Applause® WT-Amp Plus ST System
Contents
1. 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 Plus ST System No The Applause WT Amp Plus ST System is designed to amplify mRNA not DNA Has NuGEN performed reproducibility studies on the Applause WT Amp Plus 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 Plus ST System Yes In high quantities 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 Plus 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 Plus ST System for archiving cDNA Yes Amplified cDNA may be safely stored at 20 C for six months or longer How do 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 L in calculating the amplified2. 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 28000 X g 10 000 rpm and discard the flow through f Add 200 uL fresh 80 ethanol close cap spin for 30 seconds at 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 collection tube h Add 10 uL 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 28000 X g 210 000 rpm to collect the purified RNA Purification with QIAGEN 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 uL sample to the column e After closing the column spin for 15 seconds at 8000 X g 210 000 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 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
3. 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 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 5 uL 2 5 uL 5 uL 5 Add 10 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 thermal 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 teh 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 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 ro
4. 0 ST Arrays 169 format use a 90 uL hybridization volume For the GeneChip Exon 1 0 ST Arrays 49 format use a 200 uL hybridization volume We recommend a hybridization time of 18 hours 2 hours Hybridization times within this range yield comparable results Choose the appropriate fluidics script for the chosen array type as noted in Table 16 Vil Appendix 25 Applause WT Amp Plus ST System Kit Affymetrix P N 900720 Table 16 Hybridization Cocktail Assembly and Fluidics Protocol for GeneChip Gene 1 0 ST and Exon 1 0 ST Arrays using the Affymetrix HWS COMPONENT EXON 1 0 ST ARRAY 49 FORMAT GENE 1 0 ST ARRAY 169 FORMAT FINAL CONCENTRATION Fragmented biotin labeled amplified a j po M 18 2 22 7 ng pL cDNA ae HER Control Oligonucleotide B2 3 7 uL 1 8 uL 50 pM 3 nM 20X Eukaryotic hybrid 1 5 5 25 and ization controls bioB TT pL 5 5 uL 100 pM tivel bioC bioD cre i ale 2x Hybridization Mix 110 uL 55 uL 1X 100 DMSO 22 uL 11 uL 10 Water 23 3 uL 11 6 uL Final Volume 220 uL 110 uL FLUIDICS PROTOCOLS Fluidics Script FS450_0001 FS450_0007 B Performing Quantitative PCR on Amplified cDNA The amplified SPIA cDNA produced by the Applause WT Amp Plus ST System has been successfully used as template for qPCR systems including TaqMan and SYBR Green We have successfully used the following master mixes for qPCR For optimum results in
5. cDNA concentration as this is the convention we used in establishing yield guidelines in this user guide 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 Plus ST Systems con sists 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 all have been generated using this convention How many rounds of amplification are performed in the Applause WT Amp Plus ST System This System performs a single round of amplification It is not designed to sup port multiple rounds of amplification Vil Appendix 34 Applause WT Amp Plus ST System Q20 Q21 Q22 Q23 Q24 Q25 Q26 Do I need to order specific primers for the amplification No The DNA RNA primers provided in the Applause WT Amp Plus 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 Plus 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 desc
6. close the tube Vil Appendix Use nuclease free water at room temperature to elute sample 29 Applause WT Amp Plus ST System Note Use fresh 80 ethanol Lower percent ethanol mixes will reduce recovery h Spin for 2 minutes at 8000 X g 10 000 rpm and discard the flow through 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 recommendations 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 p
7. degraded RNA Using compromised samples will result in unsatisfactory and variable results How much cDNA can expect from a single reaction You should expect a minimum yield of 4 ug for the Applause WT Amp Plus 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 Plus ST System provide any labeling reagents No The Applause WT Amp Plus 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 and Exon 1 0 ST Arrays Where can 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 Plus ST System components All components of the system may be stored at 209C Ensure the vials are well sealed and do not exceed 6 freeze thaw cycles Vil Appendix 33 Applause WT Amp Plus ST System Q10 Q11 Q12 Q13 Q14 Q15 Q16 Q17 Q18 Q19
8. 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 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 Plus ST System amplifications should be significantly lower than yields for RNA inputs within the recommended input range of 50 ng to 200 ng VII Appendix ii The Bioanalyzer trace 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 ii The Bioanalyzer traces of amplification products may look significantly differ ent than the typical Applause WT Amp Plus 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
