User Manual
Contents
1. 23 Blank Plate Map 25 Introduction About This Manual Who Should Read this Manual This manual is for anyone who has purchased QuantiGene 2 0 Assay Kits from Panomics to perform the QuantiGene 2 0 Assay for any of the following sample types u Cultured cells u Whole blood a Fresh frozen or formalin fixed paraffin embedded FFPE animal tissues a Purified RNA What this Manual Covers This manual provides recommendations and step by step procedures for the following a Experimental design and data analysis QuantiGene 2 0 assay u Troubleshooting Safety Warnings and Precautions All chemicals should be considered potentially hazardous We recommend that this product and its components be handled by those trained in laboratory techniques and used according to the principles of good laboratory practice For research use only Not for use in diagnosis of disease in humans or animals Contacting Affymetrix Technical Help For technical questions please contact our technical support group by telephone at 1 877 726 6642 option 3 or email at techsupport panomics com US and Canada In Europe contact techsupport_europe panomics com For an updated list of FAQs and product support literature visit our website at www panomics com QuantiGene QuantiGene 2 0 Reagent System Basics The QuantiGene 2 0 Reagent System is designed to quantitate target specific RNA molecules2. a Dynamic range gt 8 logs u Well to well uniformity 5 Cross talk 5 x 10 stain Ex 480 nm Em 520 nm Luminescence detector with the following features Sensitivity gt 3 x 10 moles of luciferase a Fluorescent detection module optional for DNA specifications IMPORTANT Make sure your luminometer meets or exceeds minimum performance Turner BioSystems Molecular Devices Modulus Microplate Luminometer P N 9300 001 LMAX or equivalent following specifications 30 200 pL 5 volume 96 or 384 channels a Angle dispensing tip Plate stacker Automation capable a Minimal dead volume Incubator or oven capable of maintaining constant Hybaid Hybridization oven model 9270 temperatures of 50 and 55 C 1 C VWR Economy incubator model 1500E 1500EM or equivalent 4 inch soft rubber roller or Affymetrix QS0515 QuantiGene CTC Plate Sealer Affymetrix QG0400 QuantiGene Incubator Temperature Validation Kit Affymetrix QS0517 Optional Plate washer that meets or exceeds the BioTek ELx 405 model with high throughput pump option Experimental Design and Assay Optimization Overview Here we provide information and guidelines for Optimizing sample input u Replicate recommendations u Assay background controls Housekeeping genes u Data analysis Optimizing Sample Input The QuantiGene 2 0 assay has a linear dynamic range of greater than 3 5 logarithms and can detect 200
3. a Probe Set s and Blocking Reagent Thaw vortex briefly to mix then centrifuge briefly to collect contents at the bottom of the tubes a Tissue homogenates If previously frozen thaw at room temperature followed by incubation at 37 Cfor 15 30 minutes Vortex briefly then leave at room temperature until use Lysis Mixture Re dissolve any precipitates by incubating at 37 C followed by gentle swirling Remove Capture Plate from 4 C and place on the benchtop to warm completely to room temperature approximately 30 minutes Do not remove the plate from the sealed foil pouch 10 QuantiGene 2 0 Reagent System User Manual 2 If appropriate based on the expression level of the target or housekeeping RNA of interest dilute tissue homogenates with Homogenizing Solution so that the desired amount of sample is present in a volume of 40 uL assay well Use the table below as a guide and scale dilutions according to the number of assays to be run Table 3 1 Recommended input for different preparations Recommended Sample Input RNA copies per cell Tissue Homogenate uL 1 40 10 40 100 4 gt 1 000 lt 0 4 May not have sensitivity required Prepare an appropriate volume of Working Probe Set by combining the following reagents in the order listed Scale according to the number of assays to be run and include 40 overage For a guide use the table below that corresponds to the date of your
4. 1 Prepare the following reagents a Probe Set s and Blocking Reagent Thaw vortex briefly to mix then centrifuge briefly to collect contents at the bottom of the tubes RNA sample s If previously frozen thaw on ice Lysis Mixture Re dissolve any precipitates by incubating at 37 C followed by gentle swirling Remove Capture Plate from 4 C and place on the benchtop to warm completely to room temperature approximately 30 minutes Do not remove the plate from the sealed foil pouch 12 QuantiGene 2 0 Reagent System User Manual 2 Dilute RNA in nuclease free water so that the desired amount of RNA is present in a volume of 20 uL assay well based on expression level of target or housekeeping RNA of interest for total RNA or mRNA Use the table below as a guide and scale dilutions according to the number of assays to be run Table 3 1 Recommended input for different preparations Recommended Sample Input Target RNA copy number cell Total RNA ng PolyA RNA pg 1 100 2 000 10 10 200 100 1 20 gt 1 000 lt 0 1 lt 2 For in vitro transcribed IVT RNA the recommended sample input is gt 1000 RNA copies per well We recommend including 200 ng uL yeast tRNA in IVT dilutions to minimize RNA loss 3 Prepare an appropriate volume of Working Probe Set by combining the following reagents in the order listed Scale according to the number of assays to be run and include 40 o
