Applause® WT-Amp ST System
Contents
1. Vil Appendix Use nuclease free water at room temperature to elute sample Best results can be obtained by using fresh 80 ethanol in the wash step Lower percent ethanol mixes will reduce recovery 28 Applause WT Amp ST System 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 8000 X g 210 000 rpm and discard the flow through Add 200 uL wash buffer with ethanol added as per vendor s specifications After closing the column spin for 30 seconds at 8000 X g 210 000 rpm and discard the flow through Add 200 uL fresh 80 ethanol close cap spin for 30 seconds at 8000 X g 210 000 rpm and discard the flow through Place the RNA Clean and Concentrator 5 column in a fresh 1 5 mL collection tube 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 Spin for 1 minute at 8000 X g 210 000 rpm to collect the purified RNA Purification with QIAGEN RNeasy MinElute Cleanup Columns QIAGEN Cat 74204 a b C Add 80 uL ice cold Nuclease free Water D1 green cap to the sample on ice Add 350 uL Buffer RLT and mix by pipetting Add 250 uL 96 100 ethanol and mix thoroughly by pipetting Place an RNeasy2. C 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 QIAGEN 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 uL of Buffer ERC from the OIAGEN kit Add the entire volume 47 uL of the Post SPIA Modification II reaction to the tube Vortex for 5 seconds then spin briefly 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 xa e w N Centrifuge for 1 minute at maximum speed in a microcentrifuge IV Amplification Protocols 100 ethanol must be E 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 10
3. Protocol Sections IV H amp K 4 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 5 Remove the tubes from the thermal cycler spin to collect condensation and place on ice 6 Continue immediately with the SPIA Amplification protocol G SPIA Amplification 1 Obtain the SPIA Buffer Mix red C2 SPIA Primer Mix red C1 and SPIA Enzyme Mix red C3 from 20 C storage 2 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 3 Thaw reagents C1 and C2 at room temperature mix by vortexing spin and place on ice 4 Make a master mix by sequentially 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 aliquoting IV Amplification Protocols 15 Applause WT Amp ST System Table 10 SPIA Master Mix volumes listed are for a single reaction SPIA BUFFER MIX SPIA PRIMER MIX SPIA ENZYME MIX RED C2 ver 8 RED C1 ver 8 RED C3 ver 5 2 uL 2 uL 4 uL 5 Add 8 uL of the SPIA Master Mix to the Post Second Strand Enhancement reaction 6 Mix by pipetting 5 times spin and place on ice 7 Place the tubes in a pre cooled therm
4. pipetting 5 times spin and place on ice Place the tubes in a pre cooled thermal cycler programmed to run Program 3 Second Strand cDNA Synthesis see Table 6 4 C 1 min 25 C 10 min 50 C 30 min 70 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 Second Strand Enhancement proctocol Post Second Strand Enhancement Make a master mix by combining B1 and B3 in a 0 5 mL capped tube according to the volumes shown in Table 9 IV Amplification Protocols 14 Applause WT Amp ST System 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 uL 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 Add 2 uL of the Enhancement Master Mix to each Second Strand reaction tube 3 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
5. to 2 5 ug of ST cDNA the protocol for labeling employs half scale reactions of the Encore Biotin Module This means that a single 12 reaction Encore Biotin Module will perform 24 reactions when used with the Applause WT Amp ST System protocol Vil Appendix 33 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 II protocol prior to final cleanup at the points specifi cally noted in the protocol Store reaction products at 20 C 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 quan
6. vortexing then spin briefly Continue to the Measuring ST cDNA Yield and Purity protocol or store purified ST cDNA at 20 C Measuring ST cDNA Yield and Purity Note You must purify the ST cDNA before measuring yield and purity 1 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 IV Amplification Protocols 19 Applause WT Amp ST System 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 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 20 C 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 hy
7. 11 12 13 14 15 16 17 18 19 20 K 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 quantity 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 Water 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
