NuGen Applause™ WT
Contents
1. Program 6 Post SPIA Modification 49C 1 min 37 C 15 min 95 C 5 min hold at 4 C Program 7 Post SPIA Modification II 4 C 1 min 30 C 10 min 42 C 60 min 75 C 10 min hold at 4 C IV Amplification Protocols Add 3 uL of First Strand Master Mix to the 7 uL RNA Primer mix Total volume is 10 uL 12 Applause WT Amp ST System g eL Ow YU GR First Strand cDNA Synthesis Obtain the First Strand Primer Mix blue A1 First Strand Buffer Mix blue A2 First Strand Enzyme Mix blue A3 and Nuclease free Water green D1 from 20 C storage Spin down the contents of A3 and place on ice Thaw the other reagents at room temperature Mix by vortexing spin and place on ice Leave Nuclease free Water at room temperature Add 5 uL of total RNA sample 50 to 200 ng to a 0 2 mL PCR tube Add 2 uL of A1 to each reaction tube Mix by pipetting 5 times spin and place on ice Place the tubes in a pre warmed thermal cycler programmed to run Program 1 Primer Annealing see Table 6 65 C 5 min hold at 4 C Remove the tubes from the thermal cycler and place on ice Once Primer Annealing Step 7 is complete prepare a master mix by combining A2 and A3 in a 0 5 mL capped tube according to the volumes in Table 7 Table 7 First Strand Master Mix volumes listed are for a single reaction FIRST STRAND BUFFER MIX FIRST STRAND ENZYME MIX BLUE A2 ver2. RNA Storage RNA samples should be stored at 80 C Avoid frequent freeze thaw cycles of RNA as RNA degradation may result D Amplified cDNA Storage The amplified ST cDNA produced by the Applause WT Amp ST System may be stored at 20 C IV Amplification Protocols 9 Applause WT Amp ST System A Overview The Ribo SPIA amplification process used in the Applause WT Amp ST System is performed in five stages 1 First strand cDNA synthesis 1 hour 2 Second strand cDNA synthesis and enhancement 1 5 hours 3 SPIA isothermal linear amplification 1 5 hours 4 Post SPIA modification 2 hours 5 cDNA purification and quantitation 1 hour Total time to prepare amplified cDNA 7 hours Applause components are color coded with each reagent vial linked to a specific pro cess stage Performing each stage requires the simple addition of a master mix or other reagents followed by incubation Master mixes are prepared by mixing components provided for that stage B Protocol Notes It is important to set up no fewer than eight reactions at a time This will ensure that you are not pipetting very small volumes see the second strand synthesis section below the effective range of air displacement pipetting technolo gies For this reason setting up fewer than eight reactions can lead to poor performance Thaw components used in each step and immediately place them on ice as indi cated in this user guide It is best not to tha
3. 3 BLUE A3 ver 1 2 5 uL 0 5 uL 10 11 12 13 14 Add 3 uL of the First Strand Master Mix to each tube Mix by pipetting 5 times spin and place on ice Place the tubes in a pre cooled thermal cycler programmed to run Program 2 First Strand cDNA Synthesis see Table 6 49C 1 min 25 C 10 min 42 C 10 min 70 C 15 min hold at 4 C Remove the tubes from the thermal cycler spin to collect condensation and place on ice Continue immediately with the Second Strand cDNA Synthesis protocol IV Amplification Protocols Do not return B1 ver 3 buffer to the freezer as it is required for the next step as well Add 10 uL of Second Strand Master Mix to the 10 uL First Strand reaction Total volume is 20 pL 13 Applause WT Amp ST System E Second Strand cDNA Synthesis 1 Obtain the Second Strand Buffer Mix yellow B1 Second Strand Enzyme Mix yel low B2 and Enhancement Enzyme Mix yellow B3 from 20 C storage 2 Spin down the contents of B2 and B3 and place on ice 3 Thaw reagent B1 at room temperature mix by vortexing spin and place on ice 4 Make a master mix by combining B1 and B2 in a 0 5 mL capped tube according to the volumes shown in Table 8 Table 8 Second Strand Master Mix volumes listed are for a single reaction SECOND STRAND BUFFER MIX SECOND STRAND ENZYME MIX YELLOW B1 ver 3 YELLOW B2 ven 2 9 75 pL 0 25 uL 5 Add 10 uL o
4. Amp ST System is shipped on dry ice and should be unpacked imme diately upon receipt All components should be stored at 20 C in a freezer without a defrost cycle 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 NuGEN has not yet established long term storage stability for the Applause WT Amp ST System F Material Safety Data Sheet MSDS An MSDS for this product is available from NuGEN Technical Service by calling 888 654 6544 or by sending an email to techserv nugeninc com ll Kit Components 4 Applause WT Amp ST System A Reagents and Supplies Provided Table 1 First Strand cDNA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER First strand Rrimier S01262 Blue Al ver 4 Mix Fir strand Bufer S01256 Blue A2 ver 3 Mix First Strand Enzyme S01040 Blue A3 ver 1 Mix Table 2 Second Strand cDNA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER Second Strand Buffer Mix S01257 Yellow B1 ver 3 Segond Strana S01126 Yellow B2 ver 2 Enzyme Mix EU UU S01119 Yellow B3 ver 1 Enzyme Mix Table 3 SPIA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER SPIA Primer Mix S01264 Red C1 ver 8 SPIA Buffer Mix S01259 Red C2 ver 8 SPIA Enzyme Mix S01261 Red C3 ver 5 ll Kit Compone
5. genes you may need to use more cDNA per reaction VII Appendix 33 Applause WT Amp ST System Q26 What is the recommended minimum batch size Q27 We recommend a minimum batch size of eight reactions Smaller batches may result in poor performance due to the challenge of accurately pipetting small volumes Can I amplify RNA that has been isolated with the aid of a carrier Many common carriers will interfere with the amplification process Glycogen inhibits reverse transcriptase and yeast tRNA will produce cDNA in the first strand synthesis and interfere with the analysis We typically don t recom mend using a carrier but if it is unavoidable then please contact the NuGEN Technical Support Team for information on compatible carriers VII Un Appendix NuGEN imagine more from less F Update History This document the Applause WT Amp ST System user guide M01134 v4 is an update to address the following topics The table below lists the changes made to this version relative to the previous version Applause WT Amp ST and WT Amp Plus ST System user guide M01134 v3 Description Section Page s Establish independent user guides for the Applause WT Amp ST System Part No 5500 and the Applause WT Amp Plus ST Throughout System Part No 5510 Replace figure 1 with new artwork 2 Update component part numbers in Tables 3 and 4 ILA 4 Replace figure 2 with new artwork Ill 7 Increase vol