9. 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 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 4 Apply the sample up to 700 uL including any precipitate that may have formed to the column Vil Appendix 10 17 12 13 14 15 16 TZ 18 Close the tube gently and centrifuge for 15 seconds at 8000 X g 10 000 rpm Discard the flow through For volumes greater than 700 uL load aliquot
10. 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 Plus ST System e TaqMan ABsolute qPCR Mix plus ROX ABgene Cat AB 1136 B Fast Universal PCR Master Mix 2X Applied Biosystems Cat 4352042 Vil Appendix 26 Applause WT Amp Plus 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 Plus ST System
11. Applause WT Amp Plus 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 servi
12. LOW 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 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 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
13. 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 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 Plus ST System Table 9 Enhancement Master Mix volumes listed are for a single reaction SECOND STRAND BUFFER MIX REACTION ENHANCEMENT MIX YEL
14. ase 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 Sm ol e 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 8 Remove the tubes from the thermal cycler and place on ice 9 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 ver 3 BLUE A3 ver 1 2 5 uL 0 5 uL 10 Add 3 uL of the First Strand Master Mix to each tube 11 Mix by pipetting 5 times spin and place on ice 12 Applause WT Amp Plus ST System 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 Plus 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
15. ces 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
16. d subsequent hybridization to GeneChip Gene 1 0 ST and Exon 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 Plus ST System lot is tested to meet specifications of yield and array performance E Storage and Stability The Applause WT Amp Plus ST System is shipped on dry ice and should be unpacked immediately 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 Plus 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 Il Kit Components 4 Applause WT Amp Plus ST System A Reagents and Supplies Provided Table 1 First Strand cDNA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER a strand Primer 501262 Blue Al ver 4 ae strand Buffet 501256 Blue A2 ver 3 iha amana Eriam 501040 Blue A3 ver 1 Table 2 Second Strand cDNA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER a 501257 Yellow B1 v
17. eas 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 Plus ST System may be stored at 20 C IV Amplification Protocols 9 Applause WT Amp Plus ST System A Overview The Ribo SPIA amplification process used in the Applause WT Amp Plus 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 sec
18. ediately with the Post SPIA Modification II protocol Post SPIA Modification II Make a master mix by combining E2 and E3 in a 0 5 mL capped tube according to the volumes shown in Table 12 Table 12 Post SPIA Modification II Master Mix volumes listed are for a single reaction BUFFER MIX VIOLET E2 ver 2 ENZYME MIX VIOLET E3 ver 1 6 uL 6 uL IV Amplification Protocols 100 ethanol must be added to the QIAGEN Buffer PE upon first use Failure to do so will result in low amplification yields 17 Applause WT Amp Plus ST System J Add 12 uL of the Post SPIA Modification II Master Mix to the Post SPIA Modification reaction Mix by pipetting 5 times with a pipette set to 30 pL spin and place on ice Place the tubes in a pre cooled thermal cycler programmed to run Program 7 Post SPIA Modification Il see Table 6 4 C 1 min 30 C 10 min 42 C 60 min 75 10 min hold at 4 C Remove the tubes from the thermal cycler spin to collect condensation and place on ice Continue to the Purification of ST cDNA protocol or store the cDNA at 20 C 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 so OS SE ow ON 10 11 e The ERC buffer is considered hazardous according to QIAGEN and an MSDS may be consulted e Add the a
19. ee 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 in 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 ol ae o Tia i RNA Quality Continuum 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 pos
20. ensitive RNA amplification process developed by NuGEN the Applause WT Amp Plus 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 Plus ST System an ideal choice for use with whole transcript array designs such as the Gene 1 0 ST and Exon 1 0 ST Array The Applause WT Amp Plus ST System Part No 5510 provides optimized reagent formulations and a protocol to process total RNA samples B How the Applause WT Amp Plus ST System Works The Applause WT Amp Plus ST System utilizes Ribo SPIA technology that produces amplified 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 cDNA mRNA complex creates priming sites for DNA polymerase to synthesize a second strand which include