8. AND BUFFER MIX FIRST STRAND ENZYME MIX BLUE A2 ver 3 BLUE A3 ver 1 2 5 uL 0 5 pL 10 Add 3 uL of the First Strand Master Mix to each tube 11 Mix by pipetting 5 times spin and place on ice IV Amplification Protocols Do not return B1 ver 3 buffer to the freezer as it is required for the next step as well 13 Applause WT Amp ST System 12 13 14 Place the tubes in a pre cooled thermal cycler programmed to run Program 2 First Strand cDNA Synthesis see Table 6 4 C 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 Second Strand cDNA Synthesis 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 Spin down the contents of B2 and B3 and place on ice Thaw reagent B1 at room temperature mix by vortexing spin and place on ice 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 ver 2 9 75 uL 0 25 uL Add 10 uL of the Second Strand Master Mix to each First Strand reaction tube Mix by
9. Applause WT Amp ST System 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 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 commercial service
10. C SPIA AMPLIFICATION Program 5 SPIA Amplification 4 C 1 min 47 C 90 min 95 C 5 min hold at 4 C POST SPIA MODIFICATION Program 6 Post SPIA Modification 4 C 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 12 Applause WT Amp ST System D First Strand cDNA Synthesis o a ooa e 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 STR
11. DNA RNA hetero duplex at the 5 end of the first cDNA strand This exposes a DNA sequence that is available for binding to the SPIA DNA RNA chimeric primer DNA polymerase initi ates 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 Post SPIA Modification Purification and QC 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 primers 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 AAAA 3 RNA 3 TTTT ms 3 NNNNNN um 57 First Strand cDNA Synthesis RT Reverse Transcriptase 5 AAAA 3 On NNNNNNA 7 ow saw DNA SPIA Product NNNNNN WT Primer DNA RNA m SPIA Primer DNA RNA mmm Oligo dT Primer DNA RNA NNNNNNNNN Random 9 mer Seco
12. Master Mix volumes listed are for a single reaction ENHANCEMENT PRIMER MIX MASTER MIX VIOLET E1 ver 1 2uL 5 ul 6 Add 7 ul of the Post SPIA Modification Master Mix to the SPIA Amplification reaction 7 Mix by pipetting 5 times with a pipette set to 20 uL spin and place on ice 8 Place the tubes in a pre cooled thermal cycler programmed to run Program 6 Post SPIA Modification see Table 6 4 C 1 min 37 C 15 min 95 C 5 min hold at 4 C 9 Remove the tubes from the thermal cycler spin to collect condensation and place on ice 10 Continue immediately with the Post SPIA Modification II protocol l Post SPIA Modification II 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 ee the volumes shown in Table 12 pipette this mix slowly into the mix 16 Applause WT Amp ST System IV Amplification Protocols 17 Applause WT Amp ST System Table 12 Post SPIA Modification Il Master Mix volumes listed are for a single reaction BUFFER MIX ENZYME MIX VIOLET E2 ver 2 VIOLET E3 ver 1 5 ul 5 L 2 Add 10 uL of the Post SPIA Modification Il Master Mix to the Post SPIA Modification reaction 3 Mix by pipetting 5 times with a pipette set to 30 uL spin and place on ice 4 Place the tubes in a pre cooled thermal cycler programmed to run Program 7 Post SPIA Modification Il see Table 6 4
13. MinElute Spin Column into a 2 mL collection tube one column per sample and apply the 700 uL sample to the column After closing the column spin for 15 seconds at 28000 X g 210 000 rpm and discard the flow through 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 28000 X g 210 000 rpm and discard the flow through keeping the same collection tube Add 500 uL 80 ethanol to the RNeasy MinElute Spin Column and close the tube Note Use fresh 80 ethanol Lower percent ethanol mixes will reduce recovery Spin for 2 minutes at 28000 X g 210 000 rpm and discard the flow through Vil Appendix Use nuclease free water at room temperature to elute sample 29 Applause WT Amp ST System i 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 8000 X g 10 000 rpm and discard the flow through j 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 28000 X g 10 000 rpm to collect the purified RNA E Preventing Non specific Amplification Due to the high sensitivity inherent in our amplification systems we have developed a set of reco
14. 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 recommend 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 Vil Appendix 25 Applause WT Amp ST System Table 16 Hybridization Cocktail Assembly and Fluidics Protocol for GeneChip Gene 1 0 ST Arrays using the Affymetrix HWS Kit Affymetrix P N 900720 COMPONENT GENE 1 0 ST ARRAY 169 FORMAT FINAL CONCENTRATION Fragmented biotin labeled amplified cDNA 25 uL 18 2 22 7 ng pL Control ae B2 18 UL 50 pM 20X Eukaryotic