6. of the Enhancement Master Mix fresh at this point by mixing 1 85 uL B1 and 0 15 uL B3 per reaction We do not recommend storing this Master Mix overnight for use the next day Make a master mix by combining the Enhancement Master Mix and E1 in a 0 5 mL capped tube according to the volumes shown in Table 11 Table 11 Post SPIA Modification I Master Mix volumes listed are for a single reaction ENHANCEMENT PRIMER MIX MASTER MIX VIOLET E1 ver 1 2 uL 5 yl 10 Add 7 uL of the Post SPIA Modification Master Mix to the SPIA Amplification reaction Mix by pipetting 5 times with a pipette set to 20 uL spin and place on ice Place the tubes in a pre cooled thermal cycler programmed to run Program 6 Post SPIA Modification see Table 6 49C 1 min 37 C 15 min 95 C 5 min hold at 4 C Remove the tubes from the thermal cycler spin to collect condensation and place on ice Continue immediately with the Post SPIA Modification ll protocol IV Amplification Protocols E I Post SPIA Modification ll The E3 Enzyme Mix is quite 1 Make a master mix by combining E2 and E3 in a 0 5 mL capped tube according to viscas Please take cara and the volumes shown in Table 12 pipette this mix slowly into the mix Table 12 Post SPIA Modification ll Master Mix volumes listed are for a single reaction BUFFER MIX ENZYME MIX VIOLET E2 ver 2 VIOLET E3 ver 1 5 HL 5 HL Add 10 uL o
7. step after isolation in order to remove any residual organics One measure of RNA purity is the ratio of absorbance readings at 260 and 280 nm The A260 A280 ratio for RNA samples of acceptable purity should be in excess of 1 8 RNA samples with lower ratios may result in poor amplification 3 RNA Integrity Purified total RNA samples of high molecular weight with little or no evidence of degradation are required for use with this product RNA integrity can be determined using the Agilent 2100 Bioanalyzer RNA 6000 Nano LabChip or RNA 6000 Pico LabChip The RNA Integrity Number RIN avail able in the Bioanalyzer 2100 Expert Software provides an index of RNA quality that can be helpful in triaging purified RNA samples of varying integrity prior to amplification Figure 2 This illustration of RNA quality variation shows Bioanalyzer traces of three different RNAs with varying degrees of quality RNA Quality Continuum i TW i j a f ML a eaa UEN See Faaa Poor Quality Moderate Quality Good Quality RIN 2 4 RIN 6 7 RIN 9 2 7 Applause WT Amp ST System lll Planning the Experiment 8 Applause WT Amp ST System B User Quality Control Guidelines for RNA samples The inclusion of positive control RNA samples is an essential tool in evaluating the success of an amplification experiment In the absence of successful positive con trol RNA amplification it may be difficult or impossible to troubleshoo
8. 000 rpm and discard the flow through Place the RNeasy MinElute Spin Column in a fresh 2 mL collection tube and place in the microcentrifuge with the cap open Spin for 5 minutes at gt 8000 X g 210 000 rpm and discard the flow through Place the RNeasy MinElute Spin Column in a fresh 1 5 mL collection tube k Add 14 uL nuclease free water D1 green cap directly to the center of the filter in the tube and close the cap Do not use cold water Spin for 1 minute at gt 8000 X g gt 10 000 rpm to collect the purified RNA VII Appendix 30 Applause WT Amp ST System E Frequently Asked Questions FAQs Q1 Q2 Q3 Q4 Q5 Q6 Q7 os What materials are provided with the Applause WT Amp ST System The Applause WT Amp ST System provides all necessary buffers primers and enzymes for first strand cDNA synthesis second strand cDNA synthesis and amplification and all necessary buffers and enzymes for converting amplified cDNA into sense target cDNA ST cDNA For your convenience nuclease free water has also been included What additional consumables does the user need For the ST cDNA purification step the QIAGEN MinElute Reaction Cleanup Kit Catalog 28204 is required The user guide also lists recommendations for specific consumables including nuclease free pipette tips nuclease free microcentrifuge tubes 0 2 mL PCR tubes and plates RNaseZap and DNA OFF What is the minimum input requ
9. Applause WT Amp ST System Patents Licensing and Trademarks 2009 2011 NuGEN Technologies Inc The Ovation and Applause families of products and methods are covered by U S Patent Nos 6 692 918 6 251 639 6 946 251 and 7 354 717 and other issued and pending patents in the U S and other countries NUGEN the NuGEN logo Ovation SPIA Ribo SPIA WT Ovation Encore Applause Prelude 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 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 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 co
10. PIA PRIMER MIX SPIA ENZYME MIX RED C2 ver 8 RED C1 ver 8 RED C3 ver 5 2 uL 2 uL 4 uL 10 Add 8 uL of the SPIA Master Mix to the Post Second Strand Enhancement reaction Mix by pipetting 5 times spin and place on ice Place the tubes in a pre cooled thermal cycler programmed to run Program 5 SPIA Amplification see Table 6 49C 1 min 47 C 90 min 95 C 5 min hold at 4 C Remove the tubes from the thermal cycler spin to collect condensation and place on ice Optional If qPCR will be performed on the amplification products remove an aliquot of the SPIA cDNA at this point Continue immediately with the Post SPIA Modification protocol or store the SPIA reactions at 20 C overnight prior to continuing IV Amplification Protocols Add 7 uL of Post SPIA Modification I Master Mix to the 30 uL SPIA Amplification reaction Total volume is 37 uL 16 Applause WT Amp ST System Post SPIA Modification I Obtain the Primer Mix Violet E1 Buffer Mix Violet E2 and Enzyme Mix Violet E3 from 20 C storage Spin down the contents of E3 and place on ice Thaw E1 and E2 at room temperature mix by vortexing spin and place on ice Retrieve the remaining Post Second Strand Enhancement Mix from step IV F that was set aside on ice Note In cases where the reactions have been stored at 20 C overnight prior to carrying out the Post SPIA Modification Protocol make the second half
11. ach Second Strand reaction tube Mix by pipetting 5 times with a pipet set to 15 uL spin and place on ice Note Save the remaining Enhancement Master Mix on ice It will be used in the Post SPIA Modification Protocol Sections IV H amp K Place the tubes in a pre cooled thermal cycler programmed to run Program 4 Post Second Strand Enhancement see Table 6 4 C 1 min 37 C 15 min 80 C 20 min hold at 4 C Remove the tubes from the thermal cycler spin to collect condensation and place on ice Continue immediately with the SPIA Amplification protocol IV Amplification Protocols Add 8 uL of SPIA Master Mix to the 22 uL Enhancement reaction Total volume is 30 pL 15 Applause WT Amp ST System SPIA Amplification Obtain the SPIA Buffer Mix red C2 SPIA Primer Mix red C1 and SPIA Enzyme Mix red C3 from 20 C storage Thaw C3 on ice and mix the contents by inverting gently 5 times Ensure the enzyme is well mixed without introducing bubbles spin and place on ice Thaw reagents C1 and C2 at room temperature mix by vortexing spin and place on ice Make a master mix by seguentially combining C2 C1 and C3 in a 0 5 mL capped tube according to the volumes shown in Table 10 Note Ensure the addition of C3 is at the last moment and that the master mix is mixed thoroughly before aliguoting Table 10 SPIA Master Mix volumes listed are for a single reaction SPIA BUFFER MIX S
