Encore® Biotin Module
Contents
1. 95 C 2 min hold at 4 C Program 2 noes 37 C 60 min 70 C 10 min hold at 4 C Biotin Labeling E cDNA Fragmentation 1 Obtain the Fragmentation Buffer Mix Orange FL1 and Fragmentation Enzyme Mix Orange FL2 from 20 C storage Note You may thaw all reagents at once Refer to the Labeling protocol for thawing and mixing instructions for the Labeling reagents 2 Thaw FL1 at room temperature mix by vortexing spin and place on ice 3 Mix FL2 by inverting the tube 3 times spin and place on ice 4 Add 25 ul of the purified SPIA cDNA into a PCR tube on ice Refer to Table 2 to determine the amount of required amplified cDNA input Add water if necessary to bring up the volume of the cDNA sample to 25 yL 5 Make the Fragmentation Master Mix by combining FL1 and FL2 in a 0 5 mL capped tube according to the volumes shown in Table 4 Table 4 Fragmentation Master Mix volumes listed are for a single reaction FRAGMENTATION BUFFER MIX FRAGMENTATION ENZYME MIX ORANGE FL1 ORANGE FL2 5 pL 2 pL 6 Mix by pipetting spin and place on ice Use the master mix immediately IV Protocol Use Labeling Master Mix immediately after preparation 10 Encore Biotin Module 10 11 a i ee N Add 7 uL of the Fragmentation Master Mix to each sample Mix well by pipetting 8 to 10 times Vortex briefly to ensure thorough mixing spin and place on ice Place the tub2. B Using Nuclease free Techniques Nuclease contamination from equipment and work environment will lead to experimental failure Follow these guidelines to minimize contamination e Wear disposable gloves and change them frequently e Avoid touching surfaces or materials that could introduce DNases e Use only the reagents provided and recommended e Prior to initiating protocol clean and decontaminate work areas and instru ments including pipettes with commercially available decontamination reagents e Use only new DNase free pipette tips and microcentrifuge tubes C Amplified Input cDNA Storage The unlabeled cDNA product generated by NuGEN amplification system products may be stored at 20 C for up to six months prior labeling D Fragmented and Labeled cDNA Storage The labeled cDNA product produced by this kit can be used immediately after preparation or stored at 20 C for up to 6 months prior to use in array hybridization 6 Encore Biotin Module IV Protocol 7 Encore Biotin Module A Overview The cDNA labeling reaction is performed in two steps 1 cDNA fragmentation 0 5 hours 2 Biotin attachment 1 25 hours Total time to label amplified cDNA 1 75 hours B Protocol Notes This protocol should be carried out in a post amplification workspace desig nated for handling SPIA cDNA amplification products using dedicated post amplification equipment and consumables Care should be exercised to avoid the i
3. Whole Blood Solution Part Nos 3100 and 1300 e Ovation Pico WTA Systems V1 and V2 Part Nos 3300 3302 e Ovation PicoSL WTA Systems V1 and V2 Part Nos 3310 3312 e WT Ovation FFPE System V2 Part No 3400 e Ovation FFPE WTA System Part No 3403 e WT Ovation Exon Module Part No 2000 e WT Ovation One Direct System Part No 3500 e Applause WT Amp ST and WT Amp Plus ST Part Nos 5500 5510 e Applause 3 Amp System Part No 5100 The labeled cDNA target generated with the Encore Biotin Module is suitable for hybridization to Affymetrix GeneChip arrays Please refer to the amplification system user guides and www nugeninc com for appropriate sample preparation and labeling system workflows for your application and desired array platform B Fragmentation and Labeling Process This novel and proprietary two step fragmentation and labeling process is carried out by a simple add and incubate procedure and does not require purification steps The first step is a combined chemical and enzymatic fragmentation process that yields cDNA products in the 50 to 100 base range In the second step this fragmented prod uct is labeled via enzymatic attachment of a biotin labeled nucleotide to the 3 hydroxyl end of the fragmented cDNA generated in the first step C Performance Specifications The fragmentation and biotin labeling process is performed in approximately two hours and produces labeled cDNA ranging from 50 to 100