15. a good idea to keep a clean set of pipettes as a backup d Always wear gloves and don fresh gloves upon entry into this controlled area Frequently change gloves while working in the pre amplification area espe cially prior to handling stock reagents reactions and RNA samples e Stock this area with clean preferably new equipment pipettes racks consum ables that has not been exposed to post amplification workspace f Make it a policy to carry out regular cleaning of all workspaces g Capture waste generated in both pre and post amplification areas tips col umns wash solutions from beads and columns tubes everything in sealable plastic bags and dispose of promptly after each experiment to avoid waste spillage h Do not open amplified product reaction vessels in the pre amplification workspace Avoid running negative controls i e no RNA input reactions Instead use low template controls inputs of 50 pg to 100 pg in order to detect and monitor any non specific amplification issues The clearest indication that non specific amplifi cation is taking place is the appearance of higher than expected yields or irregular Bioanalyzer traces in a low template control LTC reaction a Typical amplification performance i LTC yields for Applause WT Amp ST System amplifications should be signifi cantly lower than yields for RNA inputs within the recommended input range of 50 ng to 200 ng ii The Bioanalyzer trace
16. al cycler programmed to run Program 5 SPIA Amplification see Table 6 4 C 1 min 47 C 90 min 95 C 5 min hold at 4 C 8 Remove the tubes from the thermal cycler spin to collect condensation and place on ice Do not re open the tubes in the pre amplification workspace 9 Optional If qPCR will be performed on the amplification products remove an aliquot of the SPIA cDNA at this point 10 Continue immediately with the Post SPIA Modification protocol or store the SPIA reactions at 20 C overnight prior to continuing IV Amplification Protocols H Post SPIA Modification 1 Obtain the Primer Mix Violet E1 Buffer Mix Violet E2 and Enzyme Mix Violet E3 from 20 C storage 2 Spin down the contents of E3 and place on ice 3 Thaw E1 and E2 at room temperature mix by vortexing spin and place on ice 4 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 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 5 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
17. bridization 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 mixes 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 Repea
18. cations 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 on the NuGEN website at http www nugeninc com nugen index cfm support user guides 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 Pa strano Primer S01262 Blue A1 ver 4 bie strand Buffer 501256 Blue A2 ver 3 sirana Enzyme 501040 Blue A3 ver 1 Table 2 Second Strand cDNA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER 0 501257 Yellow B1 ver 3 second Stand 501126 Yellow B2 ver 2 Enzyme Mix Eanes A 501119 Yellow B3 ver 1 Table 3 SPIA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER SPIA Primer Mix 501264 Red C1 ver 8 SPIA Buffer Mix 501259 Red C2 ver 8 SPIA Enzyme Mix 01261 Red C3 ver 5 Il Kit Components 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 01270 Violet E3 ver 1 Table 5 Additional Reag
19. com Fax 31 13 5780216 techserv nugeninc com europe 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 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 For research use only M01134 v5 1
20. csassncsssncccasenscacecvadessonasdesducassnasdsvanascenssessensasedsnvacvoaneess 23 VIL Appendix 22 ss0 224s224002024088040020240880000404240 0000 0n040n00s00dnenahtankhenesahtenuesnsseng unse 24 A Target Preparation for GeneChip Gene 1 0 ST Array Hybridization 24 B Performing Quantitative PCR on Amplified CDNA eneneneen 25 C Quality Control of Amplified CDNA Product sesennsennnenenennenenn 26 D DNase Treatment of RNA unse sea een 26 E Preventing Non specific Amplification eeenenenennnnnen 29 F Frequently Asked Questions FAQS ueesenesenesenenennnennnnnnnennn 32 G Update Histon esio rna EA AEE 35 l 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 making 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 f
21. cted cDNA yield from amplification and input into labeling protocols cited in the product materials 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 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 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 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
22. ding 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 20 C For recommendations on array hybridization refer to Appendix A VI Technical Support 23 Applause WT Amp ST System For help with any of our products please contact NUGEN Technical Support at 650 590 3674 direct or 888 654 6544 option 2 toll free U S only You may also send faxes to 888 296 6544 toll free 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 distributor for technical support Vil 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