12. all have been generated using this convention How many rounds of amplification are performed in the Applause WT Amp ST System This System performs a single round of amplification It is not designed to sup port multiple rounds of amplification Do I need to order specific primers for the amplification No The DNA RNA primers provided in the Applause WT Amp ST Systems are universal No gene specific primers are required Do I have to use the supplied DNA RNA primers Yes The Applause WT Amp ST System will not work properly with other primers Do you recommend purification of the amplified ST cDNA prior to qPCR analysis No The recommendations given in Appendix B of the user guide describe the use of diluted unpurified SPIA cDNA as the optimal template for qPCR reactions Where in my target sequence can I design qPCR primers The Applause WT Amp ST System amplifies the entire transcript so primers can be designed at any location within the mRNA In order to avoid interference from possible genomic DNA contamination we recommend treating RNA with DNase and designing amplicons to span an intron How many qPCR reactions will I get from one Applause WT Amp ST amplification The number of qPCR reactions depends on the abundance level of the genes being interrogated and the size of the SPIA cDNA aliquot set aside for this purpose For medium to high copy number genes the cDNA may be sub stantially diluted For very low copy number
13. and cDNA Synthesis o Reverse Transcriptase NNNNNNew WT Primer DNA RNA es AAA 3 mmm SPIA Primer DNA RNA O NNNNNNA E Gn br mm Oligo dT Primer DNA RNA NNNNNNNNN Random 9 mer Second Strand cDNA Synthesis Pat DNA Polymerase 5 Reverse Transcriptase a DNA Polymerase A RNAseH RNAseH cleavage of RNA Seguence 5 y 3 MSS SSL ND E N RNAseH SPIA Reaction Pol DNA Polymerase m SPIA Primer Linear Amplification MISS SSL Y Pol ynu 5 Sa RNAseH Cleavage to Free Primer Hybridization Site Primer Extension by Strand Displacement DNA Synthesis 5 x Primer Extension by 6 x DNA Polymerase Post SPIA Modification ST cDNA Production 5 3 SPIA Product NNNNNNNNN Random Primer Pol DNA Polymerase 5 y aad NNNNNNNNN r NNNNNNNNN Introduction 3 Applause WT Amp ST System C Performance Specifications The Applause WT Amp ST System synthesizes microgram quantities of ST cDNA start ing with a total RNA input of at least 50 ng In approximately seven hours the system produces sufficient cDNA for labeling and subsequent hybridization to Affymetrix Gene 1 0 ST Arrays The size of the majority of the cDNA products produced by the amplification process is between 0 1 and 2 0 kilobases D Quality Control Each Applause WT Amp ST System lot is tested to meet specifications of yield and array performance E Storage and Stability The Applause WT
14. cdsnessiedetevteateestestcnvesssands 7 B Using RNase free Techniques cece cece eee eeceeseeesscesseeetseeeseesesseseesetiees 8 Cse RNA SIORAGC asst dee anaa ennea E a E sect snessavstestoasdeagaeteessunnessanes 8 D Amplified cDNAStorag amp iei ii i Hydd YY ND ERFYN chess teers FI EST 8 IV Amplification Protocols csscccssssccsssseeesssecesesceeesssceesescesesesceeessseeeseseeeescseees 9 As OVerv eWa uan iKa tec INWY ad Lyn CR OEE EENE Fn SF E ENE YN Y Fn 5 9 Bi Protocol Notes m c su nuu add yd DD Gr Cyd fw FE a Do 9 C Programming the Thermal Cycler sssisescrsirriiiiicsriisriniirssirsinisn 11 D First Strand cDNA Synthesis 0 0 cee ect cee eee ese teeeseeeeeeeaeeeseseseseenseeaeeeenas 12 E Second Strand cDNA Synthesis cece eee ceeceeeceeeeeeeneeeseteseseeneeeeeeeeeas 13 F _ Post Second Strand Enhancement cccecceeeeeteeeeeeteeeeeeeeseseeesseeneeeeeeeeas 14 Ge SPIA Amplificatlon syinatsne lia dn a ID FFO FD GRH RD AEA 15 H PostSPlA Moditication llau sge uy Gi HN dyd GAN yn de CF0 16 I PostSPIA Modification llau i YG FC E a 17 Je Purification ot SI cDNAU uii eC GaL Ryd Fd FRY Y BYD Yd LO 17 K Measuring ST cDNA Yield and Purity cece cece nine nsnnsennsn sins noon 19 V Labeling Targets for Affymetrix GeneChip Gene 1 0 ST Arrays 20 A Encore M Biotin Module Overview eeeeeeieii i nnL YL Y LH YL FFF FFF Yn Fn on 20 B ProtoeolNoteSu G a GW nd CTRL Sd YF RS Cg Y dy
15. d GN 20 Ce Preparing cDNA Samples iia ii iii i BFG DF FE NF Wan 20 D Programming the Thermal Cycle eleni pandins a iiaiai 21 E elDNAFragmnentatiOn tus ice ii I Gu GY EER AN AERE AS 21 fe BiotinLabelinguisoe Gi en YN GYG God TO FYS YO CYFYD En 22 Table of Contents Applause WT Amp ST System VI Technical Support essesi vonese iienaa anaa e re a a aE 23 VINA PPONIX EE RYS FFYN ea FFY NF NWYF NHY FN 24 A Target Preparation for GeneChip Gene 1 0 ST Array Hybridization 24 B Performing Quantitative PCR on Amplified cDN A e eeeueeeeueeneieiiienen 25 C Quality Control of Amplified CDNA Product cece cece ete tceeteeeesees 26 D DNase Treatment of RNA in GRH ieaiaia yy GYF Eny 26 E Frequently Asked Questions FAOS 1 uinsn nin nnn nenn nenn dnn dnon noon 30 Fo Update Histonyu used sied wynned yD YG Fd YW did rn 34 Introduction 1 Applause WT Amp ST System A Background The Applause WT Amp ST System provides a fast simple and cost effective method for preparing amplified cDNA for global gene expression analysis on Affymetrix GeneChip Gene 1 0 ST Arrays Powered by Ribo SPIA technology a rapid simple and sensitive RNA amplification process developed by NuGEN the Applause WT Amp ST System enables the generation of microgram quantities of cDNA in approximately seven hours Amplification is initiated at the 3 end as well as randomly throughout the transcriptome ma
16. dix A VI Technical Support For Technical Support please contact NuGEN at U S only 888 654 6544 Toll Free Phone 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 europe nugeninc com In all other locations contact your NuGEN distributors Technical Support team 23 Applause WT Amp ST System VII Appendix 24 Applause WT Amp ST System A Target Preparation for GeneChip Gene 1 0 ST Array Hybridization Note Requires Affymetrix Hybridization Wash Stain HWS Kit for Gene 1 0 ST Arrays In general cDNA targets amplified using the Applause WT Amp ST System and labeled using the Encore Biotin Module are prepared for analysis on GeneChip Gene 1 0 ST Arrays according to the Affymetrix GeneChip Whole Transcript WT Sense Labeling Assay User Manual P N 701880 Rev 5 unless otherwise noted below To prepare target for a single array use a 1 5 mL microcentrifuge tube and mix at room temperature the target cDNA and hybridization cocktail components as indicated in Table 16 below Heat denature the hybridization cocktail at 99 C for 2 minutes not 5 minutes as specified by Affymetrix then follow the Affymetrix standard protocol 45 C in a heat block for 5 minutes then centrifuge at maximum speed for 1 minute just prior to loading For the GeneChip Gene 1 0 ST Arrays 169 format use a 90 uL hybridiza tion volume We rec