4. bases ready for hybridization to GeneChip arrays D Quality Control Each Encore Biotin Module lot is tested to meet specifications for product size and array performance 2 Introduction Encore Biotin Module E Storage and Stability The Encore Biotin Module is shipped on dry ice and should be unpacked immediately upon receipt All components should be stored at 20 C on internal shelves of a freezer without a defrost cycle Note While the Encore Biotin Module is shipped on dry ice it is critical that it not be stored long term at 80 C as this may result in poor performance This product has been tested to perform to specifications after as many as six freeze thaw cycles Kits handled and stored according to the above guidelines will perform to specifications for at least six months F Material Safety Data Sheet MSDS An MSDS for this product is available on the NuGEN website at http www nugeninc com nugen index cfm support user guides II Kit Components 3 Encore Biotin Module A Reagents and Supplies Provided Table 1 cDNA Fragmentation and Biotin Labeling Reagents 4200 12 4200 60 4200 A01 COMPONENT PART PART PART es rane NUMBER NUMBER NUMBER Fragmentation i p Buffer Mix S01182 01182 501177 Orange FL1 SA 501175 501183 501178 Orange FL2 Enzyme Mix il poer S01184 S01184 S01179 Orange FL3 Biotin Reagent S01172 01185 01180 Orange FL4 Labeling i 7 01173 011
5. damages arising from the use results of use or inability to use its products NUGEN reserves the right to change alter or modify any product to enhance its performance and design NuGEN s products are developed designed and sold FOR RESEARCH USE ONLY This product is not to be used for diagnostic or therapeutic purposes nor is it to be administered to humans or animals Except as expressly set forth herein no right to modify reverse engineer distribute offer to sell or sell NuGEN s product is conveyed or implied by buyer s purchase of this NUGEN product The buyer agrees to use NUGEN products accompanying the product insert in accordance with the intended use and the written instructions provided Table of Contents Contents li Introduetiomi ssss zi i i sicarionsiziczinzionenisiaiionionenzioneniicioneenianisia nica dacenieniziniaii 1 Ai BIcKOround iii 1 B Fragmentation and Labeling Process i 1 C Performance SpecificationSs i 1 D Quality Control nce a dea aaa 1 E Storage and Stability iaia 2 F Material Safety Data Sheet MSDS 2 Il Kit Components aaa las iaia ai 3 A Reagents and Supplies Provided uiii ina nii iana 3 B Additional Equipment Reagents and Labware 3 Ill Planning the Experiment iii 5 A Input RNA Requiem eMis isss iaia 5 B Using Nuclease free Techniques 6 C Amplified Input CDNA Storage ii 6 D Frag
6. the Encore Biotin Module be used for fragmentation and labeling of RNA No Should I purify the labeled cDNA before hybridization No Purification of the labeled cDNA is not necessary What are the recommended storage conditions for the labeled cDNA The labeled cDNA is to be stored at 20 C Ensure the vials are well sealed and avoid multiple freeze thaw cycles What types of arrays work with the Encore Biotin Module cDNA The Encore Biotin Module has been validated on Affymetrix 3 Expression and GeneChip Whole Transcript Arrays such as Gene ST or Exon ST arrays Are the array hybridization reagents included in the Encore Biotin Module No Refer to Appendix A for information on array hybridization protocols What hybridization and wash protocols do you recommend for Affymetrix GeneChip applications Refer to Appendix A for information on array hybridization protocols Where can I safely stop in the Encore Biotin Module protocol We do not recommend stopping at any step of the protocol How do I determine fragmentation success You may use an Agilent Bioanalyzer to inspect the size distribution of samples before and after fragmentation as described in Appendix B How should I qualify my cDNA for use with the Encore Biotin Module You must use cDNA generated with a validated NUGEN amplification system The concentration of starting cDNA must be determined to ensure adequate input into the labeling reaction and therefore onto th