5. Chapter 3 QuantiGene 2 0 Assay Procedure 15 Add 11 uL of 2 0 Amp to 11 mL of Amplifier Label Probe Diluent Invert to mix Keep at room temperature until use 2 Wash the Capture Plate A B C D E Remove the Capture Plate from the incubator and remove the Plate Seal Add 200 uL well of 1X Wash Buffer Invert the Capture Plate over an appropriate receptacle for example a BioHazard container and expel the contents forcibly Firmly tap the inverted plate on a clean paper towel to dry Repeat steps 2b 2d two more times using 300 uL well of 1X Wash Buffer 3 Remove all traces of 1X Wash Buffer A B Invert the Capture Plate on a clean dry paper towel Centrifuge at 240 x g for 1 minute at room temperature Use maximum acceleration and brake settings available Lu IMPORTANT Do not exceed 240 x g for 1 minute C Proceed to the next step immediately 4 Add 100 uL of 2 0 Amp Working Reagent to each well of the Capture Plate 5 Seal the Capture Plate with a Plate Seal and incubate at 55C 1 C for 60 minutes Hybridizing the Label Probe To hybridize the Label Probe 1 Prepare Label Probe Working Reagent A B C D Centrifuge Label Probe briefly to collect the contents to the bottom of the tube Add 11 uL of 2 0 Label Probe to 11 mL of Amplifier Label Probe Diluent Invert to mix Keep at room temperature until use 2 Wash the Capture Plate A Remove the Ca
6. copies of target RNA When running a sample type for the first time using a luminometer for the first time and or using new target specific Probe Sets optimize sample input to ensure that assay signals exhibit a linear dose response For each sample type we provide recommendations for typical starting sample inputs depending on expression level of target RNA see Capturing Target RNA on page 7 Using these recommendations as a guide perform a 2 to 4 fold dilution series of your sample and verify that the resulting assay signals are linearly proportional to sample input For more information see Determining Assay Linearity on page 6 Replicates Technical replicates are replicate assays from a single sample For example a cell lysate that is divided into several portions and each portion run in the same QuantiGene 2 0 assay Biological replicates are replicate assays from biologically equivalent samples For example cells grown in different wells that are subjected to the same treatment lysed independently then run as distinct samples in the QuantiGene 2 0 assay We recommend running 3 technical replicates of each distinct biological sample Recommended Assay Controls Assay Background Control Assay background is the signal generated by all assay components in the absence of sample input Include an assay background control in triplicate for each Probe Set used on an individual Capture Plate Use of Housekeeping Genes A hou
7. example a BioHazard container and expel the contents forcibly D Firmly tap the inverted plate on a clean paper towel to dry E Repeat steps 1b 1d two more times using 300 uL well of 1X Wash Buffer 2 Remove all traces of 1X Wash Buffer A Invert the Capture Plate on a clean dry paper towel B Centrifuge at 240 x g for 1 minute at room temperature Use maximum acceleration and brake settings available Lu IMPORTANT Do not exceed 240 x g for 1 minute C Proceed to the next step immediately NOTE Ensure that 2 0 Substrate is at room temperature before use 3 Add 100 uL of 2 0 Substrate to each well of the Capture Plate Seal the Capture Plate with a Plate Seal and incubate at room temperature for 5 minutes 5 Remove the Plate Seal place the Capture Plate in the luminometer and read Set integration read time to 0 2 seconds For best results read plate within 15 minutes Troubleshooting Low Assay Signal or Poor Sensitivity Table 4 1 Troubleshooting Low Assay Signal or Poor Sensitivity Probable Cause Recommended Action Number of target RNA molecules below limit of detection Increase the sample input Signal amplification reagent incorrectly prepared Dilute 2 0 PreAmp 2 0 Amp and Label Probe in Amplifier Label Probe diluent Incorrect incubation temperature Verify incubation temperatures using a QuantiGene Incubator Temperature Validation Kit temperature Ina
8. reagent shortage will occur B Using a multichannel pipette and new tips for each transfer dispense 60 uL of Working Probe Set into each assay well Avoid introducing bubbles Hi IMPORTANT Capture Probe oligonucleotides are conjugated to the surface of Capture Plate wells Do not scratch Capture Plate wells with pipette tips Using a new pipette tip for each replicate transfer 40 uL sample to each well of the Capture Plate containing Working Probe Set Avoid introducing bubbles Do not mix IMPORTANT Add 40 uL of Homogenizing Solution to 3 wells for assay background controls Bind target RNAs A Place an adhesive Plate Seal squarely on the plate and seal tightly Lu IMPORTANT Complete and uniform sealing of the overnight hybridization plate is essential Use a soft rubber roller or the QuantiGene CTC Plate Sealer Letters and numbers on the Capture Plate should be clearly defined beneath the Plate Seal B Centrifuge the Capture Plate at 240 x g for 20 seconds at room temperature to ensure the contents contact the bottom of the well C Immediately place the Capture Plate in a 55 1 C incubator to begin the overnight 16 20 hour hybridization Lu IMPORTANT Temperature must be 55 1 C Verify temperature using a QuantiGene Incubator Temperature Validation Kit Capturing Target RNA from Total RNA Purified mRNA or In Vitro Transcribed RNA To capture target RNA from purified RNA preparations
