Biotin Labeling Kit for ST/Exon Arrays
Contents
1. 25 C 37 C 42 C 47 C 65 C 70 C 75 C 93 C 99 C e Microcentrifuge e Agencourt RNAClean XP Beckman Coulter cat no A63987 e Agencourt SPRI Plate 96R Ring Magnet Plate Beckman Coulter cat no A29164 or equivalent magnetic plate e 96 well round bottom microtiter plate Costar cat no 3795 e 70 Ethanol Prepare fresh dilutions of ethanol each time RNAClean XP is used e 100 Ethanol e 65 C heat block or oven for Nuclease Free Water Vial 10 during RNAClean Purification e NanoDrop for dscDNA quantitation or equivalent quantitation instrument e Optional materials for Gel Shift assay Appendix A for Affymetrix Whole Transcript Expression Arrays e Hybridization oven e Affymetrix GeneChip Command Console Software AGCC e Thermal Cycler or heating devices for incubations at 47 C 65 C and 99 C e GeneChip Hybridization Wash and Stain Kit Affymetrix cat no 900720 e GeneChip Eukaryotic Hybridization Control Kit Affymetrix cat no 900454 e Fluidics station e Scanner Biotin Labeling Kit for ST Exon Arrays December 2011 Page 4 of 13 Important Parameters Procedural Notes e 25ug senseRNA in a volume of 23ul is recommended for this labeling procedure If 25ug senseRNA is not available proceed with 10 25ug senseRNA For each sample use as much senseRNA as possible up to 25yg and not less than 10ug e When preparing master mixes the enzyme should be added last and just prior to2. adding the master mix to the reaction After the master mix or other reagent is added to the reaction gently tap the bottom of the tube and briefly microfuge e A thermal cycler is recommended to prevent evaporation and condensation of the sample If a thermal cycler is not available heat blocks or water baths may be used Microfuge the reactions after incubations if condensation is observed Thermalcycler Programs Double stranded cDNA Synthesis Program 1 70 C 5 min 25 C 5 min 4 C 2 min Program 2 25 C 10 min 42 C 90 min 65 C 10 min hold at 4 C for at least 2 min Program 3 16 C 120 min do not use a heated lid 75 C 10 min hold at 4 C for at least 2 min Program 4 65 C 30 min hold at 25 C for at least 2 min Terminal Labeling of double stranded cDNA Program 5 37 C 60 min 93 C 2 min hold at 4 C for at least 2 min Hybridization Cocktail Program 6 99 C 5 min 47 C 5 min Biotin Labeling Kit for ST Exon Arrays December 2011 Volume 26ul 40ul 60ul 62ul 30ul or 6Oul 9Oul or 200ul Page 5 of 13 Procedure for Use Double Stranded cDNA synthesis 25ug senseRNA in a volume of 23ul is recommended for this labeling procedure If 25ug senseRNA is not available proceed with 10 25ug senseRNA For each sample use as much senseRNA as possible up to 25ug and not less than 10yg 1 Adjust the volume of senseRNA to 23ul with Nuclease Free water Vial 10 2 Onice add 3y
3. cartridge and stain the gel in 1X SYBR Gold for 30 minutes Place the gel on a UV light box and image using the appropriate filter for SYBR Gold Biotin Labeling Kit for ST Exon Arrays December 2011 Page 13 of 13
4. factor concentration of dscDNA in ng ul Calculate the A260 280 ratio to determine purity A ratio of 1 9 2 1 is desirable Optional Unlabeled dscDNA may be analyzed by Quantitative PCR do not use PCR master mixes containing Uracil N Glycosylase Proceed to Terminal Labeling with 4 8ug dscDNA for 169 format arrays or 8 16ug dScDNA for 49 64 format arrays For each sample label as much dscDNA as possible in the specified range Store remaining dscDNA at 20 C Biotin Labeling Kit for ST Exon Arrays December 2011 Page 8 of 13 Terminal Labeling of dscDNA Choose the procedure for Terminal Labeling based on the array format Terminal Labeling of dscDNA for 169 format arrays 1 Adjust the volume of 4 8ug dscDNA to 22 5ul with Nuclease Free Water Vial 10 For each sample label as much dscDNA as possible up to 8ug and not less than 4ug For each reaction prepare a Terminal Labeling reaction master mix in a separate tube on ice 6 0ul 1 Step 5X Fragment and Label Buffer Mix Vial 11 1 5ul 1 Step Fragment and Label Enzyme Mix Vial 12 7 5ul Add the 7 5ul terminal labeling reaction master mix to the 22 5ul dscDNA for a final reaction volume of 30ul Incubate the 30ul reactions for terminal labeling as follows Thermalcycler Program 5 37 C for 60 minutes 93 C for 2 minutes 4 C for 2 minutes Optional Run the gel shift assay as indicated in Appendix A Proceed to Affymetrix Whole Transcript Expression Array Hybridiza
