FlashTag® Biotin HSR
Contents
1. 1 0ul PAP Enzyme Vial 4 Note If at least 5 labeling reactions are simultaneously run a master mix may be prepared at this step Prepare one extra reaction s worth of reagents For example when 5 samples are run prepare a master mix for 6 samples 9ul 10X Reaction Buffer Vial 1 9ul 25mM MnCl Vial 2 6ul diluted ATP Mix Vial 3 dilution from step 3 6ul PAP Enzyme Vial 4 Add 5ul of master mix to the 10ul RNA Spike Control Oligos for a volume of 15ul Mix gently do not vortex and microfuge Incubate in a 37 C heat block for 15 minutes Discard any unused diluted ATP Mix from step 2 Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 6 of 17 FlashTag Biotin HSR Ligation 1 2 Briefly microfuge the 15ul of tailed RNA and place on ice Add 4ul 5X FlashTag Biotin HSR Ligation Mix Vial 5 Add 2ul of T4 DNA Ligase Vial 6 Mix gently do not vortex and microfuge Incubate at 25 C room temperature for 30 minutes Stop the reaction by adding 2 5ul HSR Stop Solution Vial 7 Mix and microfuge the 23 5 of ligated sample Remove 2ul of the biotin labeled sample and proceed to the ELOSA QC Assay Appendix A It is acceptable to store the 2ul of biotin labeled sample on ice for up to 6 hours or at 20 C for up to 2 weeks and run the ELOSA QC Assay at a convenient time The remaining 21 5 biotin labeled sample may be stored on ice for up to 6 hours or at 20 C for up t2. Journal of Biomedicine and Biotechnology 2009 Article ID 659028 Wang et al The Expression of MicroRNA miR 107 Decreases Early in Alzheimer s Disease and May Accelerate Disease Progression through Regulation of B Site Amyloid Precursor Protein Cleaving Enzyme The Journal of Neuroscience January 30 2008 28 5 1213 1223 For Research Use Only 2011 Genisphere LLC All rights reserved Genisphere 3DNA and FlashTag are registered trademarks of Genisphere LLC Affymetrix and GeneChip are registered trademarks of Affymetrix Inc mirfana and RecoverAll are trademarks of Applied Biosystems RiboGreen and TRIzol are registered trademarks Quant iT and PureLink are trademarks of Invitrogen Corp NanoDrop is a registered trademark of NanoDrop Technologies Immobilizer is a trademark of Exiqon A S Tough Spots is a registered trademark of Diversified Biotech Genisphere LLC To Order Technical Support 2801 Sterling Drive Phone 877 888 3362 Phone 877 888 3362 Hatfield PA 19440 Phone 215 996 3002 Phone 215 996 3040 Email info genisphere com FAX 877 329 3362 Email techsupport genisphere com Web www genisphere com FAX 215 996 3070 Web www genisphere com Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 12 of 17 Appendix A ELOSA QC Assay The Enzyme Linked Oligosorbent Assay ELOSA is designed to provide confirmation that the FlashTag Biotin HSR Labeling Kit has performed appropriately as a b
3. Ligase ax Biotin labeled RNA Biotin detection with Streptavidin PE Single Feature Zo on Affymetrix GeneChip miRNA Array Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 3 of 17 Components Storage and Handling FlashTag Biotin HSR RNA Labeling Kit Store all components at 20 C Vial 1 10X Reaction Buffer Vial 2 25mM MnCl Vial 3 ATP Mix Vial 4 PAP Enzyme Vial 5 5X FlashTag Biotin HSR Ligation Mix Vial 6 T4 DNA Ligase Vial 7 HSR Stop Solution Vial 8 RNA Spike Control Oligos Vial 9 ELOSA Spotting Oligos Vial 10 ELOSA Positive Control Vial 11 Nuclease Free Water Vial 12 27 5 Formamide Handling Kit Contents Vials 1 2 5 7 9 11 and 12 Thaw at room temperature vortex and briefly microfuge Vials 3 8 and 10 Thaw on ice microfuge if necessary and keep on ice at all times Vials 4 and 6 Remove from freezer just prior to use and briefly microfuge Keep on ice at all times Do not vortex Other Required Materials Refer to Appendix C for example reagent preparation and storage All materials should be nuclease free and all reagents should be prepared with nuclease free components RNA sample containing low molecular weight LMW RNA see RNA Sample and Quantitation on page 5 Nuclease free water Applied Biosystems cat no AM9932 or equivalent 1mM Tris Appendix C Reagents for analysis by Affymetrix GeneChip miRNA Array e Affymetrix miRNA Array Gen