21. er 3 second orang 501126 Yellow B2 ver 2 Enzyme Mix a K s01119 Yellow B3 ver 1 Table 3 SPIA Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER SPIA Primer Mix 501263 Red C1 ver 8 SPIA Buffer Mix 501258 Red C2 ver 8 SPIA Enzyme Mix 01260 Red C3 ver 5 Il Kit Components 5 Applause WT Amp Plus ST System Table 4 Post SPIA Modification Reagents COMPONENT PART NUMBER VIAL CAP VIAL NUMBER Primer Mix 01265 Violet E1 ver 1 Buffer Mix S01266 Violet E2 ver 2 Enzyme Mix 01383 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 Plus 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 s
22. f 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 al 27 Applause WT Amp Plus 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 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 Plus ST System 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 28000 X g 210 000 rpm
23. factor of 8 8x when calculating the master mix volumes e Components and reagents from other Ovation System WT Ovation System or Applause System products should not be used with this product e 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 e 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 amplification 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 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
24. iedeten 20 D Programming the Thermal Cycler ssasstsoosensolbtasoonsuali subasHatis tanjvnn stal sjaia 20 E EDNA Fragmentation accissnctsssspetectuacesstyesieacsag tans dustepeauedt a i E ERENER 21 E Biotin Label Giersscassctessseasntesssteasavecucssapts ach iaeveaad dass eedsiayattanastases EE 22 Table of Contents VI Technical Support cc sceicescscsassecsssscccssenscacecvadessonssdesducassnasdesanascenssessensasedsnsacsoaneess 23 VIL APP Indixieseicsccssccccesssassccecccsissscccecsisssdecceessvescececestusssdcceccabiectecacsabssct d aka b ga bk akan 24 A Target Preparation for Array Hybridization a aiaiaaiasasasasasa sasasasasanananaana 24 B Performing Quantitative PCR on Amplified CDNA aiaiaiaisasassssassssasasasasan 25 C Quality Control of Amplified CDNA Product ss seunaushsisbanoknibsasessunnnda 26 D DNase Treatment oft RNA slan ni a a ak 26 E Preventing Non specific Amplificatiohs ssassstseseooninonliohotsulkahasa b e 29 F Frequently Asked Questions FAQS v s uizaseisssunskisiarsisstursvalissavsssvisislensinun svik saka 32 G Update Histon kamar aR R EA RRR 35 I Introduction 1 Applause WT Amp Plus ST System A Background The Applause WT Amp Plus 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 and Exon 1 0 ST Arrays Powered by Ribo SPIA technology a rapid simple and s
25. itive 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 Plus 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 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 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 ar
26. lower than expected iv Contact NUGEN Technical Services when atypical performance is suspected 31 Applause WT Amp Plus ST System Vil Appendix 32 Applause WT Amp Plus ST System F Frequently Asked Questions FAQs Q1 Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 What materials are provided with the Applause WT Amp Plus ST System The Applause WT Amp Plus ST System provides all necessary buffers primers and enzymes for first strand cDNA synthesis second strand cDNA synthe sis 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 micro centrifuge 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 Plus ST System can be used with high quality puri fied total RNA in the range from 50 to 200 ng Input amounts outside this range may produce unsatisfactory and variable results Can I amplify degraded RNA with the Applause WT Amp Plus ST System The Applause WT Amp Plus ST System is not designed for use with
27. m of single stranded DNA 33 pg mL To calculate A260 A320 of diluted sample X dilution factor X 33 concentration in g mL of a 1 absorbance unit solution X 0 03 final volume in mL total yield in micrograms IV Amplification Protocols 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 6 The purified ST cDNA may be stored at 20 C 19 Applause WT Amp Plus ST System V Labeling with the Encore Biotin Module 20 Applause WT Amp Plus 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 Plus ST System in preparation for hybridization on Affymetrix GeneChip Gene 1 0 ST and Exon 1 0 ST Arrays Two Encore Biotin Module 12 reaction kits Part No 4200 12 are required to process the 24 cDNA targets from the Applause WT Amp Plus ST Syatem Follow the protocol given below when using the Applause WT Amp Plus ST System with the Encore Biotin Module 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 Always keep thawed reagents and reaction tubes on ice unless otherwise instructed e Afte
28. master mix immediately 21 Applause WT Amp Plus ST System 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 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 25 uL of the purified ST cDNA 4 to 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 5 uL 2 uL 6 Add 7 uL of the Fragmentation Master Mix to each tube 7 Mix thoroughly by pipetting 8 to 10 times spin and place on ice Place the tubes in a
29. n 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 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 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 n