17. e When using this reagent take care not to splash or contaminate gloves bench or pipettes Preferably use a dedicated pipette to measure out B3 IV Amplification Protocols 11 Applause WT Amp ST System C 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 6 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 6 Thermal Cycling Protocols FIRST STRAND cDNA SYNTHESIS Program 1 Primer Annealing 65 C 5 min hold at 4 C Program 2 First Strand Synthesis 49C 1 min 25 C 10 min 42 C 10 min 70 C 15 min hold at 4 C SECOND STRAND cDNA SYNTHESIS Program 3 Second Strand Synthesis 4 C 1 min 25 C 10 min 50 C 30 min 70 C 5 min hold at 49C POST SECOND STRAND ENHANCEMENT Program 4 Post Second Strand Enhancement 4 C 1 min 37 C 15 min 80 C 20 min hold at 4 C SPIA AMPLIFICATION Program 5 SPIA Amplification 49C 1 min 47 C 90 min 95 C 5 min hold at 4 C POST SPIA MODIFICATION
18. e process uses a SPIA DNA RNA chimeric primer DNA polymerase and RNase H in a homogeneous isothermal assay that provides highly efficient amplifi cation of DNA sequences RNase H degrades RNA in the DNA RNA heteroduplex at the 5 end of the first CDNA strand This exposes a DNA sequence that is avail able for binding to the SPIA DNA RNA chimeric primer DNA polymerase initiates replication at the 3 end of the primer displacing the existing forward strand The RNA portion at the 5 end of the newly synthesized strand is again removed by RNase H exposing the unique priming site for initiation of the next round of cDNA synthesis The process of SPIA DNA RNA primer binding DNA replication strand displacement and RNA cleavage is repeated resulting in rapid accumulation of cDNA with sequence complementary to the original mRNA Introduction 2 Applause WT Amp ST System 4 Post SPIA Modification Purification and OC 3 hours The Post SPIA Modification process completes the amplification process The first step allows the random primers to anneal to the single stranded antisense cDNA target The second step utilizes DNA polymerase to extend the annealed prim ers producing ST cDNA targets appropriate for use with GeneChip Gene 1 0 ST Arrays Figure 1 The Ribo SPIA Whole Transcriptome RNA Amplification Process The Ribo SPIA WT Amp Process 5 AAAA 3 RNA yTITT mmm DNA 3 NNNNNN mmm 57 SPIA Product First Str
19. ed the following master mixes for qPCR e TaqMan ABsolute qPCR Mix plus ROX ABgene Cat AB 1136 B Fast Universal PCR Master Mix 2X Applied Biosystems Cat 4352042 VII Appendix 26 Applause WT Amp ST System e SYBR QuantiTect SYBR Green PCR Kit QIAGEN Cat 204143 iQ SYBR Green Supermix BioRad Cat 170 8880 FastStart SYBR Green Master ROX Roche Cat 04 673 514 001 Recommendations to Achieve Optimal Results 1 Dilute the Amplified Product The SPIA cDNA aliquot should be diluted 1 10 minimum of 1 4 in 1 X TE ora buffer specified by the qPCR system manufacturer A 2 uL aliquot of diluted SPIA cDNA is typically used per 25 uL qPCR reaction Depending on the abundance of the transcripts you are measuring you may wish to dilute the cDNA further than 1 10 or use lower inputs of purified SPIA cDNA It will be necessary to empirically determine the ideal input of amplified cDNA for use in a particular qPCR system 2 Primer Design We recommend using primers and probes designed with amplicon sizes as small as possible Primers may be designed at any position along a transcript since the Applause WT Amp ST process covers the entire transcript C Quality Control of Amplified cDNA Product As a quality control test you may want to analyze the size distribution of the amplified cDNA product using an Agilent Bioanalyzer Note that the shape of this distribution trace is highly dependent on the RNA source as well as i
20. f Post SPIA 2 Add 10 pL of the Post SPIA Modification II Master Mix to the Post SPIA Modification ll Master Mix to the Modification reaction 37 uL Post SPIA Modification reaction 3 Mix by pipetting 5 times with a pipette set to 30 uL spin and place on ice Total volume is 47 pL 4 Place the tubes in a pre cooled thermal cycler programmed to run Program 7 Post SPIA Modification ll see Table 6 49C 1 min 30 C 10 min 42 C 60 min 75 10 min hold at 4 C 5 Remove the tubes from the thermal cycler spin to collect condensation and place on ice 6 Continue to the Purification of ST cDNA protocol or store the cDNA at 20 C J Purification of ST cDNA The ST cDNA should be purified using OIAGEN s MinElute Reaction Cleanup Kit Catalog 28204 Instructions are for a single reaction Important notes e The ERC buffer is considered hazardous according to QIAGEN and an MSDS may be consulted e Add the appropriate amount of 100 ethanol to Buffer PE before use see bottle label for volume e All centrifuge steps are carried out at maximum speed in a conventional table top microcentrifuge at room temperature 1 Into a clean labeled 1 5 mL microcentrifuge tube add 300 pL of Buffer ERC from the OIAGEN kit 2 Add the entire volume 47 pL of the Post SPIA Modification ll reaction to the tube 3 Vortex for 5 seconds then spin briefly 17 Applause WT Amp ST System IV Amplification Protocol
21. f the Second Strand Master Mix to each First Strand reaction tube 6 Mix by pipetting 5 times spin and place on ice 7 Place the tubes in a pre cooled thermal cycler programmed to run Program 3 Second Strand cDNA Synthesis see Table 6 49C 1 min 25 C 10 min 50 C 30 min 70 C 5 min hold at 4 C 8 Remove the tubes from the thermal cycler spin to collect condensation and place on ice 9 Continue immediately with the Post Second Strand Enhancement proctocol IV Amplification Protocols Add 2 uL of Enhancement Master Mix to the 20 uL Second Strand reaction Total volume is 22 uL 14 Applause WT Amp ST System F Post Second Strand Enhancement 1 Make a master mix by combining B1 and B3 in a 0 5 mL capped tube according to the volumes shown in Table 9 Table 9 Enhancement Master Mix volumes listed are for a single reaction SECOND STRAND BUFFER MIX REACTION ENHANCEMENT MIX YELLOW B1 ver 3 YELLOW B3 ver 1 3 7 pL 0 3 pL Note In cases where the reactions will be stored at 20 C overnight prior to carrying out the Post SPIA Modification protocol make only half the required Enhancement Master Mix at this point i e use only 1 85 uL B1 and 0 15 uL B3 per reaction The other half of this master mix will need to be made fresh on day 2 We do not recom mend storing this master mix overnight for use the next day 2 3 Add 2 uL of the Enhancement Master Mix to e