7. 33 ug mL A260 unit as the constant VI Appendix 17 Encore Biotin Module D Frequently Asked Questions FAQs Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 What materials are provided with the Encore Biotin Module The Encore Biotin Module provides all necessary buffers and enzymes for fragmentation and labeling of cDNA generated with a validated NuGEN Amplification System What equipment is required or will be useful You will need a microcentrifuge pipettes vortexer a thermal cycler and a UV Vis spectrophotometer An Agilent Bioanalyzer or a similar instrument may be used for quality control What additional reagents are required for the Encore Biotin Module No additional reagents are required What type of cDNA should I use with the Encore Biotin Module You must use SPIA cDNA generated with one of the following NuGEN products with the Encore Biotin Module e Ovation RNA Amplification System V2 Part No 3100 e Ovation Whole Blood Solution Part Nos 3100 and 1300 e Ovation Pico WTA Systems V1 and V2 Part No 3300 3302 e Ovation PicoSL WTA Systems V1 and V2 Part No 3310 3312 e Ovation FFPE WTA System Part No 3403 e WT Ovation FFPE System V2 Part No 3400 e WT Ovation Exon Module Part No 2000 e WT Ovation One Direct System Part No 3500 e Applause WT Amp ST and WT Amp Plus ST Part No 5500 5510 e Applause 3 Amp System Part No 5100 How much labeled cDNA should I hybridize to a G
8. 86 01181 Orange FL5 Enzyme Mix B Additional Equipment Reagents and Labware Required Materials e Equipment Microcentrifuge for individual 1 5 mL and 0 5 mL tubes Microcentrifuge for 0 2 mL individual and 8 X 0 2 mL strip PCR tubes 0 5 10 uL pipette 2 20 uL pipette 20 200 uL pipette 200 1000 uL pipette Vortexer Thermal cycler with 0 2 mL tube heat block heated lid and 100 uL reaction capacity Appropriate spectrophotometer and cuvettes or Nanodrop UV Vis Spectrophotometer e Labware Nuclease free pipette tips 1 5 mL and 0 5 mL RNase free microcentrifuge tubes 0 2 mL individual thin wall PCR tubes or 8 X 0 2 mL strip PCR tubes Appropriate spectrophotometer cuvettes Disposable gloves Kimwipes Ice bucket II Kit Components Optional Equipment e Agilent 2100 bioanalyzer or other equipment for electrophoretic analysis of RNA e Real time PCR system 4 Encore Biotin Module Ill Planning the Experiment A Input RNA Requirements 1 cDNA Source The most important requirement for achieving successful results with the Encore Biotin Module is to use cDNA generated with one of NuGEN s Ovation System or Applause System amplification products that have been validated for use with this module Note The Encore Biotin Module is designed solely for use with cDNA prepared using the NuGEN products listed below It is not designed for use with cDNA from other sources e Ovation RN
9. A Amplification System V2 Part No 3100 e Ovation Whole Blood Solution Part Nos 3100 and 1300 e Ovation Pico WTA Systems V1 and V2 Part Nos 3300 3302 e Ovation PicoSL WTA Systems V1 and V2 Part Nos 3310 3312 e WT Ovation FFPE System V2 Part No 3400 e Ovation FFPE WTA System Part No 3403 e WT Ovation Exon Module Part No 2000 e WT Ovation One Direct System Part No 3500 e Applause WT Amp ST and WT Amp Plus ST Part Nos 5500 5510 e Applause 3 Amp System Part No 5100 Note With the Ovation PicoSL Systems V1 and V2 use half the recommended vol umes throughout the Encore Biotin Labeling protocol Note Fragmentation and Labeling protocols for cDNA generated using the Applause products are found in the respective user guides The unlabeled cDNA product may be stored at 20 C with minimum freeze thaw cycles prior to labeling For recommendations on input cDNA quality assessment see Appendices B and C of this user guide You may also choose to qualify the start ing cDNA by performing qPCR assays as recommended in the appropriate NuGEN Amplification System user guides 2 cDNA Purity The cDNA used with the Encore Biotin Module must be purified using a supported purification method as noted in the appropriate NUGEN amplification system product user guide The adjusted 260 280 absorbance ratio of the purified SPIA cDNA should be gt 1 8 5 Encore Biotin Module Ill Planning the Experiment
10. Encore Biotin Module PART NO 4200 n imagine more GET lesse Patents Licensing and Trademarks 2009 2013 NuGEN Technologies Inc All rights reserved The Encore Ovation and Applause families of products and methods of their use are covered by several issued U S and International patents and pending applications www nugeninc com NuGEN Ovation SPIA Ribo SPIA Applause Encore Prelude Mondrian and Imagine More From Less are trademarks or registered trademarks of NUGEN Technologies Inc Other marks appearing in these materials are marks of their respective owners Specific information on patents trademarks and licenses related to the Mondrian SP Universal Cartridge the Mondrian SP Cartridge the Mondrian SP Workstation and the Mondrian SP Workstation