4. bioD cre from GeneChip Eukaryotic Hybridization Control Kit for 5 minutes at 65 C Add the following components to the 21 5 biotin labeled sample in the order listed to prepare the array hybridization cocktail 50ul 2X Hybridization Mix from GeneChip Hyb Wash and Stain Kit 15ul 27 5 Formamide Vial 12 10ul DMSO from GeneChip Hyb Wash and Stain Kit Sul 20X Eukaryotic Hybridization Controls from GeneChip Eukaryotic Hyb Control Kit 1 7ul Control Oligonucleotide B2 3nM from GeneChip Eukaryotic Hyb Control Kit The volume will be 103 2ul Incubate at 99 C for 5 minutes then 45 C for 5 minutes Aspirate 100ul and inject into an array Remove the pipet tip from the upper right septum of the array Cover both septa with 1 2 Tough Spots to minimize evaporation and or prevent leaks Place the arrays into hybridization oven trays Load the trays into the hybridization oven 10 Incubate the arrays at 48 C and 60 rpm for 16 hours Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 8 of 17 Washing and Staining For additional information about washing staining and scanning refer to the HWS Kit User Guide and the Affymetrix Command Console User Guide http www affymetrix com 1 After 16 hours of hybridization remove the arrays from the oven Remove the Tough Spots from the arrays 2 Extract the hybridization cocktail from each array and transfer it to a new tube or well of a 96 we
5. free water Ensure that the purification method retains low molecular weight species Some commercial products that have been tested successfully with FlashTag Biotin HSR include Applied Biosystems mifvana miRNA Isolation Kit Applied Biosystems RecoverAll Total Nucleic Acid Isolation Kit for FFPE Qiagen miRNeasy Mini Kit Invitrogen PureLink miRNA Isolation Kit Invitrogen TRIzol reagent total RNA only with additional overnight 20 C precipitation step during isopropanol precipitation Quantitation To accurately determine the concentration of the RNA sample Genisphere recommends the use of the Quant iT RiboGreen RNA Assay Kit Invitrogen cat no R11490 or the NanoDrop ND 1000 Spectrophotometer NanoDrop Technologies RNA Input for FlashTag Biotin HSR The table below describes general recommendations for RNA input for FlashTag Biotin HSR labeling The amount of microRNA relative to total RNA is less in cultured cells than in tissues Therefore Genisphere recommends using more RNA isolated from cultured cells RNA Sample Input for FlashTag Biotin HSR Labeling 100 1000ng total RNA from tissues Total RNa containing EMER A 500 1000ng total RNA from cultured cells 100 400ng LMW RNA from tissues eanened EMV RNA aualttated 200 400ng LMW RNA from cultured cells Enriched LMW RNA not quantitated Enriched from 100 1000ng total RNA Genisphere Technical Support 877 888 3DNA www ge
6. miRNA 2 0 array CEL files Genisphere recommends the following workflow in miRNA QC Tool Current Workflow bo Aur Genisphere Gl Detection Background Detection Background Adjustment L Ald Global Background Correction Normalization i Quantile Summarization i Median Polish Workflows Run Cancel Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 10 of 17 To edit the workflow Open QCTool software Click Cancel do not load any CEL files Select Tools Workflow Editor Select File New Type a name for the new workflow Add the procedures listed above by selecting them on the left and clicking the middle arrow button Select File Save Click OK Click Close to close the editor Using miRNA QC Tool software look for Vial 8 RNA Spike Control Oligos in the Quality Control Table Select Tables Quality Control The Affymetrix library file lists the following names for these probe sets spike in control 2_st spike in control 23_st spike in control 29_ st spike in control 31_st spike in control 36_st Each probe set should show gt 1000 units signal background Export the data into third party software for further analysis Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 11 of 17 References 220 0 11 12 13 Schembri et al MicroRNAs as modulators of smoking induced gene expression cha