5. temperature 4 Insert a pipet tip into the upper right septum to allow for venting Hybridization 1 Bring the reagents listed in step 3 below to room temperature The reagents may be found in the GeneChip Hybridization Wash and Stain Kit and the GeneChip Eukaryotic Hybridization Control Kit 2 Heat the 20X Eukaryotic Hybridization Controls for 5 minutes at 65 C 3 For each sample prepare a Master Mix 169 format 49 64 format Control Oligo B2 3nM 1 5ul 3 3ul 20X Eukaryotic Hyb Controls 4 5ul 10ul DMSO Qul 20ul Nuclease Free Water Oul 6 7ul 2X Hybridization Mix 45ul 100ul Volume of Master Mix 60ul 140ul 4 Add the Master Mix Step 3 to the biotin labeled dscDNA to prepare the Hybridization Cocktail 169 format 49 64 format Volume of Master Mix 60ul 140ul Biotin labeled dscDNA 30ul 60ul Volume of Hybridization Cocktail 90ul 200ul 5 Incubate the Hybridization Cocktail Thermalcycler Program 6 99 C for 5 minutes 47 C for 5 minutes 6 Load the appropriate amount of Hybridization Cocktail onto each array 169 format 49 64 format Volume to load on Array 90ul 200ul 7 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 Biotin Labeling Kit for ST Exon Arrays December 2011 Page 10 of 13 8 Place the arrays into hybridization oven trays Load the trays into the hybridization oven 9 Hybridize with rotation at 60rpm f
6. Genisphere SIGNAL SAMPLE AMPLIFICATION PRODUCTS Biotin Labeling Kit for ST Exon Arrays Table of Contents Page Introduction 2 Kit Specifications Components and Storage 3 Handling Kit Components 3 Additional Materials Equipment Required For Genisphere Biotin Labeling Kit for ST Exon Arrays 4 For Affymetrix Whole Transcript Expression Arrays 4 Important Parameters Procedural Notes 5 Thermalcycler Programs 5 Procedure for Use Double Stranded cDNA Synthesis 6 RNAClean XP Purification T Quantitation of dscDNA 8 Terminal Labeling of dscDNA 9 Affymetrix Whole Transcript Expression Array Hybridization 10 References 12 Appendix A Gel Shift Assay 13 Biotin Labeling Kit for ST Exon Arrays December 2011 Page 1 of 13 Introduction The Biotin Labeling Kit for ST Exon Arrays contains reagents designed to convert senseRNA from Sensation kits into labeled dscDNA for subsequent analysis on Affymetrix Whole Transcript Expression Arrays Optionally unlabeled dscDNA may be analyzed by Quantitative PCR see page 8 Biotin Labeling for ST ExonArrays Double Stranded cDNA Synthesis 3 5 hours then 1 hour for purification SenseRNA is reverse transcribed using random 12mer primer and dT12 V primer dNTP mix dUTP and Reverse Transcriptase Second Strand cDNA is made with DNA Polymerase ana RNAse H The dscONAts punfied with RNAClean XP senseRNA from Sensation Random and dT Primer Reverse Transcriptase a dNTPs dUT
7. P NY dscD NWA Temminal Labeling of double stranded cDNA T how 4 10ug of dscDNA is fragmented and end labeled with 1 Step Fragment and Label Buffer and Enzyme Biotin dscD NA Analysis of Biotin ddscDNA The biatin dscDNA is ready for hybridization to Affymetrix Vvhole Transcript Expression Arrays Biotin Labeling Kit for ST Exon Arrays December 2011 Page 2 of 13 Kit Specifications Components and Storage Vial 1 Labeling RT Primer Mix Vial 2 5X MMLV RT Buffer Vial 3 MMLV RT Enzyme Vial 4 WT dNTP mix 10mM dNTP dUTP Vial 5 1M MgCl Vial 6 DNA Polymerase Vial 7 RNaseH Vial 8 dscDNA Stop Solution Vial 9 1M Tris HCl pH 7 0 Vial 10 Nuclease Free Water Vial 11 1 Step 5X Fragment and Label Buffer Mix Vial 12 1 Step Fragment and Label Enzyme Mix Store all vials at 20 C Handling Kit Components Vials 1 2 5 8 9 10 Thaw at room temperature vortex and briefly microfuge Keep at room temperature until use Vials 3 6 7 12 Briefly centrifuge then keep on ice at all times Do not vortex Vials 4 11 Thaw on ice briefly microfuge if necessary and keep on ice at all times Do not vortex Biotin Labeling Kit for ST Exon Arrays December 2011 Page 3 of 13 Additional Materials Equipment Required for Genisphere Biotin Labeling Kit for ST Exon Arrays e 10 25ug senseRNA in a volume of 23ul from Sensation kits from Genisphere e Thermal Cycler or heating devices for incubations at 4 C 16 C