may be found in the Mondrian SP Universal Cartridge User Guide M01265 the Mondrian SP Cartridge User Guide M01344 the Mondrian SP Workstation User Manual Part No M01264 and the Mondrian SP Workstation User Manual M01322 The purchase of this product conveys to the buyer the limited non exclusive non transferable right without the right to modify reverse engineer resell repackage or further sublicense under these patent applications and any patents issuing from these patent applications to use this prod uct and methods accompanying this user guide for research and development purposes solely in accordance with the intended use described and the written
11. abeled cDNA may be processed immediately for array hybridization or stored at 20 C For recommendations on array hybridization see Appendix A 11 Encore Biotin Module V Technical Support For Technical Support please contact NuGEN at U S only 888 654 6544 Toll Free Phone or 888 296 6544 Toll Free Fax or email techserv nugeninc com In Europe contact NUGEN at 31 0 135780215 Phone or 31 0 135780216 Fax or email at europe nugeninc com In all other locations contact your NuGEN distributors Technical Support team 12 Encore Biotin Module VI Appendix 13 Encore Biotin Module A Target Preparation for Affymetrix GeneChip Eukaryotic Array Analysis In general cDNA targets labeled using the Encore Biotin Module are prepared for analysis on Affymetrix GeneChip arrays according to the manufacturer s guidlines Some specific exceptions are noted below Using the Affymetrix Hybridization Wash and Stain Kit with GeneChip 3 Expression Ar rays or Whole Transcript Arrays Guidelines for processing GeneChip Whole Transcript Arrays e g Gene ST and Exon ST arrays can be found in the Affymetrix GeneChip Whole Transcript WT Sense Labeling Assay User Manual Affymetrix Part No 701880 Rev 5 unless otherwise noted below Guidelines for processing GeneChip 3 Expression Arrays e g HG U133 arrays can be found in the Affymetrix GeneChip Expression Analysis Technical Manual Affymetrix Part No 702232 Rev 3 u
12. e arrays Please refer to Table 2 for cDNA input requirements You may choose to further qualify the input cDNA by performing qPCR assays as recommended in the appro priate NuGEN amplification system user guides VI Appendix Q20 Which protocol should I use for cDNA produced using Applause 3 Amp Applause WT Amp ST and WT Amp Plus ST RNA Amplification Systems The protocols for labeling cDNA amplified with Applause 3 Amp Applause WT Amp ST and WT Amp Plus ST are found in the respective user guides for these products 19 Encore Biotin Module VI Appendix CA NUGEN imagine more from lesse E Update History This document the Encore Biotin Module user guide M01111 v6 is an update to address the following topics Description Section Page s Corrected component name of FL4 to Biotin Reagent IV F 10 NuGEN Technologies Inc Headquarters USA Europe 201 Industrial Road Suite 310 P O Box 109 For our international distributors contact San Carlos CA 94070 USA 9350 AC Leek information visit our website Toll Free Tel 888 654 6544 Toll Free Fax 888 296 6544 custserv nugeninc com techserv nugeninc com The Netherlands Tel 31 13 5780215 Fax 31 13 5780216 europe nugeninc com www nugeninc com 2009 2013 NuGEN Technologies Inc All rights reserved The Encore Ovation and Applause families of products and methods of their use are covered by several issued U S and Internati
13. em V2 Part No 3100 3 75 ug 17 ng pL Ovation Whole Blood Solution Part No 1300 amp 3100 4 4 ug 20 ng uL WT Ovation Exon Module 5 ug uni Part No 2000 Applause WT Amp ST and WT Amp Plus ST Part No 5500 5510 Refer to the appropriate Applause user guide Refer to the appropriate Applause user guide Applause 3 Amp System Part No 5100 Refer to the appropriate Applause user guide Refer to the appropriate Applause user guide All cDNA concentrations are assessed using 33 yg mL A260 as the constant WTA System user guide for specific guidelines Requires performing half volume Encore Biotin Module reactions Refer to the appropriate Ovation PicoSL IV Protocol Use Fragmentation Master Mix immediately after 9 preparation Encore Biotin Module D Programming the Thermal Cycler Use a thermal cycler with a heat block designed for 0 2 mL tubes equipped with a heated lid and with a capacity of 100 uL reaction volume Prepare the programs shown in Table 3 following the operating instructions provided by the manufacturer For ther mal cyclers with an adjustable heated lid set the lid temperature at 100 C For thermal cyclers with a fixed temperature heated lid e g ABI GeneAmp PCR 9600 and 9700 models use the default settings typically 100 to 105 C Table 3 Thermal Cycler Programming PROGRAMMING DETAILS Program 1 EDNA Fragmentation 37 C 30 min