23. e 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 1 2 3 4 gi 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 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 Over
24. ed cDNA product including bead removal final purification post SPIA modification array hybridization and any other analytical work c Ideally the pre amplification workspace will be in a separate room If this is not possible ensure the pre amplification area is sufficiently isolated from post amplification work d PCR Workstation enclosures with UV illumination for use as pre amplification workspaces can be an option in situations where conditions preclude physical separation of pre and post amplification activities 2 Establish and maintain a clean work environment Vil Appendix 30 Applause WT Amp ST System a Initially clean the entire lab thoroughly with DNA OFF Follow this treatment with a thorough rinse with water to ensure no residual cleaning agents are left behind b In the pre amplification area remove all small equipment and then clean every surface that may have been exposed to amplified SPIA cDNA surfaces drawer handles key pads etc Before reintroducing any equipment clean every piece of equipment thoroughly e Clean thermal cycler blocks by heating to 99 C for 15 minutes then wipe down exposed surfaces and keypad with cleaning solution e Clean magnets by immersion in cleaning solution or use a cotton swab c Carry out a thorough external and internal cleaning of all pipettes with DNA OFF Carefully follow the manufacturer instructions for this process to avoid damaging the pipettes It is
25. entrifuge tubes 0 2 mL PCR tubes and plates RNaseZap and DNA OFF What is the minimum input required 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
26. ents 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 s 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 Equipment Required 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 microcentrifuge 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 electrophor
27. ents 8 D Amplified cDNA Storage u eu neben 8 IV Amplification Protocolsi uses an ae uralte ale 9 A OveriieW aneenn ans ri ENER p a a ENE SS ESNA 9 B Protocol NOtES rederne e a e ea e Eaa 9 C Programming the Thermal Cycler ics ccscccsncsspesiesccsstesncecssstovnes sansa nenn 11 D First Strand CDNA Synthesis suc sc sascssmeesapdeseiesanstencosgyendeodessesstasussedesientesdagsues 12 E Second Strand cDNA Synthesis 0 00 eee eee cee ete teseeeeeseseeeseeeneeeeeeeeas 13 F Post Second Strand Enhancemernt esnsenenesneensenennenesneennnnennee nenn 13 Ge SPIAR Amplifieation zusenden 14 Ho Post SPIA Modification sessioni act 5er ann 16 I PostSPIA Modification Inseln ala 16 J Purification of SISCDNA vc cse cevs cucvsenasaticasevesacentdeguasveascesuedsarsuspaitad nu al 17 K Measuring ST cDNA Yield and Purity ueesesesesesensennensensensennennennennnnnn 18 V Labeling Targets for Affymetrix GeneChip Gene 1 0 ST Arrays sscssseseeees 20 A Encore Biotin Module Overview nnsennensennennsennenneena 20 B ProtocollNotes serorei nien E E Gael he a aa 20 Ce Preparing cDNA Samples u a kennen eleenn 20 D Programming the Thermal Cycler eseseneenenneneennennne nennen 21 Ex eDN A FrFAgMENTAtlONn zu cenecacdvesgs aduan gees sagcadsedeevenccc avs A EEES 21 E Biotin Label Girsscassctes seas seta nalen bank 22 Table of Contents VI Technical Support cc scicescs
28. etic 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 giagen com e Sigma Aldrich Inc www sigmaaldrich com e USB Corporation www usbweb com Ill Planning the Experiment 7 Applause WT Amp ST System A Input RNA Requirements It is important to assess the quality of your RNA sample prior to planning your amplifi cation The Applause WT Amp ST System is designed to be used with high quality 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 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 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 i
29. fer 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 MNEFOMUCHON ss scsssssscsscacsssssessssvssasesssesssstecasessessasssssoosesassvsasssosussesasesssvscassossseasssssess 1 PAG Backgrounder E R E E A E E SPEER IPEETER LE 1 B How the Applause WT Amp ST System Works uesesenennesnennennennennennennennn 1 C Performance Specifications ueneesesennesennenennnnennnnennnnnnnnnnnnnnnennnnennnennn nenn 3 D Quality Control sense nn 3 E Storage and Stability ersehen nennen 3 F Material Safety Data Sheet MSDS neeeeneenneeneenneenen 3 ll Kit Components 22sssssssessennssonsennnnnnsonsennnnnnnonsssnnnnsnnnssensnsssnnssensnssnsnssensnssnnnnne 4 A Reagents and Supplies Provided u u uu u naienenrneienennn 4 B Additional Reagents Supplies and Equipment uneeeeenenenenennennenn 5 Il Planning the Experiment 4 224222452 525 800050 42248 28420042208 00020 0480088 04000458 anaser 7 A Input RNA Requirements 3 ccsscndcesteaiesascssnsasedeestaivessanssentenessenestisa arrainean 7 B Using RNase free Techniques c ccc e cece eects cee eecessceeseesetsesetseseseneceeaees 8 Ge TRINA SIOTAG SG Maren nana nA dons na te ninesientenies cansbunsssadesauecesunsv
