFN gamma mRNA AF750 Not for further distribution without written consent 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com
30. preparation of signal amplification reagents 4 Incorrect incubator temperature 5 Incorrect Wash Buffer 6 Incorrect diluents used 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 using trypan blue or other viability dye before beginning the assay 1 Confirm settings are for RCF xg not RPM 2 Set centrifuge to the speed specified in the protocol Only use the 1 5 mL tubes provided with the kit Tubes from other vendors may result in significant cell loss Ensure vacuum setting for aspirator is low Aspirate no faster than 0 5 mL sec Place a 10 uL pipette tip on the tip of the aspirator to help reduce aspiration rate Follow the meniscus while aspirating down to the 100 uL mark on the side of the tube Avoid excessive vortexing to prevent cell damage Vortex in short pulses Verify the flow rate of cytometer using beads of known concentration such as the Flow Cytometry Absolute Count Standard Bangs Laboratories cat 580 Check that your instrument is properly set up according to the manufacturer s recommendations Use samples labeled with one each of the included Positive Control Probe Sets to set compensation Do not use fluorochrome conjugated antibodies to set compensation for RNA detection Make sure PreAmp Mix
31. r Spin cells at 800xg for 5 minutes Wash once with Wash Buffer with RNase Inhibitor 1 Spin cells at 800xg for 5 minutes Store samples overnight PreAmp hybridization for 1 5 hours at 40 C Wash three times with Wash Buffer Spin cells at 800xg for 5 minutes Amp hybridization for 1 5 hours at 40 C Wash twice with Wash Buffer Spin cells at 800xg for 5 minutes Label Probe hybridization for 1 hour at 40 C Wash twice with Wash Buffer Spin cells at 800xg for 5 minutes Wash once with Storage Buffer Spin cells at 800xg for 5 minutes Analyze samples on a flow cytometer Not for further distribution without written consent O 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 e Fax 858 642 2046 e www ebioscience com info ebioscience com 9 affymetrix eBioscience PrimeFlow RNA Assay Protocol Page 9 of 20 PrimeFlow RNA Assay User Manual and Protocol Research Use Only Appendix A1 Troubleshooting A Poor Cell Recovery Low Cell Count on Cytometer B Weak No Signal 1 Cell lysis due to cells in poor physiological condition during processing 2 Improper centrifuge settings 3 Improper tubes used 4 High aspiration rate by pipetting or vacuum aspirator when removing supernatant 5 Rough handling 6 Incorrect cell count by flow cytometer 1 Cytometer not set up properly 2 Incorrect samples used for setting compensation 3 Incorrect
32. racellular staining is not desired skip this step and proceed to Step 12 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 Add 1 mL of 1X Permeabilization Buffer with RNase Inhibitors to each sample invert to mix and spin down at 800xg for 5 minutes then discard supernatant and resuspend cells in the residual volume by vortexing gently Prepare 1X Fixation Buffer 2 by combining 125 uL of Fixation Buffer 2 8X with 875 uL of 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 Fixation Buffer 2 8X with 8 75 mL of Wash Buffer Avoid vortexing or vigorously shaking this buffer This buffer should be prepared fresh Dispose of any unused buffer Add 1 mL of 1X Fixation Buffer 2 to each sample invert to mix and then incubate for 60 minutes in the dark at room temperature Note It is important to fix the samples at room temperature Do not perform this step on ice OPTIONAL Cells
33. rtexing Reduce voltage settings of your cytometer Note that this assay may require lower settings than typical antibody staining Follow the protocol for proper fixation time Dilute Target Probe at a 1 20 dilution Not for further distribution without written consent 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 Fax 858 642 2046 e www ebioscience com info ebioscience com PrimeFlow RNA Assay Protocol Page 11 of 20 9 affymetrix eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only A2 Instrument and Equipment Setup Guide This guide illustrates the setup of typical equipment and their specifications for the PrimeFlow RNA 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 Specifications 1 Incubator with heat block inside 1 Validated to maintain 40 1 C 2 Metal heat block for 1 5 mL microcentrifuge tube Example shown Incubator Affymetrix cat QS0704 Metal heat block VWR cat 13259 002 Metal heat block 2 Refrigerated swinging bucket centrifuge Swinging bucket with adaptors for 15 mL conical tubes and 1 5 mL microcentrifuge tubes Optional with refrigeration to 4 C wm Examples shown Geier Centrifuge Eppendorf model 5810R
34. say User Manual and Protocol Research Use Only 4 Assess incubator temperature uniformity a Repeat Step 3 to measure the temperature at multiple positions in a the incubator to determine temperature uniformity sa D Note The temperature for all positions should be 40 1 T z go 5 Assess temperature ramp up time a Remove the 1 5 mL tube containing the probe from the prewarmed heat block from Step 3 and allow it to return to room temperature b Open the incubator door for 1 minute then place the 1 5 mL tube containing the probe from Step 5a 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 c Repeat Steps 5a b two more times Note 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 Not for further distribution without written consent 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 Fax 858 642 2046 e www ebioscience com info ebioscience com
35. te 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 21 Thaw Target Probes including Positive Control Probes at room temperature Pre warm Target Probe Diluent to 40 C Not for further distribution without written consent 2000 eBioscience Inc an Affymetrix company Tel 888 999 1371 or 858 642 2058 Fax 858 642 2046 e www ebioscience com info ebioscience com PrimeFlow RNA Assay Protocol Page 7 of 20 9 affymetrix eBioscience PrimeFlow RNA Assay User Manual and Protocol Research Use Only 22 23 24 20 26 gr Dilute Target Probes 1 20 in 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 Target Probe Diluent accordingly so that the final volume remains 100 uL per sample Add 100 uL of diluted Target Probe s directly into the cell suspension for the appropriate samples briefly vortex to mix and then incubate for 2 hours at 40 C Invert samples to mix after 1 hour Add 1 mL of Wash Buffer to each sample invert to mix and spin down at 800xg for 5 minutes Aspirate all but 100 uL of supernatant and resuspend cells in the residual volume by vortexing gently Prepare Wash Buffer with RNase Inhi
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