14. eneChip array Refer to Appendix A for guidance regarding the use of the Encore Biotin Module with Affymetrix GeneChip Arrays Can vary the amount of cDNA input to fragmentation and labeling You may use from 2 to 6 ug of cDNA input with the Encore Biotin Module Refer to Table 2 to determine the specific recommendations for your sample type array type and amplification kit used We recommend using the same cDNA input across all samples in a single experiment where a range of inputs is given Can use any cDNA as starting material in the Encore Biotin Module No the cDNA must be generated using a validated NuGEN amplification system Use of other cDNAs will result in poor performance How much fragmented and labeled cDNA yield can expect Since this module does not require any purification the final yield is equal to the amount of cDNA input VI Appendix 18 Encore Biotin Module Q9 Q10 Q11 Q12 Q13 Q14 Q15 Q16 Q17 Q18 Q19 What is the size range of fragmented cDNA generated by the Encore Biotin Module As measured with an Agilent Bioanalyzer 80 of product falls below 200 bases with an average peak at 85 bases Has NuGEN performed reproducibility studies on the Encore Biotin Module Yes our studies have included sample to sample lot to lot and operator to operator reproducibility Refer to the Encore Biotin Module Technical Report 1 for a summary of our performance data Can
15. ented and Fragmented cDNA Product HeLa RNA amplified with the Ovation Pico WTA System Part No 3300 was processed with the Encore Biotin Module and analyzed on an Agilent Bioana lyzer Unfragmented cDNA traces will vary depending on amplification method and sample type 12 54 RNA 6000 Ladder 10 0 SPIA cDNA Fragmented and Labeled cDNA E 7 5 U N 2 o 5 07 2 LL 2 5 0 0 T T T T T T T T I 19 24 29 34 39 44 49 54 59 64 69 Time seconds VI Appendix 16 Encore Biotin Module Input cDNA Analysis Measuring Concentration and Purity Before using the Encore Biotin Module it is highly recommended to determine the concentration of your sample to ensure sufficient cDNA input for the labeling process Mix the sample by brief vortexing and spinning prior to checking the concentration Measure the absorbance of the amplified cDNA product at 260 280 and 320 nm You may need to make a 1 20 dilution of the cDNA in water prior to measuring the absorbance Purity Subtract the A320 value from both A260 and A280 values The adjusted A260 A320 A280 A320 ratio should be gt 1 8 Yield Assume 1 A260 unit 33 pg mL To calculate A260 A320 of diluted sample X dilution factor X 33 concentration in ug mL A260 unit X 0 03 final volume in mL total yield in micrograms Alternatively you may measure the concentration and purity of cDNA with a Nanodrop using
16. es in a pre warmed thermal cycler programmed to run Program 1 cDNA Fragmentation see Table 3 37 C 30 min 95 C 2 min hold at 4 C Remove the tubes from the thermal cycler spin to collect condensation and place on ice Continue immediately with the Biotin Labeling protocol Biotin Labeling Obtain the Labeling Buffer Mix Orange FL3 Biotin Reagent Orange FL4 and the Labeling Enzyme Mix Orange FL5 from the product box stored at 20 C Place all reagents on ice Thaw FL3 and FL4 at room temperature mix by vortexing spin and place on ice Mix FLS by inverting the tube 3 times spin and place on ice Make a Labeling Master Mix as outlined below Table 5 Labeling Master Mix volumes listed are for a single reaction LABELING BUFFER MIX BIOTIN REAGENT LABELING ENZYME MIX ORANGE FL3 ORANGE FLA ORANGE FL5 15 pL 1 5 uL 1 5 uL 5O GO i Oy Mix by pipetting spin and place on ice Use the master mix immediately Add 18 uL of the Labeling Master Mix to each fragmented cDNA sample tube Mix well by pipetting 8 to 10 times Vortex briefly to ensure thorough mixing spin and place on ice Place tubes in a pre warmed thermal cycler programmed to run Program 2 Biotin Labeling see Table 3 37 C 60 min 70 C 10 min hold at 4 C IV Protocol 11 After completion remove tubes from thermal cycler spin to collect condensation and place on ice 12 The fragmented and l