7. Genisphere SIGNAL SAMPLE AMPLIFICATION PRODUCTS FlashTag Biotin HSR RNA Labeling Kit for Affymetrix GeneChip miRNA Arrays Table of Contents Introduction Background Information Procedure Overview Components Storage and Handling Other Required Materials RNA Sample and Quantitation FlashTag Biotin HSR RNA Labeling Procedure Poly A Tailing FlashTag Biotin HSR Ligation Affymetrix GeneChip miRNA Array Procedure Preparation of Ovens Arrays and Sample Registration Files Hybridization Washing and Staining Scanning Analysis References Appendix A ELOSA QC Assay Appendix B Array Rehybridization Procedure Appendix C Example Reagent Preparation and Storage Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 10 12 13 16 17 Page 1 of 17 Introduction Background Information The FlashTag Biotin HSR kit will label any RNA sample including total RNA severely degraded RNA plant RNA and low molecular weight RNA This protocol describes labeling total RNA or low molecular weight LMW RNA for analysis by Affymetrix GeneChip miRNA Arrays with an in process ELOSA QC Assay LMW RNA molecules snRNA hnRNA piRNA miRNA etc have recently been shown to be involved in important biological processes such as mRNA degradation transcriptional gene silencing TGS and translational repression As a result these newly discovered biomolecules are gaining the interest of the scientifi
8. c community as possible new drug targets and for use in diagnostics FlashTag Biotin HSR provides the necessary tools to identify such targets FlashTag Biotin HSR labeling is fast simple accurate highly sensitive and reproducible Starting with 100 1000ng of total RNA the process begins with a brief tailing reaction followed by ligation of the biotinylated signal molecule to the target RNA sample see page 3 The labeling process is complete in less than one hour The high sensitivity of FlashTag Biotin HSR is due to Genisphere s proprietary 3DNA dendrimer signal amplification technology The 3DNA dendrimer is a branched structure of single and double stranded DNA conjugated with numerous labels The 3DNA molecule in the FlashTag Biotin HSR kit provides ultrasensitive biotin labeling Please review this product manual before beginning experiments Materials needed for Affymetrix GeneChip miRNA Arrays are listed on page 4 Materials needed for the ELOSA QC Assay are listed in Appendix A Note that ELOSA wells must be coated with DNA Spotting Oligos and incubated overnight before the ELOSA assay may be run Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 2 of 17 FlashTagHSR Procedure Overview 5 3 RNAmolecule Poly A Tailing ATP 15 minutes Poly APolymerase Poly A tail 5 3 Poly A tailed RNA Biotin labeled 3DNA 6 Ligation Flash Tag Ligation Mix 30 minutes
9. eChip miRNA Array Affymetrix cat no 901324 901325 or 901326 GeneChip miRNA 2 0 Array Affymetrix cat no 901753 901754 or 901755 e Affymetrix GeneChip Command Console Software AGCC e GeneChip Eukaryotic Hybridization Control Kit Affymetrix cat no 900454 If necessary Control Oligonucleotide B2 3nM included in Hybridization Control Kit can be ordered separately Affymetrix cat no 900301 e GeneChip Fluidics Station 450 Affymetrix cat no 00 0079 e GeneChip Hybridization Wash and Stain Kit Affymetrix cat no 900720 If necessary Wash Buffer A included in Hybridization Wash and Stain Kit can be ordered separately Affymetrix cat no 900721 If necessary Wash Buffer B included in Hybridization Wash and Stain Kit can be ordered separately Affymetrix cat no 900722 e Laser Tough Spots 3 8 diameter Diversified Biotech cat no SPOT 1000 e Laser Tough Spots 1 2 diameter Diversified Biotech cat no SPOT 2000 e Reagents for ELOSA QC Assay Refer to Appendix A and Appendix C Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 4 of 17 RNA Sample and Quantitation Either Total RNA or LMW Low Molecular Weight RNA can be labeled with FlashTag Biotin HSR Using total RNA can save time and money and prevent sample loss 11 RNA Isolation Any kit for purification of total RNA or LMW RNA will be compatible with FlashTag Biotin HSR Elute or resuspend the RNA in nuclease
10. ere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 16 of 17 Appendix C Example Reagent Preparation and Storage For all of the reagents below it is important to remove the amount that is needed for the day or step of the protocol by carefully pouring off or using a long pipette to avoid contamination of the stock buffer All components should be nuclease free and stored in nuclease free tubes or bottles Recommended suppliers and catalog numbers are listed Equivalent suppliers may be used for reagents other than BSA We strongly recommend BSA from Sigma Sigma cat no A3294 1mM Tris 50mL Transfer 50mL nuclease free water Applied Biosystems