30. fication reactions The two steps to accomplish this are 1 Designating separate workspaces for pre amplification and post amplification steps and materials and 2 Implementing routine clean up protocols for workspaces as standard operating procedure A detailed set of these recommendations is given in the Appendix 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 4 C 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 4 C 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
31. gmentation 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 7 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 after preparation Mix by pipetting and spin down EI the master mix briefly at low speed Place on ice Use master mix immediately 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 accor
32. hybridization 1 5 5 25 and 100 pM controls bioB bioC bioD cre le respectively 2x Hybridization buffer 55 uL 1X 100 DMSO 11 uL 10 Water 11 6 uL Final Volume 110 uL FLUIDICS PROTOCOLS Gene 1 0 ST Array FS450_0007 We have successfully used the following master mixes for qPCR B Performing Quantitative 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 qPCR 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 e TaqMan ABsolute qPCR Mix plus ROX ABgene Cat AB 1136 B Fast Universal PCR Master Mix 2X Applied Biosystems Cat 4352042 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 Vil Appendix 26 Applause WT Amp ST System 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 d
33. iluted 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 input 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 RNea
34. medium to high copy number genes the cDNA may be sub stantially diluted For very low copy number genes you may need to use more cDNA per reaction What is the recommended minimum batch size 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 Vil Appendix E Q27 Can 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 G Update History This document the Applause WT Amp ST System user guide M01134 v5 1 is an update to address the following topics Description Section Page s Updated SPIA technology description 1B 1 Updated contact information for NUGEN Technical Support Vi 23 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 The Netherlands U Toll Free Fax 888 296 6544 Tel 31 13 5780215 un imagine more from less custserv nugeninc
35. mmendations designed to minimize the potential for the generation of non specific amplification products through the carry over of previously amplified SPIA cDNA We strongly recommend implementing these procedures especially for the high throughput and low RNA input environments typical in today s gene expression laboratories We have two general recommendations First designate separate workspaces for pre amplification and post amplification steps and materials This provides the best work environment for processing RNA using our highly sensitive amplification protocols Our second recommendation is to implement routine clean up protocols for workspaces as standard operating procedure This will prevent non specific amplifica tion products from spreading through the laboratory Details regarding establishing and maintaining a suitable work environment are listed below 1 Designate a pre amplification workspace separate from the post amplification workspace or general lab areas a Pre amplification includes all steps and materials related to RNA sample handling and dilution NuGEN s first strand reaction second strand reac tion second strand cleanup and SPIA amplification reaction setup After SPIA incubation the reactions are immediately removed from the pre amplification workspace and opened only in the post amplification area b Post amplification includes all steps and materials related to the handling of the final amplifi
36. n poor amplification 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 f all Ut l a at in a RNA Quality Continuum ze fer TIEI Poor Quality Moderate Quality Good Quality RIN 2 4 RIN 6 7 RIN 9 2 4 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 Ill Planning the Experiment 8 Applause WT Amp ST System B trol RNA amplification it may be difficult or impossible to troubleshoot 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 DNas
37. nd Strand cDNA Synthesis Pol DNA Polymerase RT Reverse Transcriptase Pol DNA Polymerase A RNAseH 5 3 3 5 RNAseH cleavage of RNA Sequence 5 Eu aan Ay RNAseH SPIA Reaction Pol DNA Polymerase um SPIA Primer Amplification SRR Rn as RNAseH Cleavage to Free Primer Hybridization Site Primer Extension by Strand Displacement DNA Synthesis 5 3 Primer Extension by Gn Bi ET 5 DNA Polymerase 5 7 P a Post SPIA Modification ST cDNA Production 5 3 SPIA Product NNNNNNNNN Random Primer Pol DNA Polymerase 5 BW NNNNNNNINN Ble NNNNNNNNN 3 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 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 specifi