17. instructions provided in this user guide No license to make or sell products by use of this product is granted to the buyer whether expressly by implication by estoppels or otherwise In particular the purchase of this product does not include or carry any right or license to use develop or otherwise exploit this product commercially and no rights are conveyed to the buyer to use the product or components of the product for purposes including commercial services or clinical diagnostics For information on purchasing a license to the NuGEN patents for uses other than in conjunction with this product or to use this product for purposes other than research please contact NuGEN Technologies Inc 201 Industrial Road Suite 310 San Carlos CA 94070 Phone 888 654 6544 or 650 590 3600 FAX 888 296 6544 or 650 590 3630 Warranty NuGEN warrants that this product meets the performance standards described in the Company s product and technical literature for a period of six months from the date of purchase provided that the product is handled and stored according to published instructions and that the product is not altered or misused If the product fails to meet these performance standards NuGEN will replace the product free of charge or issue a credit for the purchase price NUGEN s liability under this warranty shall not exceed the purchase price of the product NUGEN shall assume no liability for direct indirect consequential or incidental
18. mented and Labeled cDNA Storage eee eeeeeeeteeeteeeeeeteeees 6 IV Protocol PRA 7 Ao QUGIVIOW sioe Rare a thats bucdcuncds suchiduedsdedvacetsncpssndesad esunds iecgeseasseerds 7 B Protocol Notes eoii et eaaa ia 7 C Preparing cDNA Samples secans auinen naa aa aai EAE AREE iEn 8 D Programming the Thermal Cycler eccecceeeeeeeeeseeeeeseeeseeeseeeneeteeees 9 E CDNA Fragmentation ace iii 9 F Biotinabeling ee iii ANA ES E E ENESES 10 V Technical Support sisicsciii r svssioiieniinieisiricisizionicaionisioniaianicicizinia 12 VE Appendix iaia ia 13 A Target Preparation for Affymetrix GeneChip Eukaryotic Array Analysis ssianccaia anal lnaniraro EEE SEEEN EES 13 B Quality Control of Amplified Fragmented and Labeled CDNA PROGUGEE sein Rata a Die sbacsiPtesseeesiet 15 C Input cDNA Analysis Measuring Concentration and Purity iii 16 D Frequently Asked Questions FAQS iii 17 E Update Histoyirpa rare lee a 20 1 Introduction Encore Biotin Module A Background NuGEN s proprietary fragmentation and labeling process patent pending combines enzymatic and chemical processes for the preparation of labeled cDNA to generate labeled targets suitable for hybridization to Affymetrix GeneChip arrays The Encore Biotin Module is validated for use with amplified cDNA generated using the following NuGEN products e Ovation RNA Amplification System V2 Part No 3100 e Ovation
19. nless otherwise noted below e Refer to Table 6 for guidelines on hybridization cocktail formulation and hybrid ization cocktail loading volume with NuGEN prepared samples e Heat denature the hybridization cocktail at 99 C for 2 minutes not 5 minutes as specified by Affymetrix then follow the Affymetrix protocol 45 C in a heat block for 5 minutes then centrifuge at maximum speed for 1 minute just prior to loading e We recommend a hybridization time of 18 hours 2 hours Hybridization for 16 to 20 hours yields comparable results e Refer to Table 6 for guidelines on selection of the appropriate fluidics scripts Note that in some cases the optimal fluidics script will be different for NUGEN targets than for Affymetrix labeled targets Using Target Prepared With the WT Ovation One Direct System We recommend extending the array hybridization time to 40 hours when using the WT Ovation One Direct System to maximize detection sensitivity with the exceedingly small samples used VI Appendix 14 Encore Biotin Module Table 6 Hybridization Cocktail Assembly and Fluidics Protocols for Single GeneChip Arrays using Affymetrix HWS kit Affymetrix P N 900720 For ST arrays Exon arrays Gene arrays STANDARD ARRAY MIDI ARRAY MINI ARRAY FINAL COMPONENT 49 or 64 100 FORMAT 169 FORMAT CONCENTRATION FORMAT Fragmented kes biotin labeled 50 uL 34 pL 25