cat no AM9932 to a 50mL conical tube Remove and discard 50ul water Add 50uL of 1M Tris HCl pH 8 USB cat no 22638 After this dilution is made do not take a pH reading Store at room temperature up to 3 months 25 dextran sulfate 10mL Slowly pour 5mL 50 dextran sulfate Millipore cat no S4030 into a 15mL conical tube Add 5mL nuclease free water Applied Biosystems cat no AM9932 and vortex thoroughly Store at room temperature up to 3 months 25 dextran sulfate may also be ordered from Genisphere cat no V25DEX 1X PBS 1L 100mL 10X PBS pH 7 4 Applied Biosystems cat no AM9625 900mL nuclease free water Applied Biosystems cat no AM9932 Store at room temperature up to 3 months 1X PBS 0 02 Tween 20 1L 100mL 10X PBS pH 7 4 A
11. iotin labeling process Specifically the ELOSA is designed to detect the RNA Spike Control Oligos Vial 8 included in all FlashTag Biotin HSR labeling reactions Only 2ul of the labeling reaction is required for the ELOSA assay Successful biotin labeling is verified via a simple colorimetric ELOSA assay through the hybridization of the biotin labeled RNA Spike Control Oligos Vial 8 to complementary ELOSA Spotting Oligos Vial 9 immobilized onto microtiter plate wells The ELOSA Positive Control Vial 10 confirms the ELOSA assay is working properly This assay should be run prior to the use of any labeling reaction on microarrays to assure the FlashTag Biotin HSR labeling process worked appropriately with known controls Please note that this procedure does not assure the performance of any RNA sample on a microarray Additional Required Materials Refer to Appendix C for example preparation and storage e Flat bottom Immobilizer Amino 8 well strips Nunc cat no 436013 30 plates or Genisphere cat no FTSELOSA 5 plates Do not use strips or plates from other manufacturers e Adhesive plate sealers VWR cat no 62402 921 or equivalent e Squirt wash bottle or washing instrument for vigorous washing e 1XPBS e 1X PBS 0 02 Tween 20 e 5XSSC 0 05 SDS 0 005 BSA If a precipitate forms in this buffer warm at 42 C to dissolve Use at room temperature e 5 BSA in 1X PBS e 25 dextran sulfate Genisphere cat no V25DEX
12. ll plate in order to save the hybridization cocktail Store on ice during the procedure or at 80 C for long term storage Refer to Appendix B Array Rehybridization Procedure if necessary 3 Fill each array completely with Array Holding Buffer 4 Allow the arrays to equilibrate to room temperature before washing and staining NOTE Arrays can be stored in the Array Holding Buffer at 4 C for up to 3 hours before proceeding with washing and staining Equilibrate arrays to room temperature before washing and staining 5 Place vials into sample holders on the fluidics station a Place one amber vial containing 600ul Stain Cocktail 1 in sample holder 1 b Place one clear vial containing 600uI Stain Cocktail 2 in sample holder 2 c Place one clear vial containing 800ul Array Holding Buffer in sample holder 3 6 Wash and stain with Fluidics Station 450 using fluidics script FS450_ 0003 Post Hyb Wash 1 10 cycles of 2 mixes cycle with Wash Buffer A at 25 C Post Hyb Wash 2 8 cycles of 15 mixes cycle with Wash Buffer B at 50 C 1st Stain Stain the probe array for 10 minutes with Stain Cocktail 1 Vial Position 1 at 25 C Post Stain Wash 10 cycles of 4 mixes cycle with Wash Buffer A at 30 C 2nd Stain Stain the probe array for 10 minutes with Stain Cocktail 2 Vial Position 2 at 25 C 3rd Stain Stain the probe array for 10 minutes with Stain Cocktail 1 Vial Position 1 at 25 C Final Wash 15 cycles of 4 mixes cycle with Wash Buffer A a
13. nges in human airway epithelium PNAS 2009 vol 106 no 7 2319 2324 Taylor and Gant Emerging fundamental roles for non coding RNA species in toxicology Toxicology 2008 vol 246 Issue 1 34 39 Ronemus M et al MicroRNA Targeted and Small Interfering RNA Mediated mRNA Degradation Is Regulated by Argonaute Dicer and RNA Dependent RNA Polymerase in Arabidopsis The Plant Cell 2006 18 7 1559 1574 Morel JB et al Hypomorphic ARGONAUTE ago7 Mutants Impaired in Post Transcriptional Gene Silencing and Virus Resistance The Plant Cell 2002 Vol 14 3 629 639 Krichevsky AM et al A microRNA array reveals extensive regulation of microRNAs during brain development RNA 2003 9 10 1274 1281 Schmittgen TD et al A high throughput method to monitor the expression of microRNA precursors Nucleic Acids Res 2004 32 4 e43 Thomson JM et al A Custom Microarray Platform for Analysis of MicroRNA Gene Expression Nature Methods 2004 1 1 47 53 Ambros V The functions of animal microRNAs Nature 2004 431 350 Nilsen TW et al Dendritic Nucleic Acid Structures J Theor Biol 1997 187 273 284 Stears RL et al A novel sensitive detection system for high density microarrays using dendrimer technology Physiol Genomics 2000 3 93 99 http www genetics pitt edu forms flyers miRNAextractionevaluation pdf Masotti et al Quantification of Small Non Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles
14. nisphere com FlashTag HSR Feb2011 Page 5 of 17 FlashTag Biotin HSR RNA Labeling Procedure Genisphere recommends running an ELOSA QC Assay to verify this labeling procedure prior to array hybridization Refer to Appendix A Note that ELOSA wells must be coated with DNA Spotting Oligos and incubated overnight before the ELOSA assay may be run and that Plate Washing and Blocking steps may be completed prior to or during the FlashTag Biotin HSR labeling procedure Poly A Tailing 1 2 Adjust the volume of RNA to 8ul with Nuclease Free Water Vial 11 Transfer the 8ul RNA to ice Add 2ul RNA Spike Control Oligos Vial 8 and return to ice Dilute the ATP mix Vial 3 in 1mM Tris as follows For total RNA samples dilute the ATP Mix 1 500 For enriched quantitated samples calculate the dilution factor according to the following formula 5000 ng input LMW RNA Example If using 100ng of enriched LMW RNA the dilution factor is 5000 100 50 Dilute the ATP Mix 1 50 For enriched samples that are not quantitated calculate the dilution factor according to the following formula 1000 ug input total RNA Example If the sample was enriched from 500ng total RNA the dilution factor is 1000 0 5 2000 Dilute the ATP Mix 1 2000 Add the following components to the 10ul RNA Spike Control Oligos for a volume of 15ut 1 5ul 10X Reaction Buffer Vial 1 1 5ul 25mM MnCl Vial 2 1 0ul diluted ATP Mix Vial 3 dilution from step 3
15. o 2 weeks prior to hybridization on Affymetrix GeneChip miRNA Arrays Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 7 of 17 Affymetrix GeneChip miRNA Array Procedure Preparation of Ovens Arrays and Sample Registration Files 1 Download and install the miRNA Array library file package if not performed previously into Affymetrix GeneChip Command Console AGCC software using the Command Console Library File Importer tool The files can be found at http www affymetrix com Array Type Library File GeneChip miRNA Array miRNA 1_0_2Xgain GeneChip miRNA 2 0 Array miRNA 2_0 Turn Affymetrix Hybridization Oven 640 or 645 on and set the temperature to 48 C Set the RPM to 60 Turn the rotation on and allow the oven to preheat Upload the sample and array information Sample names barcode IDs etc into AGCC Unwrap the arrays and place on the bench top Allow the arrays to warm to room temperature 10 15 minutes Mark each array with a meaningful designation Insert a 20ul or 200 pipet tip unfiltered type recommended into the upper right septum to allow for proper venting when hybridization cocktail is injected For more information refer to the Affymetrix Command Console User Guide http www affymetrix com Hybridization 1 2 Bring the reagents listed in step 3 below to room temperature Completely thaw and then heat the 20X Eukaryotic Hybridization Controls bioB bioC
16. oating Wells with ELOSA Spotting Oligos Vial 9 1 Dilute the ELOSA Spotting Oligos Vial 9 1 50 in 1X PBS according to the table below Number of Wells Total Volume Required ELOSA Spotting Oligos 1X PBS 3 225ul 4 5ul 220 5ul 12 900uI 18ul 882 ul 24 1800 36ul 1764 ul 2 Add 75ul of the diluted ELOSA Spotting Oligos to each well of the plate or strip 3 Cover with an adhesive plate sealer and incubate overnight at 2 8 C The plates or wells may be stored at 2 8 C for up to 2 weeks if covered tightly with an adhesive plate sealer and no evaporation occurs but for best results incubate overnight Washing and Blocking These steps may be completed prior to or during the FlashTag Biotin HSR labeling procedure 1 Remove the ELOSA Spotting Oligos by expelling the liquid into a sink 2 Wash 2 times with 1X PBS 0 02 Tween 20 blot dry 3 Add 150nuI of 5 BSA in 1X PBS to each well 4 Cover the wells and incubate for 1 hour at room temperature Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 14 of 17 Sample Hybridization 1 2 0ul of each biotin labeling reaction page 7 step 6 will be used in the ELOSA Add the following components and gently vortex until the dextran sulfate is in solution Briefly microfuge 2 0ul FlashTag Biotin HSR labeled RNA sample or negative control no Vial 8 labeling reaction 48 0ul 5X SSC 0 05 SDS 0 005 BSA 2 5ul 25 Dextran sulfate For the positive c