22. ge tubes 0 2 mL individual thin wall PCR tubes 8 X 0 2 mL strip PCR tubes or 0 2 mL thin wall PCR plates QIAGEN MinElute Reaction Cleanup Kit Cat 28204 Disposable gloves Kimwipes Ice bucket Optional Materials e Agilent 2100 Bioanalyzer or materials and equipment for electrophoretic analysis of RNA e Real Time PCR system e Cleaning solutions such as RNaseZap Ambion Cat AM9780 and DNA OFF MP Biomedicals Cat QD0500 To Order e Ambion Inc www ambion com e MP Biomedicals www mpbio com e New England BioLabs www neb com e QIAGEN Inc www qiagen com e Sigma Aldrich Inc www sigmaaldrich com e USB Corporation www usbweb com lll Planning the Experiment A Input RNA Reguirements It is important to assess the guality of your RNA sample prior to planning your amplifi cation The Applause WT Amp ST System is designed to be used with high guality RNA samples 1 RNA Quantity The Applause WT Amp ST System is designed to use purified total RNA samples in the input range from 50 to 200 ng 2 RNA Purity Purified total RNA samples must be free of contaminating proteins and other cellu lar material organic solvents including phenol and ethanol and salts used in many RNA isolation methods Use of a commercially available system for preparation of RNA that does not require organic solvents is recommended If a method such as TRizol is used we recommend employing an additional column purification
23. ibute 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 Applause WT Amp ST System Contents k ntroductiOnu ueeeuoue eesiEocn Aeccceveseeedesvassdeestedsuesdaeccessundubiecdsssuseliccasssssotiseicesesetasee 1 As Ba ei lt o o S ao sees w RATA NAETH a i EN A EAA En REER 1 B How the Applause WT Amp ST System Works cccccescesesseesceseeeeseeeeeseeneeas 1 C Performance Specifications cece eee c cee ceee teens cseeetscsecaeeesseeecsesesesecetiees 3 D Quality Control pensiunan DF Tn FI FT E HD AUST FC 3 E Storage and Stability issseicssaceccesiceesesseehscesnseeceugespeedengsdenanedaapadeassesdsaacaseestcbssessevee 3 F Material Safety Data Sheet MSDS cccecceeceesceeceeececeeeeeceeeeeeeeeeeeeaeeeneeeeees 3 ll Kit Component ccccccccccsssssssseessesssseeeeesesssseesesseesseecessssesseeseessesseeesesseensgees 4 A Reagents and Supplies Provided ccc ccc ceeeseseeseeseeneeseeseseesenseneeeenias 4 B Additional Reagents Supplies and Equipment c cece eeeeceeeeeeeeeeeeeneees 5 lll Planning the Experiment cccccccsssssssssesssssessssseesssseesssseesseseessssseesseseeseseeens 7 A Input RNA REQUIFEIMENTS icciiccctcscesccssiesheoucadecssoesceessssa
24. ired for amplification Is there a maxi mum input The Applause WT Amp ST System can be used with high quality purified total RNA in the range from 50 to 200 ng Input amounts outside this range may produce unsatisfactory and variable results Can amplify degraded RNA with the Applause WT Amp ST System The Applause WT Amp ST System is not designed for use with degraded RNA Using compromised samples will result in unsatisfactory and variable results How much cDNA can expect from a single reaction You should expect yields of 2 5 to 5 ug for the Applause WT Amp ST System when used as directed What equipment is required or will be useful Required equipment includes a microcentrifuge pipettes vortexer thermal cycler and a UV Vis spectrophotometer An Agilent Bioanalyzer or similar instrument may be used for quality control Does the Applause WT Amp ST System provide any labeling reagents No The Applause WT Amp ST System is used to generate ST cDNA from total RNA for use in gene expression experiments The resulting ST cDNA may be processed further using the Encore Biotin Module for labeling and analysis on Affymetrix GeneChip Gene 1 0 ST Arrays Why is a single 12 reaction Encore Biotin Module kit sufficient for label ing 24 cDNA samples from the WT Amp ST System Since the Affymetrix GeneChip Gene 1 0 ST Array requires only 2 to 2 5 pg of ST cDNA the protocol for labeling employs half scale reactions of the Encore Biotin M
25. king the Applause WT Amp ST System an ideal choice for use with whole transcript array designs such as the Gene 1 0 ST Array The Applause WT Amp ST System Part No 5500 provides optimized reagent formula tions and a protocol to process total RNA samples B How the Applause WT Amp ST System Works The Applause WT Amp ST System utilizes Ribo SPIA technology that produces ampli fied cDNA from total RNA see Figure 1 1 Generation of First Strand cDNA 1 hour First strand cDNA is prepared from a minimum of 50 ng of high quality total RNA sample using a unique first strand DNA RNA chimeric primer mix and reverse transcriptase RT The primers have a DNA portion that hybridizes either to the 5 portion of the poly A seguence or randomly across the transcript RT extends the 3 DNA end of each primer generating first strand cDNA The resulting cDNA mRNA hybrid molecule contains a unique RNA sequence at the 5 end of the cDNA strand 2 Generation of a DNA RNA Heteroduplex Double stranded cDNA 1 5 hours Fragmentation of the mRNA within the cbNA mRNA complex creates priming sites for DNA polymerase to synthesize a second strand which includes DNA complementary to the 5 unique sequence from the first strand chimeric primers The result is a double stranded cDNA with a unique DNA RNA heteroduplex at one end 3 SPIA Amplification 1 5 hours SPIA amplification is a linear isothermal DNA amplification process developed by NuGEN Th
26. lowing the operating instructions provided by the manufacturer For thermal 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 13 Thermal Cycler Programming PROGRAMMING DETAILS Program 8 cDNA Fragmentation 37 C 30 min 95 C 2 min hold at 4 C Program 9 Biotin Labeling 37 C 60 min 70 C 10 min hold at 4 C E cDNA Fragmentation 1 Obtain the Fragmentation Buffer Mix Orange FL1 and Fragmentation Enzyme Mix Orange FL2 from 20 C storage 2 Spin down the contents of FL2 and FL5 and place on ice 3 Thaw the other reagents at room temperature mix by vortexing spin and place on ice 4 Add 12 5 uL of the purified ST cDNA 2 to 2 5 ug to a 0 2 mL PCR tube 5 Make a master mix by combining FL1 and FL2 in a 0 5 mL capped tube according to the volumes shown in Table 14 Table 14 Fragmentation Master Mix volumes listed are for a single reaction FRAGMENTATION BUFFER MIX ORANGE FL1 FRAGMENTATION ENZYME MIX ORANGE FL2 2 5 uL 1 uL 6 Add 3 5 uL of the Fragmentation Master Mix to each tube Mix thoroughly by pipetting 8 to 10 times spin and place on ice V Labeling Targets for Affymetrix GeneChip Gene 1 0 ST Arrays Use Labeling Master Mix immediately af
27. may measure the concentration and purity of ST cDNA with a Nanodrop using 1 absorbance unit at 260 nm of single stranded DNA 33 pg mL as the constant The purified ST cDNA may be stored at 209C V Labeling Targets for Affymetrix GeneChip Gene 1 0 ST Arrays 20 Applause WT Amp ST System A Encore Biotin Module Overview The Encore Biotin Module Part No 4200 is used to label the ST cDNA generated by the Applause WT Amp ST System in preparation for hybridization on Affymetrix GeneChip Gene 1 0 ST Arrays A single Encore Biotin Module 12 reaction kit Part No 4200 12 is sufficient to process 24 cDNA targets from the Applause WT Amp ST kit due to the use of smaller labeling reaction volumes than those recommended in the Encore Biotin Module user guide It is important to follow the protocol given below when using the Applause WT Amp ST System The cDNA labeling procedure is performed in two stages 1 cDNA fragmentation 0 5 hours 2 Biotin labeling 1 25 hours Total time to label amplified cDNA 1 75 hours B Protocol Notes e Thaw only the components used in each step and immediately place them on ice e Always keep thawed reagents and reaction tubes on ice unless otherwise instructed e 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 2 minutes at room temperature followed by brief vortexing Do not warm any enzyme mi