9. Aspirate delay Crosswise aspirate No No Crosswise aspirate on Crosswise height Crosswise horizontal position Final aspirate Yes Yes Final aspirate delay 2 seconds 2 seconds Capture Plate Dimensions About Capture Plate Dimensions We provide the Capture Plate dimensions to enable you to setup and validate alternative automated plate washers NOTE The Capture Plate construction adheres to the Society for Biomolecular screening standards Figure B 1 oi Ene bottom View A 10 8 mm Well depth 14 3 mm Plate height 14 0 mm Well A1 x offset D 11 2 mm Well A1 y offset b D 9 0 mm Well to well offset D 85 5 mm Plate width 127 8 mm Plate length Top View 24 QuantiGene 2 0 Reagent System User Manual Blank Plate Map 26 QuantiGene 2 0 Reagent System User Manual
10. KG Affymetrix User Manual QuantiGene 2 0 Reagent System For research use only Not for use in diagnostic procedures Trademarks a Affymetrix and AMG are trademarks of Affymetrix Inc QuantiGene is a registered trademark exclusively licensed to Affymetrix Inc All other trademarks are the property of their respective owners Limited License Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products Affymetrix grants you a non exclusive non transferable non sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix You understand and agree that except as expressly set forth in the Affymetrix terms and conditions no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product In particular no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided licensed or specifically recommended by Affymetrix for such use Citing QuantiGene 2 0 Reagent System in Publications When describing a procedure for publication using this product please refer to it as the QuantiGene 2 0 Reagent System If a paper cites a QuantiGene product and is published in a research journal the lead author s may receive a travel stipend for use at a technology conference or tradeshow by sending a cop
11. Prepare PreAmp Working Reagent A Thaw 2 0 PreAmp then centrifuge briefly to collect the contents at the bottom of the tube B Add 11 uL of 2 0 PreAmp to 11 mL of Amplifier Label Probe Diluent C Invert to mix D Keep at room temperature until use Wash the Capture Plate A Remove the Capture Plate from the incubator and remove the Plate Seal B Add 200 uL well of 1X Wash Buffer C Invert the Capture Plate over an appropriate receptacle for example a BioHazard container and expel the contents forcibly D Firmly tap the inverted plate on a clean paper towel to dry E Repeat steps 2b 2d two more times using 300 pL well of 1X Wash Buffer NOTE For recommendations for automated plate washing see Alternative Capture Plate Washing Method on page 21 Remove all traces of 1X Wash Buffer A Invert the Capture Plate on a clean dry paper towel B Centrifuge at 240 x g for 1 minute at room temperature Use maximum acceleration and brake settings available Lu IMPORTANT Do not exceed 240 x g for 1 minute C Proceed to the next step immediately Add 100 uL of 2 0 PreAmp Working Reagent to each well of the Capture Plate Seal the Capture Plate with a Plate Seal and incubate at 55 1 C for 60 minutes Hybridizing the 2 0 Amplifier To hybridize the 2 0 Amp 1 Prepare 2 0 Amp Working Reagent A Thaw 2 0 Amp then centrifuge briefly to collect the contents at the bottom of the tube B C D
12. Probe Set Lu IMPORTANT Include 3 wells for assay background controls Table 3 2 For QuantiGene 2 0 Probe Sets Received Before December 31 2007 Reagent 1 Well pL 48 Wells pL 96 Wells pL Nuclease free water 25 4 1 646 3 293 Lysis Mixture 33 3 2 238 4 476 Blocking Reagent 1 67 134 CEs 0 1 6 7 13 4 LEs 0 1 6 7 13 4 BLs 0 1 6 7 13 4 Total 60 4 032 8 064 a Includes 40 overage bOmit for 18S or 28S RNA robe Sets Substitute volume with nuclease free water Table 3 3 For QuantiGene 2 0 Probe Sets Received After January 1 2008 Reagent 1 Well pL 48 Wells pL 96 Wells pL Nuclease free water 25 4 1 646 3 293 Lysis Mixture 33 3 2 238 4 476 Blocking Reagent 1 67 134 2 0 Probe Set 0 3 20 1 40 2 Total 60 4 032 8 064 a Includes 40 overage Omit for 18S or 28S RNA robe Sets Substitute volume with nuclease free water Prepare the Capture Plate A Open the sealed foil pouch and remove the Capture Plate Chapter 3 QuantiGene 2 0 Assay Procedure 11 B Vortex Working Probe Set briefly to mix then dispense into the Capture Plate For fewer than 48 wells Using a single channel pipette and a new tip for each transfer dispense 60 uL Working Probe Set into each assay well Avoid introducing bubbles For 48 wells or more A Using a single channel pipette transfer Working Probe Set to a 25 mL divided reagent reservoir NOTE Do not pour or