17. of the ELOSA wells with a squirt bottle filled with washing buffer is a simple and inexpensive method that works well when performed over a sink alternatively an automated washing instrument capable of vigorous washing may be used Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 13 of 17 Experimental Design Recommendations To understand the validity of this ELOSA method appropriate controls should be included in all ELOSA assays Negative controls should include a FlashTag Biotin HSR labeling reaction that does not contain any RNA Spike Control Oligos Vial 8 It is optional to include Total RNA in the negative control This type of control should result in a negative reaction in the ELOSA assay and will define any baseline non specific background signals If a Negative control FlashTag Biotin HSR reaction is not run another acceptable negative control is 50ul 5X SSC 0 05 SDS 0 005 BSA 2 5ul 25 Dextran sulfate Spike controls should include a FlashTag Biotin HSR labeling reaction containing both total RNA and the RNA Spike Control Oligos Vial 8 Labeled samples that have previously demonstrated appropriate reactivity for the ELOSA assay should be used Labeled samples that have shown appropriate performance on microarrays may also be of value Positive controls should include the ELOSA Positive Control Vial 10 an oligo which is already biotinylated and confirms the ELOSA is working properly C
18. ontrol add the following components and gently vortex until the dextran sulfate is in solution Briefly microfuge 2 0ul ELOSA Positive Control Vial 10 48 0ul 5X SSC 0 05 SDS 0 005 BSA 2 5ul 25 Dextran sulfate Remove the BSA blocking solution by expelling the liquid into a sink Blot dry Add all 52 5 of hybridization solution to a designated well Cover the wells and incubate for 1 hour at room temperature SA HRP Binding 1 Dilute SA HRP in 5 BSA in 1X PBS If using Thermo Scientific SA HRP a dilution of 1 4000 to 1 8000 is recommended Remove the hybridization solution by expelling the liquid into a sink Vigorously wash 3 4 times with 1X PBS 0 02 Tween 20 blot dry Add 75ul of the diluted SA HRP from step 1 to each well Cover the wells and incubate for 30 minutes at room temperature Signal Development 1 2 Remove the SA HRP by expelling the liquid into a sink Vigorously wash 3 4 times with 1X PBS 0 02 Tween 20 blot dry Remove any bubbles from the wells with a forced air duster or equivalent device Add 100ul of TMB Substrate to each well Cover the wells and incubate at room temperature for 5 30 minutes in the dark or covered with aluminum foil The blue substrate color indicates a positive result and may be used as qualitative results Optional For instrument quantitation remove the adhesive plate sealer and add 100ul Stop Reagent or equivalent acidic TMB stop reagent to each well This
19. or equivalent see Appendix C e Streptavidin HRP Thermo Scientific Pierce cat no N100 or equivalent e TMB Substrate Solution Thermo Scientific Pierce cat no N301 or equivalent e Optional TMB Stop Reagent Thermo Scientific Pierce cat no N600 or equivalent e Optional Plate reader or instrument capable of reading absorbance at 450nm Procedural Notes e All materials should be nuclease free and all reagents should be prepared with nuclease free components e 2u ul of each biotin labeling reaction page 7 step 6 will be used in the ELOSA It is acceptable to store the 2ul of biotin labeled sample on ice up to 6 hours or at 20 C up to 2 weeks and run the ELOSA at a convenient time e The ELOSA Positive Control Vial 10 is already labeled with biotin and should be added to its own well each time the ELOSA assay is run e Bring all solutions to room temperature before using them in the ELOSA e During all incubation steps cover the plate with an adhesive plate sealer e To blot dry expel the liquid into a sink and repeatedly tap the inverted plate on a stack of paper towels Do not insert laboratory wipes into the ELOSA wells e A multichannel pipette 8 or 12 tip is recommended but not required e Do not touch pipette tips to the bottom of the ELOSA wells at any step of the procedure e Vigorous washing is required to minimize non specific background signals in negative control wells Vigorous manual washing