28. mmercial 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 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 drug purposes nor is it to be administered to humans or animals Except as expressly set forth herein no right to modify reverse engineer distr
29. nput RNA integrity We recom mend using an RNA 6000 Nano LabChip Agilent Cat 5065 4476 and the Eukaryotic Total RNA Nano program Nano assay in the Expert 2100 software following the manufacturer s instructions Depending on the availability of amplified product you may choose to load less than 100 ng of purified ST cDNA product on the Bioanalyzer chip D DNase Treatment of RNA DNase Treatment During Purification Using the QIAGEN RNase Free DNase Set and the RNeasy Mini RNA Purification Kit 1 Homogenize the sample in RLT buffer including B mercaptoethanol according to the type of sample as described in the RNeasy Mini Kit protocol 2 Add 1X volume of 70 ethanol to the homogenized lysate pipet up and down to mix sample well Do not centrifuge 3 Place an RNeasy mini column in a 2 mL collection tube VII Appendix 10 11 12 13 14 15 16 17 18 Apply the sample up to 700 uL including any precipitate that may have formed to the column Close the tube gently and centrifuge for 15 seconds at gt 8000 X g gt 10 000 rpm Discard the flow through For volumes greater than 700 pL load aliquots onto the RNeasy column succes sively and centrifuge as before Add 350 uL Buffer RW1 into the RNeasy mini column to wash and centrifuge for 15 seconds at gt 8000 X g gt 10 000 rpm Discard the flow through Add 10 uL DNase I to 70 uL Buffer RDD Gently invert the tube to mix Note Other DNase e
30. nts 5 Applause WT Amp ST System Table 4 Post SPIA Modification Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER Primer Mix S01268 Violet E1 ver 1 Buffer Mix S01269 Violet E2 ver 2 Enzyme Mix S01270 Violet E3 ver 1 Table 5 Additional Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER Nuclease free Water S01001 Green D1 Note The reagents in the Applause WT Amp ST System are similar to reagents in NuGEN other kits However unless the part numbers are identical these reagents do not have exactly the same composition and therefore are not interchangeable Do not exchange reagents between different kits as it will adversely affect performance B Additional Reagents Supplies and Eguipment Reguired Materials e Equipment Microcentrifuge for individual 1 5 mL and 0 5 mL tubes Microcentrifuge or centrifuge for individual 0 2 mL tubes strip tubes and PCR plates 0 5 10 uL pipette 2 20 uL pipette 20 200 uL pipette and 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 ora Nanodrop UV Vis Spectrophotometer e Reagents Ethanol Sigma Aldrich Cat E7023 for purification steps ll Kit Components 6 Applause WT Amp ST System e Supplies and Labware Nuclease free pipette tips 1 5 mL and 0 5 mL RNase free microcentrifu
31. nzymes we can recommend to use in this step are the Shrimp DNase recombinant from USB Corp use 10 uL or the DNase I RNase free from New England BioLabs use 10 uL See the Additional Reagent section of this user guide for ordering information Pipet the DNase incubation mix 80 uL directly onto the membrane inside the RNeasy mini column Incubate on the bench top 25 C for 15 min Add 350 uL Buffer RW1 into the RNeasy mini column and centrifuge for 15 sec onds at 28000 X g 210 000 rpm to wash Discard the flow through Transfer the RNeasy column to a fresh 2 mL collection tube Add 500 uL Buffer RPE with the added ethanol to the RNeasy column Close the tube gently and centrifuge for 15 seconds at gt 8000 X g 210 000 rpm Discard the flow through Add another 500 uL Buffer RPE to the RNeasy column Close the tube gently and centrifuge for 2 minutes at gt 8000 X g 210 000 rpm Discard the flow through Transfer the RNeasy column to a new 1 5 mL collection tube Pipet 30 50 uL RNase free water directly onto the RNeasy membrane Close the tube gently and centrifuge for 1 minute at gt 8000 X g 210 000 rpm to elute If yields of greater than 30 ug are expected repeat elution step and collect in the same collection tube DNase Treatment of RNA Post Purification Using RNase free DNase and Either the RNA Clean and Concentrator 5 Columns or the RNeasy MinElute Columns Note If you are unable to quantify y
32. odule This means that a single 12 reaction Encore Biotin Module will perform 24 reactions when used with the Applause WT Amp ST System protocol VII Appendix 31 Applause WT Amp ST System Q9 Q10 Q11 Q12 Q13 Q14 Q15 Q16 Q17 Q18 Where can I safely stop in the protocol You may stop immediately following the SPIA Amplification protocol or after Post SPIA Modification ll protocol prior to final cleanup at the points specifi cally noted in the protocol Store reaction products at 209C What are the recommended storage conditions for the Applause WT Amp ST System components All components of the system may be stored at 20 C Ensure the vials are well sealed and do not exceed 6 freeze thaw cycles What are the recommended storage conditions for the amplified ST cDNA The amplified ST cDNA may be stored at 20 C Can DNA be used as input for the Applause WT Amp ST System No The Applause WT Amp ST System is designed to amplify mRNA not DNA Has NuGEN performed reproducibility studies on the Applause WT Amp ST System Yes Sample to sample lot to lot and operator to operator reproducibility studies are routinely conducted according to NuGEN S internal Quality Control metrics Can contaminating genomic DNA interfere with the performance of the Applause WT Amp ST System Yes In high quantities genomic DNA will interfere with amplification Does NuGEN recommend DNase treatment of pu
33. ommend a hybridization time of 18 hours 2 hours Hybridization times within this range yield comparable results Use fluidics protocol FS450_0007 on the GeneChip Fluidics Station 450 See Table 16 VII Appendix 25 Applause WT Amp ST System Affymetrix P N 900720 Table 16 Hybridization Cocktail Assembly and Fluidics Protocol for GeneChip Gene 1 0 ST Arrays using the Affymetrix HWS Kit GENE 1 0 ST FINAL COMPONENT ARRAY 169 FORMAT CONCENTRATION Fragmented biotin labeled amplified 25 uL 18 2 22 7 ng pL cDNA Control oligonucle otide B2 3 nM neue eM 20X Eukaryotic hybrid 1 5 5 25 and ization controls bioB 5 5 uL 100 oM eval bioC bioD cre elena ey 2x Hybridization buffer as ne 100 DMSO 11 uL 10 Water 11 6 uL Final Volume 110 uL FLUIDICS PROTOCOLS Gene 1 0 ST Array FS450_0007 B Performing Ouantitative PCR on Amplified cDNA The amplified SPIA cDNA produced by the Applause WT Amp ST System has been suc cessfully used as template for qPCR systems including TaqMan and SYBR Green For optimum results in gPCR applications an aliquot of the amplified SPIA cDNA should be removed prior to Post SPIA Modification where specified and used as template for qPCR as described here Refer to pg 15 step IV G 9 Note RT PCR master mixes containing the enzyme Uracil N Glycosylase UNG are not compatible with the Applause WT Amp ST System We have successfully us