13. ackground controls Table 3 2 For QuantiGene 2 0 Probe Sets Received Before December 31 2007 Reagent 1 Well pL 48 Wells pL 96 Wells pL Nuclease free water 12 1 813 1 626 Lysis Mixture 6 6 444 887 Blocking Reagent 1 67 134 CEs 0 1 6 7 13 4 LEs 0 1 6 7 13 4 BLs 0 1 6 7 13 4 Total 20 1 344 2 688 a Includes 40 overage bOmit for 18S or 28S RNA robe Sets Substitute volume with nuclease free water Table 3 3 For QuantiGene 2 0 Probe Sets Received After January 1 2008 Reagent 1 Well pL 48 Wells pL 96 Wells pL Nuclease free water 12 1 813 1 626 Lysis Mixture 6 6 444 887 Blocking Reagent 1 67 134 2 0 Probe Set 0 3 20 1 40 2 Total 20 1 344 2 688 a Includes 40 overage Omit for 18S or 28S RNA robe Sets Substitute volume with nuclease free water Prepare the Capture Plate A Open the sealed foil pouch and remove the Capture Plate Chapter 3 QuantiGene 2 0 Assay Procedure 9 B Vortex Working Probe Set briefly to mix then dispense into the Capture Plate For fewer than 48 wells Using a single channel pipette and a new tip for each transfer dispense 20 uL Working Probe Set into each assay well Avoid introducing bubbles For 48 wells or more A Using a single channel pipette transfer Working Probe Set to a 25 mL divided reagent reservoir NOTE Do not pour or reagent shortage will occur B Using a multichannel pipette and ne
14. directly from a Cultured cell lysates u Whole blood PAXgene blood RNA or dried blood spot lysates Fresh or frozen animal tissue homogenates FFPE animal tissue homogenates u Total RNA mRNA or in vitro transcribed RNA preparations Please refer to the QuantiGene Sample Processing Kit Package Inserts for instructions on preparing cultured cell or blood lysates or animal tissue homogenates To prepare RNA follow standard laboratory methods 2 QuantiGeneG 2 0 Reagent System User Manual The QuantiGene 2 0 assay is a hybridization based assay performed on 96 well plates The ability to quantify specific RNA molecules within a sample lies in the design of a QuantiGene 2 0 Probe Set Each oligonucleotide probe set contains three types of synthetic probes Capture Extenders CEs Label Extenders LEs and Blockers BLs that hybridize to contiguous sequences of the target RNA The CEs bind to the capture oligonucleotides conjugated to the well surface and via cooperative hybridization capture the associated target RNA Signal amplification is mediated by DNA amplification molecules that hybridize to the tails of the LEs Each amplification unit contains hybridization sites for 400 alkaline phosphatase conjugated Label Probes which can then be detected by the alkaline phosphatase mediated degradation of a chemiluminescent substrate Luminescence is reported as relative light units RLUs on a microplate luminometer The amount of luminescen
15. e QuantiGene 2 0 Reagent System Kit Components The components of the QuantiGene 2 0 Assay Kit and their recommended storage conditions are listed below The QuantiGene 2 0 Assay Kit is available in 4 sizes Refer to the product insert for quantities of individual components supplies Kit components have a shelf life of 6 months from date of receipt and contains the following components Table 1 1 QuantiGene 2 0 Reagent System Kit Components and Their Storage Conditions Component Description Storage 2 0 PreAmplifier PreAmp 1 DNA in aqueous buffered solution 20 C 2 0 Amplifier Amp 1 DNA in aqueous buffered solution 20 C Blocking Reagent Aqueous buffered solution 20 C containing a preservative Capture Plate 96 well polystyrene plate coated 2 8 C with capture probes Label Probe Oligonucleotide alkaline 2 8 C phosphatase conjugate in aqueous buffered solution 2 0 Substrate Chemiluminescent substrate 2 8 C Amplifier Label Probe Diluent Aqueous buffered solution with a 15 30 C protein containing preservative Lysis Mixture Aqueous buffered solution 15 30 C containing a preservative Plate Seals Adhesive backed foil seal 15 30 C Wash Buffer Component 1 Aqueous solution 15 30 C Wash Comp 1 Wash Buffer Component 2 Aqueous buffered solution 15 30 C Wash Comp 2 2Lumigen APS 5 QuantiGene 2 0 Reagent System Accessory Reagents In addition to QuantiGene 2 0 Assa
16. g to the next step in the procedure Scratching of the capture well surface Minimize contact with the Capture Plate well surfaces during all addition and washing steps Cross talk among neighboring wells during reading Only use luminometers with cross talk 0 001 Variable salt concentrations Hybridization is affected by salt When diluting samples always use the appropriate diluent Inaccurate pipetting Only use calibrated precision pipettes a Affix tips securely a Use a new tip for each transfer a Pipet slowly and carefully avoiding bubbles Non homogenous samples Warm samples to 37 C to dissolve any precipitates and vortex briefly before use Samples too viscous to pipet accurately Dilute samples 1 2 in the appropriate diluent before use Chapter 4 Troubleshooting 19 Day To Day Variation Table 4 5 Troubleshooting High Inter Plate CVs Probable Cause Recommended Action Variable incubation temperatures Keep incubation temperatures consistent Variable incubation times Keep incubation times consistent especially for incubation with 2 0 Substrate Non constant time between Make sure that time between addition of 2 0 Substrate and plate read is addition of 2 0 Substrate and plate consistent read 20 QuantiGene Reporter Gene Assay User Manual Alternative Capture Plate Washing Method Automated Washing Procedure NOTE Automated wa
17. ired Materials Not Provided 4 Experimental Design and Assay Optimization 5 OI Re hrhnrnna xDxx xonmmi 5 Optimizing Sample Inp t ee eras a halala b a sister Rc EROR Rabe tes 5 R DIICAT S oid cs ded nn nee epe ede bbb ee det bbb die rote eee d 5 Recommended Assay Controls 5 Assay Background Contrl see ck ea Wo H l ee b b Se Ok REA PEST 5 Use of Housekeeping Genes 2 5 Data Analysis Guidelines 6 Calculating Assay Precision 6 Calculating Assay Limit of Detection 1 6 Determining Assay Linearity 6 Normalizing Gene Expression Data 6 Calculating Fold Change of Gene Expression 6 QuantiGene 2 0 Assay Procedure 7 noA A PTLC LMPET 7 Capturing Target RINAC 224 me uet Rp 0008 8 de ede RR AU diet o e ode ets A bee eS 7 About Capturing Target RNA 43 ahaa tent eens 7 Capturing Target RNA from Cultured Cell or Blood Lysates 7 Capturing Target RNA from Fresh Frozen or FFPE Tissue Homogenates 9 Capturing Target RNA from T