20. pplied Biosystems cat no AM9625 0 2mL Tween 20 200uL Sigma cat no P 9416 Add water to a final volume of 1L Store at room temperature up to 3 months 5 BSA in 1X PBS 40mL Transfer 2g of powdered BSA Sigma cat no A3294 to a 50mL conical tube Slowly add 1XPBS to a final volume of 40mL Shake or vortex to mix Make 8 aliquots of 5mL Store each aliquot at 20 degrees C up to 6 months Do not freeze thaw each 5mL aliquot more than 4 times Once thawed store one aliquot at 4 degrees C for 1 week 5X SSC 0 05 SDS 0 005 BSA 10mL 2 5mL 20X SSC Applied Biosystems cat no AM9763 0 05mL 10 SDS 50uL Applied Biosystems cat no AM9823 0 01mL 5 BSA in 1XPBS 10uL Add water to a final volume of 10mL Make 10 aliquots of 1mL Store each aliquot at 20 degrees C up to 6 months Do not freeze thaw each 1mL aliquot more than 4 times Once thawed store one aliquot at 4 degrees C for 1 week If a precipitate forms in this buffer warm at 42 C to dissolve Use at room temperature Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 17 of 17
21. t 35 C Array Holding Buffer Fill the probe array with Array Holding Buffer Vial Position 3 7 Check for air bubbles If there are air bubbles manually fill the array with Array Holding Buffer If there are no air bubbles cover both septa with 3 8 Tough Spots Inspect the array glass surface for dust and or other particulates and if necessary carefully wipe the surface with a clean lab wipe before scanning Scanning The instructions for using the scanner and scanning arrays can be found in the Affymetrix Command Console Software User Manual in Chapter 6 http Awww affymetrix com Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 9 of 17 Analysis Use the free miRNA QC Tool software for data summarization normalization and quality control There are two versions of the tools available on the website Please choose the correct version depending on the array type Array Type miRNA QC Tool version GeneChip miRNA Array 1 0 33 0 GeneChip miRNA 2 0 Array 1 1 1 0 Both versions of the miRNA QC Tool cannot exist on the same computer If you are planning on using both versions of the miRNA arrays please install versions 1 0 33 0 and 1 1 10 on separate computers If you have previously installed miRNA QC Tool version 1 0 33 0 and are now running miRNA 2 0 arrays please uninstall remove the program before installing miRNA QC Tool version 1 1 10 After installation reboot your computer before analyzing
22. will convert the blue substrate to a yellow color Read the absorbance at 450nm on a plate reader Readings of greater than 0 10 OD 450nm over a negative control should be considered positive Typically this assay generates positive results of at least 0 15 1 00 OD when working appropriately After a successful ELOSA QC Assay proceed to Affymetrix GeneChip miRNA Array Procedure on page 8 Genisphere Technical Support 877 888 3DNA www genisphere com FlashTag HSR Feb2011 Page 15 of 17 Appendix B Array Rehybridization Procedure Follow the procedure below if it is necessary to rehybridize another Affymetrix GeneChip miRNA Array 1 2 Record the volume of recovered hybridization cocktail from page 9 Washing and Staining Step 2 Prepare a 1X Hyb Mix 21 5ul Nuclease Free Water Vial 11 50ul 2X Hybridization Mix from GeneChip Hyb Wash and Stain Kit Affymetrix cat no 900720 15ul 27 5 Formamide Vial 12 10ul DMSO from GeneChip Hyb Wash and Stain Kit Affymetrix cat no 900720 Sul 20X Eukaryotic Hybridization Controls bioB bioC bioD cre from GeneChip Eukaryotic Hybridization Control Kit Affymetrix cat no 900454 1 7ul Control Oligonucleotide B2 3nM Affymetrix cat no 900301 Adjust the volume of recovered hybridization cocktail Step 1 to 103 21 with 1X Hyb Mix Step 2 Follow the hybridization instructions on page 8 to complete the hybridization process Continue with Washing and Staining on page 9 Genisph
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