38. of the LTC amplification product is consistent with that seen with higher input b Atypical amplification performance i LTC yields may be similar to those obtained using higher inputs of total RNA Vil Appendix ii The Bioanalyzer traces of amplification products may look significantly dif ferent than the typical Applause WT Amp ST System reaction traces The LTC reaction is designed to be an especially sensitive indicator of atypical amplification performance iii Sensitivity on arrays may be lower than expected iv Contact NUGEN Technical Services when atypical performance is suspected 31 Applause WT Amp ST System Vil Appendix 32 Applause WT Amp ST System F Frequently Asked Questions FAQs Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 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 microc
39. ormula 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 sequence 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 cCDNA 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 is a robust isothermal strand displacement amplification process developed by NuGEN The process uses a SPIA DNA RNA chimeric primer DNA polymerase and RNase H in a homogeneous isothermal assay that provides highly efficient amplification of DNA sequences RNase H degrades RNA in the
40. s 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 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 of
41. sy 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 4 Apply the sample up to 700 uL including any precipitate that may have formed to the column 5 Close the tube gently and centrifuge for 15 seconds at 8000 X g 210 000 rpm Discard the flow through 6 For volumes greater than 700 pL load aliquots onto the RNeasy column succes sively and centrifuge as before 7 Add 350 uL Buffer RW1 into the RNeasy mini column to wash and centrifuge for 15 seconds at 28000 X g 210 000 rpm Discard the flow through Vil Appendix 27 Applause WT Amp ST System 10 11 12 13 14 19 16 17 18 Add 10 uL DNase to 70 uL Buffer RDD Gently invert the tube to mix Note Other DNase enzymes we can recommend to use in this step are the Shrimp DNase recombinant from USB Corp use 10 uL or the DNase RNase free from New England BioLabs use 10 pL 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 collec
42. t 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 Bi down the master mix briefly Place on ice Use master mix immediately 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 following 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 ABl 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 Fra
43. tion tube Add 500 uL Buffer RPE with the added ethanol to the RNeasy column Close the tube gently and centrifuge for 15 seconds at 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 28000 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 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 your RNA because the sample is contaminated with DNA we recommend DNase treatment followed by purification R 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 yL 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
44. tities genomic DNA will interfere with amplification Does NuGEN recommend DNase treatment of purified total RNA samples Yes For DNase treatment of RNA samples refer to Appendix D of the user guide Can 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 quantitate 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 uL in calculating the amplified cDNA concentration as this is the convention we used in establishing yield guidelines in this user guide Vil Appendix 34 Applause WT Amp ST System Q19 Q20 Q21 Q22 Q23 Q24 Q25 Q26 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 dou ble stranded character to this mixture we have developed and optimized the kit using the single stranded cDNA conversion factor 1 A260 unit 33 ng L of cDNA Expe
45. 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 enzyme When using this reagent take care not to splash or contaminate gloves bench or pipettes Preferably use a dedicated pipette to measure out B3 Due to the high sensitivity inherent in this amplification system we strongly recommend taking measures to minimize the potential for the carryover of previ ously amplified SPIA cDNA into new ampli
46. view 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 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 thaw 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
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