18. otal RNA Purified mRNA or In Vitro Transcribed RNA 11 Signal Amplification and Detection 0 0 kk kk kk kK KK KK KK KI KK K K KK K KK KIRI KI KK KI KK 13 About Signal Amplification and Detection 13 Preparing 1X Wash Buffer 14 Hybridizing the 2 0 PreAmplifier 2 14 Aybridizing the 2 0 AmpliflE auc s cama as 3d bre ad ani So ed ere a ee v dun ui 14 iv QuantiGene 2 0 Reagent System User Manual Chapter 4 Appendix A Appendix B Appendix C Hybridizing the Label Probe 15 Adding Substrate and Detecting Signal 16 NPOUDIESHOOMNG qas a be assis bine 32805 8 Alaya ak ROW NUR AEE E K3 den 17 Low Assay Signal or Poor Sensitivity 17 Non Uniform Signal Across the Plate 17 High Background Signal 18 Well To Well Variation 2 0 0 0 ls RR e 18 Day To Day Variation xxx rate n ace ene bb kwa a dn d r Og A o gc ote n y E an 19 Alternative Capture Plate Washing Method 21 Automated Washing Procedure 21 Capture Plate Dimensions 23 About Capture Plate Dimensions
19. ppropriate hybridization Hybridization reactions must be carried out at 40 1 C Use a QuantiGene Incubator Temperature Validation Kit to verify and monitor the temperature phosphatase Inactivation of alkaline Do not exceed 50 C after the addition of Label Probe Do not allow the Capture Plate to stand dry for more than 5 minutes once the signal amplification and detection procedure has started Expired reagents were used Reagents are good for up to 6 months from date of receipt Non Uniform Signal Across the Plate Luminometer does not have the required sensitivity Only use luminometers that meet or exceed the minimum performance specifications see page 9 Table 4 2 Troubleshooting Non Uniform Signal Probable Cause Recommended Action incubator Temperature gradients within the Verify that the incubator maintains a constant even temperature Avoid opening and closing the incubator door during hybridization steps Temperature gradients on Capture Plate at time of reading Read plate at room temperature If luminometer has heating capability ensure that this function is turned off Incomplete sealing during overnight hybridization Use the CTC Plate Sealer for robust plate sealing Affymetrix P N QG0400 Ensure numbers and letters are clearly visible from under the foil seal Verify that the supplied plate seal was used prior to the assay Capture Plates exposed to moi
20. pture Plate from the incubator and remove the Plate Seal Li IMPORTANT If you are using a single incubator adjust the temperature to 50 1 C Verify the temperature using a QuantiGene Incubator Temperature Validation Kit B Add 200 uL well of 1X Wash Buffer C Invert the Capture Plate over an appropriate receptacle for example a BioHazard container and D E expel the contents forcibly Firmly tap the inverted plate on a clean paper towel to dry Repeat steps 2b 2d two more times using 300 uL well of 1X Wash Buffer 3 Remove all traces of 1X Wash Buffer A Invert the Capture Plate on a clean dry paper towel Centrifuge at 240 x g for 1 minute at room temperature Use maximum acceleration and brake settings available Lu IMPORTANT Do not exceed 240 x g for 1 minute 16 QuantiGene 2 0 Reagent System User Manual C Proceed to the next step immediately 4 Add 100 uL of Label Probe Working Reagent to each well of the Capture Plate 5 Seal the Capture Plate with a Plate Seal and incubate at 50 1 C for 60 minutes Lu IMPORTANT During this incubation remove 2 0 Substrate from 4 C and allow it to warm to room temperature Adding Substrate and Detecting Signal To detect signal 1 Wash the Capture Plate A Remove the Capture Plate from the incubator and remove the Plate Seal B Add 200 uL well of 1X Wash Buffer C Invert the Capture Plate over an appropriate receptacle for
21. r Working Probe Set to a 25 mL divided reagent reservoir NOTE Do not pour or reagent shortage will occur B Using a multichannel pipette and new tips for each transfer dispense 80 uL of Working Probe Set into each assay well Avoid introducing bubbles Lu IMPORTANT Capture Probe oligonucleotides are conjugated to the surface of Capture Plate wells Do not scratch Capture Plate wells with pipette tips 5 Using a new pipette tip for each replicate transfer 20 uL sample to each well of the Capture Plate containing Working Probe Set Avoid introducing bubbles Do not mix Lu IMPORTANT Add 20 uL of nuclease free water to 3 wells for assay background controls 6 Bind target RNAs A Place an adhesive Plate Seal squarely on the plate and seal tightly Lu IMPORTANT Complete and uniform sealing of the overnight hybridization plate is essential Use a soft rubber roller or the QuantiGene CTC Plate Sealer Letters and numbers on the Capture Plate should be clearly defined beneath the Plate Seal B Centrifuge the Capture Plate at 240 x g for 20 seconds at room temperature to ensure the contents contact the bottom of the well C Immediately place the Capture Plate in a 55 1 C incubator to begin the overnight 16 20 hour hybridization Lu IMPORTANT Temperature must be 55 1 C Verify temperature using a QuantiGene Incubator Temperature Validation Kit Signal Amplification and Detection Abou