30. offer to sell or sell NUGEN s product is conveyed or implied by buyer s purchase of this NUGEN product The buyer agrees to use NUGEN products accompanying the product insert in accordance with the intended use and the written instructions provided Table of Contents Contents l MNEPOMUCHON ss scsssssscsscacsssssessssvecsascssssssssaacasessesssscscsoosesassosasssosussesasasssvseasssssseasssssess 1 As 2712 0 010 clo esera Peter wert ter E E r tenet reer weree rere etre eer ye 1 B How the Applause WT Amp Plus ST System Works iiiaiaiiaiaiasasasassssssasasaaai 1 C Performance SpetilficationSse scsosnaoumulssbalnununnssnnum 3 B Quality Control ccscssccseeccieteciacesuersteve a k ka sa la sT a a S Saa ala ml a aaj Vina 3 E Storage and Stabilitt yc 2 0 cs2 acnesacasestessenesindosasustas dopeedcenndoadeassesdeedecaacoonsteseeten easy 3 F Material Safety Data Sheet MSDS aaaaiasaasassassasasvasasaasaaaaasaavasakaaaaaaaa 3 Il Kit Component cccccccssssssssecssesssseseessenssceeeessesesceeeceseseseeeeeesssensceesesssenseees 4 A Reagents and Supplies Provided ccicccccsscticsietsccussidscsste cacacueecacesdtestssesteres 4 B Additional Reagents Supplies and Equipment aiiaiaisiasasassssssssasasasssaai 5 lll Planning the Experiment ccciscscescccscsesstscececisssesescoctsastesctactiasssescacsissvesocactiassesoceces 7 A Input RNAIREQUIFEMENTS 12 32sssalasa inasina st ssa sss
31. om temperature mix by vortexing spin and place on ice 15 Applause WT Amp Plus ST System IV Amplification Protocols The E3 Enzyme Mix is quite viscous Please take care and pipette this mix slowly into the mix 16 Applause WT Amp Plus ST System 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 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 Master Mix volumes listed are for a single reaction ENHANCEMENT PRIMER MIX MASTER MIX VIOLET E1 ver 1 2 uL 6 uL 6 Add 8 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 imm
32. ort 23 Applause WT Amp Plus 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 Plus ST System A Target Preparation for Array Hybridization Note Requires Affymetrix Hybridization Wash Stain HWS Kit for Gene 1 0 ST and Exon 1 0 ST Arrays In general cDNA targets amplified using the Applause WT Amp Plus ST System and labeled using the Encore Biotin Module are prepared for analysis on GeneChip Gene 1 0 ST and Exon 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
33. pectrophotometer and cuvettes ora Nanodrop UV Vis Spectrophotometer e Reagents Ethanol Sigma Aldrich Cat E7023 for purification steps Il Kit Components 6 Applause WT Amp Plus 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 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 giagen com e Sigma Aldrich Inc www sigmaaldrich com e USB Corporation www usbweb com III Planning the Experiment 7 Applause WT Amp Plus 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 Plus ST System is designed to be used with high quality RNA samples 1 RNA Quantity The Applause WT Amp Plus 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 fr
34. physical separation of pre and post amplification activities Establish and maintain a clean work environment 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 Carry out a thorough external and internal cleaning of all pipettes with DNA OFF Carefully follow the manufacturer s instructions for this process to avoid damaging the pipettes It is a good idea to keep a clean set of pipettes as a backup Always wear gloves and don fresh gloves upon entry into this controlled area Frequently change gloves while working in the pre amplification area especially prior to handling stock reagents reactions and RNA samples Stock this area with clean preferably new equipment pipettes racks consum ables that has not been exposed to post amplification workspace Make it a policy to carry out regular cleaning of all workspaces Capture waste
35. ppropriate 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 Into a clean labeled 1 5 mL microcentrifuge tube add 300 uL of Buffer ERC from the OIAGEN kit Add the entire volume 52 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 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 IV Amplification Protocols Use nuclease free water at room temperature to elute sample 18 Applause WT Amp Plus ST System 12 13 14 5 16 17 18 19 20 K 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 colum
36. 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 V Labeling with the Encore Biotin Module Use Labeling Master Mix immediately after preparation Mix by pipetting and spin down HD the master mix briefly at low speed Place on ice Use master mix immediately 22 Applause WT Amp Plus ST System 10 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 15 pL 1 5 uL 1 5 pL Add 18 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 Supp
37. r 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 three 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 Plus ST System is ready for labeling with the Encore Biotin Module protocol Use 4 to 5 ug of ST cDNA as input into the Encore Biotin Module D Programming the Thermal Cycler Use a thermal cycler with a heat block designed for 0 2 mL tubes equipped with a heated lid with a capacity of 100 uL reaction volume Prepare the 2 programs shown V Labeling with the Encore Biotin Module Use Fragmentation Master Mix immediately after preparation Mix by pipetting and spin E down the master mix briefly Place on ice Use