8. n Arrays December 2011 Page 6 of 13 12 13 Incubate the 62ul samples at 65 C for 30 minutes Thermalcycler Program 4 Note during this time shake the Agencourt RNAClean XP Reagent bottle to resuspend the magnetic particles thay may have settled Aliquot the appropriate amount of RNACleanXP Reagent and keep at room temperature For each reaction 126ul will be needed Add 8ul of 1M Tris HCl pH 7 0 Vial 9 to each reaction for a volume of 7Oul Gently mix and briefly microfuge RNAClean XP Purification Note Prepare fresh dilutions of ethanol each time RNAClean XP is used 1 10 11 12 If not already done shake the RNAClean XP Reagent bottle to resuspend the magnetic particles that may have settled Allow samples and RNAClean XP Reagent to reach room temperature Place Nuclease Free Water Vial 10 in a 65 C heat block or oven For each reaction add 126ul of RNAClean XP reagent to a well of a Costar 96 well round bottom plate Transfer each 7Oul dscDNA sample to a well containing RNAClean XP and mix well by pipetting up and down 10 20 times Add 65ul of 100 Ethanol to the dscDNA sample RNAclean XP mixture and mix well by pipetting up and down 10 20 times Incubate for 10 minutes at room temperature 20 25 C Place the plate onto an Agencourt SPRIPlate 96 Ring magnet or equivalent for 10 minutes to separate the beads from the solution Slowly aspirate and discard the cleared solution being careful
9. not to disturb the magnetic beads While on the magnet add 170ul of 70 ethanol to each well and incubate for 30 seconds at room temperature With a pipette set to 200ul slowly aspirate the ethanol wash solution being careful not to disturb the magnetic beads Repeat for a total of 3 washes Completely remove the final wash solution Air dry on the magnetic plate for 10 minutes or until all ethanol has evaporated It may be necessary to extend the air dry step for an additional 5 minutes until all ethanol has evaporated Under or over drying may result in lower sample recovery Remove the plate from the magnet Add 27ul of the 65 C Nuclease Free Water to each well Once water is added to all wells incubate at room temperature 20 25 C for 2 3 minutes to elute the sample from the magnetic beads During this time beads can be mixed by gentle shaking of the plate or by pipetting up and down until resuspended Place the plate onto the magnet for 2 5 minutes to separate the beads from the solution Slowly aspirate the purified dscDNA sample and transfer to a new tube being careful not to disturb the magnetic beads Record the volume recovered Biotin Labeling Kit for ST Exon Arrays December 2011 Page 7 of 13 Quantitation of dscDNA Quantitate the dscDNA using a NanoDrop or other instrument From the OD determine the nucleic acid concentration of each labeled sample A260nm x 50 double stranded DNA extinction coefficient x dilution
10. or 16 18 hours at 47 C 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 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 well plate in order to save the hybridization cocktail Store on ice during the procedure or at 80 C for long term storage 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 600ul Stain Cocktail 2 in sample holder 2 c Place one clear vial containing 800ul Array Holding Buffer in sample holder 3 6 Wash the arrays according to array type and components used for Hybridization Wash and Stain For HWS kits the protocols are 169 format 49 64 format Fluidics Protocol FS450 0007 FS450 0001 7 Check for air bubbles If there are air bubbles manually fill the array with Array Holding Buffe
11. r 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 www affymetrix com Biotin Labeling Kit for ST Exon Arrays December 2011 Page 11 of 13 References 1 Roberts L et al Identification of methods for use of formalin fixed paraffin embedded tissue samples in RNA expression profiling Genomics 2009 94 5 341 8 2 Pillai R etal Validation and Reproducibility of a Microarray Based Gene Expression Test for Tumor Identification in Formalin Fixed Paraffin Embedded Specimens Journal of Molecular Diagnostics January 2011 Vol 13 No 1 For Research Use Only 2012 Genisphere Inc All rights reserved Genisphere is a registered trademark Sensation is a trademark of Genisphere LLC Affymetrix GeneChip and Command Console are registered trademarks of Affymetrix Inc Agencourt RNAClean and SPRI are registered trademarks of Beckman Coulter Inc NanoDrop is a trademark of Thermo Fisher Scientific Inc SYBR is a registered trademark of Life Technologies Patents pending Genisphere LLC To Order Technical Support 2801 Sterling Drive Toll Free 877 888 3362 Toll Free 877 888 3362 Hatfield PA 19440 info geni