22. raight line R2 0 95 indicates you are operating in the linear range of the assay a Calculate the ratio of background subtracted AVG RLU from sequential sample dilutions Observed values should be within 20 of the expected ratio For example for a 2 fold sample dilution the expected ratio of background subtracted AVG RLU is 2 20 so the observed ratio should be between 1 6 and 2 4 Normalizing Gene Expression Data To normalize gene expression data 1 For the gene of interest subtract the AVG assay background signal from the AVG signal of technical replicates 2 Divide the background subtracted AVG signals by the background subtracted AVG signal of the housekeeping RNA NOTE If multiple housekeeping RNAs are measured the geometric mean of background subtracted AVG housekeeping RNA signals may be used for data normalization Calculating Fold Change of Gene Expression To calculate fold change of gene expression of target RNA in treated versus untreated samples 1 Normalize gene expression data as described Normalizing Gene Expression Data on page 6 2 Divide the normalized value for the treated sample by the normalized value for the untreated sample QuantiGene 2 0 Assay Procedure Assay Workflow Table 3 4 Step Tasks For a procedure refer to 1 Prepare samples Appropriate QuantiGene Sample Processing Kit package insert for preparing cultured cell whole blood lysates and tissue homogena
23. sekeeping gene is a target gene that is stably expressed under all experimental conditions evaluated Signals from housekeeping genes can be used to normalize gene expression data across samples Measure one or more housekeeping genes in triplicate for each sample For a list of available 2 0 Housekeeping Gene Probe Sets please go to www panomics com 6 QuantiGene 2 0 Reagent System User Manual Data Analysis Guidelines Calculating Assay Precision The Coefficient of Variation CV is a measure of assay precision QuantiGene 2 0 Assay CVs are typically less than 15 for technical replicates To determine the assay CV 1 Run technical replicates n 3 of each sample 2 Calculate the average signal AVG of technical replicates 3 Calculate the standard deviation SD for the triplicates of signals from technical replicates 4 Calculate the CV CV SD AVG 100 Calculating Assay Limit of Detection Calculate assay limit of detection LOD as follows LOD AVG RLU of assay background control wells 3X SD of assay background signals Assay signals below LOD should not be used to draw quantitative conclusions about gene expression Determining Assay Linearity To determine assay linearity 1 Runa dilution series of your sample 2 Subtract the AVG assay background signal from the AVG signal of technical replicates 3 Use one of the following methods a Plot background subtracted AVG signal versus the amount of sample used A st
24. shing of plates might require the purchase of additional Wash Buffer Program the BIO TEK ELx405R washer with settings for the dispense program D3 and the wash programs 44 and 45 Link the dispense program D3 to the wash programs 44 and 45 to yield Link 1 and 2 respectively Use Link 1 to wash the Capture Plates after the overnight hybridization of the sample with the target specific Probe Set after the 2 0 Pre Amplifier hybridization and the 2 0 Amplifier hybridization Use Link 2 to wash the Capture Plates after the Label Probe hybridization Table A 1 ELx405R Washer Settings Parameter Program D3 44 45 Method Number of cycles 3 5 Soak Shake Yes Yes Soak duration 10 seconds 10 seconds Shake before soak No No Prime after soak No No Prime volume Prime flow rate Dispense Dispense volume 290 395 395 Dispense flow rate 5 5 5 Dispense height 115 115 115 Horizontal dispense position 10 10 10 Horizontal Y dispense position 0 0 0 Bottom wash first No No No Bottom dispense volume Bottom flow rate Bottom dispense height Bottom dispense position Prime No No No Prime volume Prime flow rate Aspiration Aspirate height 32 32 22 QuantiGene 2 0 Reagent System User Manual Table A 1 ELx405R Washer Settings Horizontal aspirate position 45 45 Horizontal Y aspirate position Aspirate rate 5 5
25. sture Allow the Capture Plate to come to room temperature for 30 minutes before opening the sealed foil pouch to avoid condensation Variable salt concentrations Hybridization is affected by salt When diluting samples always use the appropriate diluent 18 QuantiGene Reporter Gene Assay User Manual High Background Signal Table 4 3 Troubleshooting High Background Signal Probable Cause Recommended Action Residual Wash Buffer Ensure that the plate wash method completely removes all residual Wash Buffer prior to moving to the next step in the procedure Incorrect temperature in the incubator Verify incubation temperatures using a QuantiGene Incubator Temperature Validation Kit Expired reagents were used Reagents are good for 6 months from the date of receipt Capture Plate sat at room temperature longer than 20 minutes after the addition of sample Do not let the Capture Plate sit at room temperature for longer than 20 minutes after the addition of the overnight hybridization mixture Capture Plate sat at room temperature for longer than 10 minutes before washing 2nd day Well To Well Variation Table 4 4 Troubleshooting Assay CVs Wash the Capture Plate within 10 minutes after removal from the incubator Probable Cause Recommended Action Residual Wash Buffer Ensure that the plate wash method completely removes all residual Wash Buffer prior to movin