38. ribe 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 Plus ST System amplifies the entire transcript so prim ers can be designed at any location within the mRNA In order to avoid inter ference 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 Plus 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 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 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 carrier
39. rocessing 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 NUGENS 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 amplified 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 Vil Appendix 30 Applause WT Amp Plus ST System d PCR Workstation enclosures with UV illumination for use as pre amplification workspaces can be an option in situations where conditions preclude
40. s Vil Appendix NuGEN imagine more from less G Update History This document the Applause WT Amp Plus ST System user guide M01199 v2 1 is an update to a previous version to address the following topics Description Section Page s Updated SPIA technology description I B 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 San Carlos CA 94070 USA 9350 AC Leek Toll Free Tel 888 654 6544 The Netherlands Toll Free Fax 888 296 6544 Tel 31 13 5780215 custserv nugeninc com Fax 31 13 5780216 echserv nugeninc com europe nugeninc com For research use only Technologies Inc Other marks appearing in these materials are marks of their respective owners www nugeninc com For our international distributors contact information visit our website 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 M01199 v2 1
41. s 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 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 I Introduction 2 Applause WT Amp Plus ST System 4 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 e
42. s 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 8000 X g 210 000 rpm Discard the flow through 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 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 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 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 8000 X g 210 000 rpm to elute I
43. tandingaaskanban tas ads lgdsndansnandinunna ups 7 B Using RNase free Techniques aiaaaauasaaanaaaiaiinanananananannnnnnnnnnananananannnndnanini 8 G TRINA Slogan 8 D Amplified cDNA Storage sss nangstsdssd kstlsb sskand avsk ka s kk sklavstkask na a 8 IV Amplification Protoco 5s 22 54550025lsalsssdsinnasladasi kassandaiianaanisaanabuanaidununbunndnanaaabakadainua 9 As OVaWVleWiiasaondsbstddiioekanis vanlod iaia ENR ska lla aa G b t SS Esie 9 B Protocol N ls un dsb a a taa l taal aS ska EERTE 9 C Programming the Thermal Cycler 111 12 00uussosodoncobsnoswondundio tita naiai 10 B irst Strand cDINA Synthesist aeinn enntre arai e oie 12 E Second Strand cDNA Synthesis siasa nastala iktai sskkdssinaav lds ana saat 13 F Post Second Strand Enhancement aaaaaaiaaaaaasassasaaasvassasasassasasaavaaaaa 13 G SA AVI aaa 14 H PostSPIA Modification seserinis aeae and jg l pga alla al 15 I PostSPIA Modification I iresi A 16 J Purification of ST CDNA vadsske usdi las sss ana 17 K Measuring ST cDNA Yield and Purity a iaiiaiaiiaaasasasasassasasasasaasasasasansaaaaaa 18 V Labeling with the Encore Biotin Module aaaaaaaaaaassnsssnsnnnnnnnnnnnnnnnnnnnnnnnnn 20 A Encore Biotin Module Overview a iasiaiaisasaaaassasasaassaaasassasakaaaaaa 20 B Protocol Notes sisieun r a a 20 GC Preparing cDNA Samples sis scccisteicsentthesesstessasnisdvisutias eatin ele
44. tion 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 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 Plus ST System e 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
45. typically 100 to 105 C IV Amplification Protocols 11 Applause WT Amp Plus ST System 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 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 D First Strand cDNA Synthesis 1 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 2 Spin down the contents of A3 and place on ice o Thaw the other reagents at room temperature Mix by vortexing spin and place on ice Leave Nucle
46. xtend the annealed primers producing ST cDNA targets appropriate for use with GeneChip Gene 1 0 ST and Exon 1 0 ST Arrays Figure 1 The Ribo SPIA Whole Transcriptome RNA Amplification Process The Ribo SPIA WT Amp Process First Strand cDNA Synthesis 5 O NNNNNNA oma 5 5 Second Strand cDNA Synthesis 5 3 RNAseH cleavage of RNA Sequence 5 AAAA 3 RNA 3 TTTT 5 xas DNA 3 NNNNNN u 5 as SPIA Product Reverse Transcriptase NNNNNN WT Primer DNA RNA AAAA 3 exam SPIA Primer DNA RNA Oligo dT Primer DNA RNA NNNNNNNNN Random 9 mer Pol DNA Polymerase Reverse Transcriptase Pol DNA Polymerase A RNAseH 3 5 3 3 PO LS SPIA Reaction Amplification FARRAR RRs Primer Extension by Strand Displacement DNA Synthesis 5 Eni Post SPIA Modification ST cDNA Production 5 5 z A RNAseH Pol DNA Polymerase Cam SPIA Primer RNAseH Cleavage to Free Primer Hybridization Site gt BN RNAseH Primer Extension by DNA Polymerase 3 3 SPIA Product NNNNNNNNN Random Primer Pol DNA Polymerase 3 EB NNNNNNNNN Ble NNNNNNNNN Il Introduction 3 Applause WT Amp Plus ST System C Performance Specifications The Applause WT Amp Plus ST System synthesizes microgram quantities of ST cDNA starting with a total RNA input of at least 50 ng In approximately seven hours the system produces sufficient cDNA for labeling an
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