34. on tube h Add 10 pL nuclease free water green D1 directly to the center of the filter in the tube and close the cap Do not use cold water i Spin for 1 minute at gt 8000 X g gt 10 000 rpm to collect the purified RNA Purification with OIAGEN RNeasy MinElute Cleanup Columns QIAGEN Cat 74204 a Add 80 uL ice cold nuclease free water D1 green cap to the sample on ice b Add 350 uL Buffer RLT and mix by pipetting c Add 250 uL 96 100 ethanol and mix thoroughly by pipetting d Place an RNeasy MinElute Spin Column into a 2 mL collection tube one column per sample and apply the 700 pL sample to the column e After closing the column spin for 15 seconds at gt 8000 X g gt 10 000 rpm and discard the flow through f Place the RNeasy MinElute Spin Column into a fresh 2 mL collection tube Add 500 uL Buffer RPE to the column and close the tube Spin for 15 seconds at VII Appendix Best results can be obtained by using fresh 8026 ethanol in the wash step Lower percent ethanol mixes will reduce recovery Use nuclease free water at room temperature to elute sample 29 Applause WT Amp ST System 28000 X g 210 000 rpm and discard the flow through keeping the same collection tube g Add 500 uL 80 ethanol to the RNeasy MinElute Spin Column and close the j tube Note Use fresh 8096 ethanol Lower percent ethanol mixes will reduce recovery Spin for 2 minutes at gt 8000 X g gt 10
35. our RNA because the sample is contaminated with DNA we recommend DNase treatment followed by purification 27 Applause WT Amp ST System VII Appendix Use nuclease free water at room temperature to elute sample 28 Applause WT Amp ST System On ice mix together 2 5 uL 10X DNase Reaction buffer Roche Cat 04716728001 or USB PN 78316 with 1 uL rDNase 10 Units Roche Cat 04716728001 or 2 Units USB PN 78311 Add RNA sample up to 500 ng and add nuclease free water D1 green cap to bring the final volume to 25 pL Incubate at 25 C for 15 minutes followed by 37 C for 15 minutes and return to ice After the DNase treatment the sample must be purified We recommend either of the two purification procedures below Purification with RNA Clean and Concentrator 5 Zymo Research Cat R1015 a Add 4 volumes 100 uL of RNA binding buffer to the sample b Obtain one RNA Clean and Concentrator 5 column and apply sample to column c Spin column for 30 seconds at gt 8000 X g gt 10 000 rpm and discard the flow through d Add 200 uL wash buffer with ethanol added as per vendor s specifications e After closing the column spin for 30 seconds at gt 8000 X g gt 10 000 rpm and discard the flow through f Add 200 uL fresh 8096 ethanol close cap spin for 30 seconds at gt 8000 X g 210 000 rpm and discard the flow through g Place the RNA Clean and Concentrator 5 column in a fresh 1 5 mL collecti
36. r green D1 from the NuGEN kit to the center of each column Note Ensure that the water is dispensed directly onto the membrane for complete elution of bound cDNA Let the column stand for 1 minute at room temperature Centrifuge for 1 minute at maximum speed If two columns were used per sample pool the eluates Discard the column and measure the volume recovered There should be approxi mately 12 to 15 uL of purified SPIA cDNA Mix the sample by vortexing then spin briefly Continue to the Measuring ST cDNA Yield and Purity protocol or store purified ST cDNA at 20 C IV Amplification Protocols 19 Applause WT Amp ST System K Measuring ST cDNA Yield and Purity Note You must purify the ST cDNA before measuring yield and purity iE Mix the purified ST cDNA sample by brief vortexing and spinning prior to checking the concentration Measure the absorbance at 260 280 and 320 nm of your ST cDNA product You may need to make a 1 20 dilution of the ST 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 absorbance unit at 260 nm of single stranded DNA 33 pg mL To calculate A260 A320 of diluted sample X dilution factor X 33 concentration in pg mL of a 1 absorbance unit solution X 0 03 final volume in mL total yield in micrograms Alternatively you
37. rified total RNA samples Yes For DNase treatment of RNA samples refer to Appendix D of the user guide Can I use the Applause WT Amp ST System on bacterial RNA samples The amplification process theoretically will work with many bacterial species however the kit has not been optimized for this purpose and NuGEN cannot guarantee success with such samples Can I use the Applause WT Amp ST System for archiving cDNA Yes Amplified cDNA may be safely stored at 20 C for six months or longer How do I guantitate the amplified cDNA product You may use a standard UV Vis spectrophotometer or a NanoDrop Be sure to use the single stranded cDNA conversion factor of 1 A260 unit 33 ng pL in calculating the amplified cDNA concentration as this is the convention we used in establishing yield guidelines in this user guide VII Appendix 32 Applause WT Amp ST System Q19 Q20 Q21 Q22 Q23 Q24 Q25 Why do I need to use the single stranded cDNA conversion factor when converting my A260 reading to cDNA concentration The amplified cDNA product of the Applause WT Amp ST Systems consists of both sense and antisense cDNA strands While there may be some double stranded character to this mixture we have developed and optimized the kit using the single stranded cDNA conversion factor 1 A260 unit 33 ng uL of cDNA Expected cDNA yield from amplification and input into labeling protocols cited in the product materials
38. s 100 ethanol must be El added to the QIAGEN Buffer PE upon first use Failure to do so will result in low amplification yields Use nuclease free water at room temperature to elute sample 18 Applause WT Amp ST System 30 ON 9 oe 10 11 12 13 14 15 16 17 18 19 20 Obtain and label a MinElute spin column and place it into a collection tube Load the entire volume of sample buffer mixture onto the column Centrifuge for 1 minute at maximum speed in a microcentrifuge Discard the flow through and replace the column in the same collection tube Add 750 uL of Buffer PE the column Centrifuge for 1 minute at maximum speed Discard the flow through and replace the column in the same collection tube Centrifuge for an additional 2 minutes at maximum speed to remove all residual Buffer PE Note Residual ethanol from the wash buffer will not be completely removed unless the flow through is discarded before this additional centrifugation Discard the flow through with the collection tube Blot the column onto clean absorbant paper to remove any residual wash buffer from the tip of the column Note Blotting the column tip MUST be done prior to transferring the column to a clean tube Failure to do so may result in a small guantity of wash buffer in your final eluted sample Place the column into a clean labeled 1 5 mL microcentrifuge tube Add 15 uL of room temperature nuclease free wate