26. t Signal Amplification and Detection These instructions are for processing a single Capture Plate using multichannel pipettes and reagent reservoirs To process more than one Capture Plate scale reagents accordingly If using a 50 plate kit scale reagent preparations for a minimum of 10 plates per run or reagent shortages will occur If you are processing fewer than 96 wells use the protocol for processing a partial plate located on the Panomics website at www panomics com Do not let the Capture Plate s stand dry for more than 5 minutes at any point in this procedure Do not disturb the contents of the Capture Plate s or open the incubator door during incubation steps Incubation temperatures must be 55 1 C 2 0 PreAmp and 2 0 Amp hybridization or 50 1 C Label Probe hybridization Verify temperatures using a QuantiGene Incubator Temperature Validation Kit 14 QuantiGene 2 0 Reagent System User Manual Preparing 1X Wash Buffer To prepare 1X Wash Buffer 1 Add to a 500 mL graduated cylinder in this order u 496 mL nuclease free water 1 5 mL Wash Comp 1 u 2 5 mL Wash Com 2 NOTE Scale preparation according to the number of plates to be processed One half liter is sufficient for processing 1 Capture Plate Transfer to a 500 mL bottle and invert to mix Do not store unused 1X Wash Buffer Make 1 X Wash Buffer fresh daily Hybridizing the 2 0 PreAmplifier To hybridize the 2 0 PreAmp 1
27. t signal is linearly proportional to the number of RNA molecules present in the sample How it Works Figure 1 1 QuantiGene QuantiGene 2 0 Reagent System Basics Capture Extender CE 2 0 Substrate 2 0 PreAmplifier e Label Extender LE 2 0 Amplifi Na O PO 5 c Dried Blood Spots r APAT er AY e e Blocking Probe BL Label Probe L Whole Blood or E T PAXgene Blood lm FFPE Sections gt C i P P Animal Tissues RNA A Le rj Cultured Cells 7 Capture Plate Capture Probe Step 1 Release Target RNA Step 2 Target RNA Capture Step 3 Signal Amplification Step 4 Detection Cells are lysed to release RNA Probe Set design determines the Signal amplification is performed Addition of a chemilumigenic 2 0 specificity of the target RNA capture via sequential hybridization of 2 0 Substrate generates a luminescent Probe Set oligonucleotides CEs LEs Pre Amplifier to Probe Set LEs Label signal that is proportional to the BLs bind a contiguous region of the Extenders 2 0 Amplifier to the 2 0 amount of target mRNA present in target RNA and the CEs Capture Pre Amplifier and Label Probe to the the sample Extenders by cooperative hybridiza 2 0 Amplifier The number of LEs Lumigen APS 5 tion selectively capture target RNA determines assay sensitivity to the 96 well Capture Plate during an overnight incubation Chapter 1 Introduction 3 Required Materials QuantiGen
28. tes Follow standard laboratory methods for purification of RNA Use samples immediately or store at 80 C until ready to use 2 Capture RNA u Dilute samples a Prepare Working Probes Sets Dispense Working Probe Sets samples and controls into Capture Plate u Hybridize overnight a Capturing Target RNA from Cultured Cell or Blood Lysates on page 7 u Capturing Target RNA from Fresh Frozen or FFPE Tissue Homogenates on page 9 u Capturing Target RNA from Total RNA Purified mRNA or In Vitro Transcribed RNA on page 11 3 Amplify and detect signal u Wash away unbound material Sequentially hybridize 2 0 PreAmp Amp and Label Probe Add 2 0 Substrate incubate and read signal Capturing Target RNA About Capturing Target RNA Signal Amplification and Detection on page 13 This following provides procedures for capturing target RNA based on the following sample type a Cultured cell and blood lysates u Fresh frozen or FFPE tissue homogenates u Total RNA mRNA or in vitro transcribed RNA preparations Refer to the appropriate procedure for your sample type Capturing Target RNA from Cultured Cell or Blood Lysates To capture target RNA from cultured cell or blood lysates 1 Prepare the following reagents a Probe Set s and Blocking Reagent Thaw vortex briefly to mix then briefly centrifuge to collect contents at the bottom of the tubes a Cultured cell or whole blood lysate s If previo
29. usly frozen thaw at room temperature followed by incubation at 37 C for 15 30 minutes Vortex briefly then leave at room temperature until use Lysis Mixture Re dissolve any precipitates by incubating at 37 C followed by gentle swirling Remove Capture Plate from 4 C and place on the benchtop to warm completely to room temperature approximately 30 minutes Do not remove the plate from the sealed foil pouch 8 QuantiGene 2 0 Reagent System User Manual 2 If appropriate based on the expression level of target or housekeeping RNA of interest dilute samples with Dilute Lysis Mixture 1 volume of Lysis Mixture plus 2 volumes nuclease free water prepared fresh so that the desired amount of sample is present in volume of 80 uL assay well Use the table below as a guide and scale dilutions according to the number of assays to be run Table 3 1 Recommended input for different preparations Recommended Sample Input RNA copies per cell Cultured Cells number of cells Whole Blood Lysate uL 1 6 000 802 10 600 80 100 60 8 gt 1 000 lt 6 lt 0 8 May not have sensitivity required Prepare an appropriate volume of Working Probe Set by combining the following reagents in the order listed Scale according to the number of assays to be run and include 40 overage For a guide use the table below that corresponds to the date of your Probe Set Lu IMPORTANT Include 3 wells for assay b