20. ntroduction of SPIA cDNA into workspaces used to set up SPIA amplifica tion reactions For more information on this topic please refer to the NuGEN RNA amplification product user guide or contact NUGEN Technical Services Thaw only components used in each step and immediately place them on ice Always keep thawed reagents and reaction tubes on ice unless otherwise instructed After thawing and mixing buffer mixes if any precipitate is observed re dissolve it completely prior to use You may gently warm the buffer mix for two minutes at room temperature followed by brief vortexing Do not warm any enzyme mixes FL3 labeling buffer may appear to have pink coloration This is normal Spin down labeling master mix briefly at low speed High speed spins for long periods can cause formation of a precipitate The reagent volumes recovered greatly depend on the number of batches processed with each kit Set up no fewer than three reactions at a time with the 4200 12 kit no fewer than 10 reactions at a time with 4200 60 and no fewer than 48 reactions at a time with 4200 A01 The 4200 A01 kit has been designed for use with automation protocols requiring large batch sizes For information on automation solutions contact NUGEN Technical Services When placing small amounts of reagents into reaction mix gently pipet up and down several times to ensure complete transfer When instructed to pipet mix gently aspirate and dispense at least half of total reac
21. onal patents and pending applications www nugeninc com NuGEN Ovation SPIA Ribo SPIA Applause Encore Prelude Mondrian and Imagine More From Less are trademarks or registered trademarks of NuGEN Technologies Inc Other marks appearing in these materials are marks of their respective owners For research use only M01111 v6
22. tion mix volume Repeat a minimum of five times to ensure complete mixing Allow the thermal cycler to reach incubation temperature before placing samples in the block When working with more than one sample excess master mix may be needed Components of this NuGEN product should not be used or combined with any other Ovation Systems or Applause products and vice versa IV Protocol 8 Encore Biotin Module C Preparing cDNA Samples Final Hybridization Cocktail Concentrations The amount of amplified cDNA required for each fragmentation and labeling reaction depends on the method of cDNA generation Specific NUGEN sample preparation systems have been validated for use with the Encore Biotin Module and the required cDNA input from each are listed in Table 2 Ensure that the correct input is used in sec tion IV E step 6 of the Fragmentation protocol Table 2 cDNA Input Requirements for Fragmentation and Labeling Reactions and NUGEN AMPLIFICATION SYSTEM cDNA INPUT FINAL HYB Part No PER REACTION COCKTAIL CONCENTRATION WT Ovation One Direct RNA Amplification System Cat 3500 5 6 pg 23 27 ng pL Ovation FFPE WTA System Part No 3403 4 5 ug 18 23 ng uL WT Ovation FFPE System V2 Part No 3400 4 5 ug 18 23 ng L Ovation Pico WTA Systems V1 and V2 5 ees Part No 3300 3302 ug g uL Ovation PicoSL WTA Systems V1 and V2 ni Part No 3310 3312 2 5 ug 23 ng pL Ovation RNA Amplification Syst
23. uL amplified cDNA amplification P method Control oligo nucleotide B2 3 7 yL 2 5 pL 1 9 pL 50 pM 3 nM 20X Eukaryotic 155 25 hybridization SEL controls bioB 11 uL 7 5 uL 5 5 ul and 190 pM bioC bioD cre respectively 2X Hybridization buffer 110 uL 75 uL 55 uL 1X 100 DMSO 22 uL 15 uL 11 uL 10 Water 23 3 uL 16 uL 11 6 uL N A Total Volume 220 uL 150 uL 110 pL Array Loading Wale 200 pL 130 pL 90 uL FLUIDICS PROTOCOLS For 3 arrays FS450_0004 FS450_0002 FS450_0001 FS450_0007 Refer to Table 2 for cDNA input requirements into fragmentation and labeling reactions and final hybridization cocktail concentrations VI Appendix 15 Encore Biotin Module B Quality Control of Amplified Fragmented and Labeled cDNA Product The size distribution of the final fragmented and biotinylated product may be viewed on an Agilent Bioanalyzer by loading 100 ng of each sample before and after the fragmentation and labeling process on an RNA 6000 Nano LabChip Agilent Part No 5065 4476 using the Total RNA program following the manufacturer s instructions Product that is not sufficiently fragmented has been shown to yield poor results on GeneChip arrays For good results on GeneChip arrays 80 or greater of the frag mented cDNA product should be smaller than 200 bases in length For examples of Bioanalyzer traces of unfragmented and fragmented cDNA product refer to Figure 1 Figure 1 Bioanalyzer Trace of Amplified Un fragm
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