12. sphere com techsupport genisphere com www genisphere com Biotin Labeling Kit for ST Exon Arrays December 2011 Page 12 of 13 Appendix A Gel Shift Assay Materials Required 10 11 12 13 NeutrAvidin 4 to 20 TBE gel Electrophoresis system with power supply 1X TBE running buffer DNA Ladder Gel loading dye SYBR Gold Nucleic Acid Gel Stain Transilluminator Dilute 1ul of each terminal labeling reaction 3ul Nuclease Free water 1ul terminal labeling reaction Note 2ul of this dilution will be used for the gel shift Save the remaining 2ul for repeat gel shift analysis if necessary Prepare a NeutrAvidin solution of 2 mg mL following the manufacturer s recommendation Place a 4 to 20 TBE gel into the gel holder and load system with 1X TBE Buffer For each sample to be tested remove two 1ul aliquots of fragmented and biotinylated sample to fresh tubes Heat the aliquots of samples at 70 C 2 minutes Add 5ul of 2 mg mL NeutrAvidin to one of the two tubes for each sample tested Mix and incubate at room temperature for 5 minutes Bring the volume of DNA ladders to 6ul with water Add 4ul loading dye to all samples and DNA ladders Carefully load 10ul samples and ladders on gel Run the gel at 150 volts until the front dye almost reaches the bottom approximately 1 hour While the gel is running prepare 100mL of a 1X solution of SYBR Gold for staining SYBR Gold may be diluted in 1X TBE running buffer Break open
13. tion Terminal Labeling of dscDNA for 49 64 format arrays 1 Adjust the volume of 8 16ug dscDNA to 45ul with Nuclease Free Water Vial 10 For each sample label as much dscDNA as possible up to 16ug and not less than 8ug For each reaction prepare a Terminal Labeling reaction master mix in a separate tube on ice 12ul 1 Step 5X Fragment and Label Buffer Mix Vial 11 3ul 1 Step Fragment and Label Enzyme Mix Vial 12 15ul Add the 15ul terminal labeling reaction master mix to the 45ul dscDNA for a final reaction volume of 60ul Incubate the 60ul reactions for terminal labeling as follows Thermalcycler Program 5 37 C for 60 minutes 93 C for 2 minutes 4 C for 2 minutes Optional Run the gel shift assay as indicated in Appendix A Proceed to Affymetrix Whole Transcript Expression Array Hybridization Biotin Labeling Kit for ST Exon Arrays December 2011 Page 9 of 13 Affymetrix Whole Transcript Expression Array Hybridization Preparation of Ovens Arrays and Sample Registration Files 1 Turn Affymetrix Hybridization Oven on and set the temperature to 47 C Set the RPM to 60 Turn the rotation on and allow the oven to preheat 2 Mark each array package with a meaningful designation and upload the sample and array information Sample names barcode IDs etc into AGCC 3 Unwrap the arrays and place on the bench top and mark each array with the corresponding designation from the wrapper Allow the arrays to warm to room
14. ul Labeling RT Primer Mix Vial 1 to the 23ul senseRNA to make a 26ul senseRNA Primer Mix 3 Incubate the 26ul senseRNA Primer Mix Thermalcycler Program 1 70 C for 5 minutes 25 C for 5 minutes 4 C for 2 minutes 4 For each reaction prepare a First Strand Master Mix in a separate tube on ice Sul 5X MMLV RT Buffer Vial 2 Sul WT dNTP mix 10mM dNTP dUTP Vial 4 3yul MMLV RT Enzyme Vial 3 14 ul 5 Add the 14ul First Strand Master Mix to the 26ul senseRNA Primer Mix for a volume of 40ul 6 Incubate the 40ul reactions for first strand synthesis Thermalcycler Program 2 25 C for 10 minutes 42 C for 90 minutes 65 C for 10 minutes 4 C for 2 minutes 7 Prepare a fresh solution of 17 5mM MgCl by combining 2 8ul 1M MgCl Vial 5 with 157 2ul Nuclease Free Water Vial 10 8 For each reaction prepare a Second Strand Master Mix in a separate tube on ice 12ul 17 5mM MgCl Vial 5 dilution from above 1ul Nuclease Free Water Vial 10 2ul WT dNTP mix 10mM dNTP dUTP Vial 4 3yul DNA Polymerase Vial 6 _2ul RNaseH Vial 7 20ul 9 Add 20ul Second Strand Master Mix to each 40ul sample for a volume of 60ul 10 Incubate the 60ul reactions for second strand synthesis Thermalcycler Program 3 16 C for 120 minutes for thermalcyclers do not use a heated lid 75 C for 10 minutes 4 C for 2 minutes 11 Add 2ul of dscDNA Stop Solution Vial 8 to each reaction Proceed to page 7 Biotin Labeling Kit for ST Exo
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