30. verage For a guide use the table below that corresponds to the date of your Probe Set Lu IMPORTANT Include 3 wells for assay background controls Table 3 2 For QuantiGene 2 0 Probe Sets Received Before December 31 2007 a Includes 40 overage bOmit for 18S or 28S RNA robe Sets Substitute volume with nuclease free water Table 3 3 For QuantiGene 2 0 Probe Sets Received After January 1 2008 Reagent 1 Well pL 48 Wells pL 96 Wells pL Nuclease free water 45 4 3 051 6 102 Lysis Mixture 33 3 2 238 4 476 Blocking Reagent 1 67 134 CEs 0 1 6 7 13 4 LEs 0 1 6 7 13 4 BLs 0 1 6 7 13 4 Total 80 5 376 10 752 a Includes 40 overage Omit for 185 or 28S RNA robe Sets Substitute volume with nuclease free water 4 Prepare the Capture Plate Reagent 1 Well pL 48 Wells pL 96 Wells pL Nuclease free water 45 4 3 051 6 102 Lysis Mixture 33 3 2 238 4 476 Blocking Reagent 1 67 134 2 0 Probe Set 0 3 20 1 40 2 Total 80 5 376 10 752 Chapter 3 QuantiGene 2 0 Assay Procedure 13 A Open the sealed foil pouch and remove the Capture Plate B Vortex Working Probe Set briefly to mix then dispense into the Capture Plate For fewer than 48 wells Using a single channel pipette and a new tip for each transfer dispense 80 uL Working Probe Set into each assay well Avoid introducing bubbles For 48 wells or more A Using a single channel pipette transfe
31. w tips for each transfer dispense 20 uL of Working Probe Set into each assay well Avoid introducing bubbles Hi IMPORTANT Capture Probe oligonucleotides are conjugated to the surface of Capture Plate wells Do not scratch Capture Plate wells with pipette tips Using a new pipette tip for each transfer add 80 uL sample to each well of the Capture Plate containing Working Probe Set Avoid introducing bubbles Do not mix IMPORTANT Add 80 pL of Dilute Lysis Mixture 1 volume Lysis Mixture plus 2 volumes nuclease free water to 3 wells for assay background controls Bind target RNAs A Place an adhesive Plate Seal squarely on the plate and seal tightly Lu IMPORTANT Complete and uniform sealing of the overnight hybridization plate is essential Use a soft rubber roller or the QuantiGene CTC Plate Sealer Letters and numbers on the Capture Plate should be clearly defined beneath the Plate Seal B Centrifuge the Capture Plate at 240 x g for 20 seconds at room temperature to ensure the contents contact the bottom of the well C Immediately place the Capture Plate in a 55 1 C incubator to begin the overnight 16 20 hour hybridization F IMPORTANT Temperature must be 55 1 C Verify temperature using a QuantiGene Incubator Temperature Validation Kit Capturing Target RNA from Fresh Frozen or FFPE Tissue Homogenates To capture target RNA from tissue homogenates 1 Prepare the following reagents
32. y Kits two accessory reagents are required to perform QuantiGene 2 0 assays For ordering information please visit our website at www panomics com Table 1 2 QuantiGene 2 0 Reagent System Accessory Components Accessory Reagent Description QuantiGene Sample Processing Kit Contains reagents and instructions for processing different sample types Specify sample type cultured cells fresh or frozen animal tissue FFPE samples or blood samples QuantiGene 2 0 target specific and housekeeping Probe Sets Each Probe Set contains CEs LEs and BLs 4 QuantiGene 2 0 Reagent System User Manual Required Materials Not Provided Other materials required to perform the QuantiGene QuantiGene 2 0 Reagent System that are not included in the are listed here Table 1 3 Required Materials Not Provided Required Material Source Part Number or Model 1000 uL Adjustable single and multi channel precision pipettes for dispensing 1 20 pL 20 200 uL and 200 Major laboratory supplier MLS Reagent reservoirs 25 mL capacity divided 25 mL capacity 100 mL capacity VistaLab Technologies P N 3054 1004 or equivalent P N 3054 1002 or equivalent Corning Costar P N CLS 4873 or equivalent Microcentrifuge Eppendorf 541D or equivalent Microplate centrifuge that can achieve 240 x g Eppendorf 5804R and rotor A 2 DWP or equivalent Vortex mixer MLS Nuclease free water MLS
33. y of the paper to our technical support group at pqbhelp affymetrix com or via fax at 510 818 2610 Disclaimer Affymetrix Inc reserves the right to change its products and services at any time to incorporate technological developments This manual is subject to change without notice Although this manual has been prepared with every precaution to ensure accuracy Affymetrix Inc assumes no liability for any errors or omissions nor for any damages resulting from the application or use of this information Copyright 2010 Affymetrix Inc All rights reserved Contents Chapter 1 Chapter 2 Chapter 3 ai dde e 5 eio ME 1 About This Manual i xta elds Kehoe booa doo e C6 Rose de Ead 1 Who Should Read this Manual 1 What this Manual Cov rs Lucem ane antl ee CR VU e wan eo ea 1 Safety Warnings and Precautions 1 Contacting AYME lxi x 3545 needs SSE ww dw ue e RTS Ne dre ee 1 Technical Help SR SR Ea ee eth st eee eh d eae Re nerd 1 QuantiGene QuantiGene 2 0 Reagent System Basics 1 FIOW IE V V OS AS ee ae o san DAQMA a aie CLE 2 Required Materials 2 2 kk kk kk kk kk kk KK kK KK KK KK KK KK KK KK KK KI KK KK KK KK KK KK KK KK KK 3 QuantiGene QuantiGene 2 0 Reagent System Kit Components 3 QuantiGene 2 0 Reagent System Accessory Reagents 3 Requ
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