39. t amplifica tion issues DNase Treatment The use of DNase treatment is highly recommended when using purified RNA samples Contaminating genomic DNA will interfere with accurate quantitation of RNA samples and may negatively impact detection sensitivity and data quality Refer to the Appendix for examples of DNase treatment protocols that have been used successfully Carrier use for RNA isolation We strongly recommend against the use of nucleic acid based carriers during RNA purification because many have been shown to produce cDNA product in first strand synthesis We also advise against the use of glycogen in RNA isolation as it inhibits reverse transcription For the latest information regarding other carriers contact our technical services team Using RNase free Techniques RNase contamination through reagents and work environment will lead to experimental failure Follow these guidelines to minimize RNases in the workspace iP 2 3 4 ail C Wear disposable gloves and change them frequently Avoid touching surfaces or materials that could introduce RNases Use reagents provided Substitutions may introduce RNases Clean work areas and instruments including pipettes with commercially available cleaning reagents such as RNaseZap Use only new RNase free pipette tips and microcentrifuge tubes Use a work area specifically designated for RNA work and do not use other high copy number materials in the same area
40. ter preparation Mix by pipetting and spin down the master mix briefly at low speed Place on ice Use master mix immediately Add 9 uL of Labeling Master Mix to the 16 uL Fragmentation reaction Total volume is 25 pL 22 Applause WT Amp ST System 10 Place the tubes in a pre warmed thermal cycler programmed to run Program 8 cDNA Fragmentation see Table 13 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 Make a master mix by combining FL3 FL4 and FL5 in a 0 5 mL capped tube according to the volumes shown in Table 15 Table 15 Labeling Master Mix volumes listed are for a single reaction LABELING BUFFER MIX LABELING REAGENT LABELING ENZYME MIX ORANGE FL3 ORANGE FL4 ORANGE FL5 7 5 uL 0 75 uL 0 75 uL Add 9 uL of the Labeling Master Mix to each cDNA Fragmentation reaction tube Mix thoroughly by pipetting 8 to 10 times spin and place on ice Place the tubes in a pre warmed thermal cycler programmed to run Program 9 Labeling see Table 13 37 C 60 min 70 C 10 min hold at 4 C Remove the tubes from the thermal cycler spin to collect condensation and place on ice The labeled cDNA may be used immediately for array hybridization or stored at 209C For recommendations on array hybridization refer to Appen
41. ume in the SPIA Amplification and Post SPIA IV 15 17 Modification steps to stabilize yield Add margin notes tracking reaction volumes throughout 2 9 3 IV and V 12 23 protocol NuGEN Technologies Inc Headquarters USA Europe 201 Industrial Road Suite 310 P O Box 149 For our international distributors contact San Carlos CA 94070 USA 6680 AC Bemmel information visit our website Toll Free Tel 888 654 6544 The Netherlands Toll Free Fax 888 296 6544 Tel 31 13 5780215 sla a custserv nugeninc com Fax 31 13 5780216 techserv nugeninc com europe nugeninc com 2009 2011 NuGEN Technologies Inc All rights reserved The Ovation and Applause families of products and methods are covered by U S Patent Nos Patent Nos 6 692 918 6 251 639 6 946 251 and 7 354 717 and other issued and pending patents in the U S and other countries NuGEN the NuGEN logo Ovation SPIA Ribo SPIA WT Ovation Applause Encore Prelude 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 M01134 v4
42. w reagents for all steps at once The reagent color coding can be a guideline for appropriate reagent grouping Always keep thawed reagents and reaction tubes on ice unless otherwise instructed After thawing and mixing buffer mixes in rare instances a precipitate is observed It is important that it be re dissolved 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 or primer mixes When placing small amounts of reagents into the reaction mix pipet up and down several times to ensure complete transfer When instructed to pipet mix gently aspirate and dispense a volume that is at least half of the total volume of the reaction mix Always allow the thermal cycler to reach the initial incubation temperature prior to placing the tubes or plates in the block IV Amplification Protocols 10 Applause WT Amp ST System When preparing master mixes use the minimal amount of extra material to ensure there are sufficient reagents for 24 reactions Typically an overage factor of 10 is acceptable For example if making a master mix for eight reactions use a factor of 8 8x when calculating the master mix volumes Components and reagents from other Ovation System WT Ovation System or Applause System products should not be used with this product Caution The Enhancement Enzyme Mix B3 patent pending contains a heat labile RNase enzym
43. xes e FL3 labeling buffer may appear to have pink coloration this is normal e The reagent volumes recovered greatly depend on the number of batches pro cessed with each kit Set up no fewer than eight reactions at a time e When placing small amounts of reagents into reaction mix gently pipet up and down several times to ensure complete transfer e When instructed to pipet mix gently aspirate and dispense a volume at least half of total reaction mix volume Repeat a minimum of five times to ensure complete mixing e Allow the thermal cycler to reach the initial incubation temperature before plac ing samples in the block C Preparing cDNA Samples Purified ST cDNA from the Applause WT Amp ST System is ready for labeling with the Encore Biotin Module protocol Use 2 to 2 5 ug of ST CDNA per sample for hybridization to Affymetrix GeneChip Gene 1 0 ST Arrays V Labeling Targets for Affymetrix GeneChip Gene 1 0 ST Arrays Use Fragmentation Master Mix immediately after preparation Mix by pipetting and spin down the master mix briefly Place on ice Use master mix immediately Add 3 5 uL of Fragmentation Master Mix to the 12 5 uL of purified ST cDNA Total volume is 16 uL 21 Applause WT Amp ST System D Programming the Thermal Cycler Use a thermal cycler with a heat block designed for 0 2 mL tubes equipped with a heated lid with a capacity of 100 uL reaction volume Prepare the 2 programs shown in Table 13 fol
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