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1. Decrease the concentration of template RNA Product detected at lower than expected cycle number and or positive signal from no template controls Template or PCR carry over contamination Isolate source of contamination and replace reagent s Use separate dedicated pipettors for reaction assembly and post PCR analysis Assemble reactions except for target addition in a DNA free area Use aerosol resistant pipet tips or positive displacement pipettors Unexpected bands after RNA is contaminated with DNA Pre treat RNA with DNase I electrophoresis Oligo dT or random primers used Use gene specific primers Low specificity in PCR Optimize PCR conditions as described above References 1 Kotewicz M L D Alessio J M Driftmier K M Blodgett K P and Gerard G F 1985 Gene 35 249 2 Gerard G F D Alessio J M Kotewicz M L and Noon M C 1986 DNA 5 271 3 Chou Q Russel M Birch D Raymond J and Bloch W 1992 Nucl Acids Res 20 1717 4 Sharkey D J Scalice E R Christy K G Atwood S M and Daiss J L 1994 BioTechnology 12 506 5 Westfall B Sitaraman K Hughes J and Rashtchian A 1997 Focus 19 46 6 Lowe B Avila H A Bloom F Gleeson M and Kusser W 2003 Anal Biochem 315 95 7 Nazarenko I Lowe B Darfler M Ikonomi P Schuster D and Rashtchian A 2002 Nucleic Acids Res 30 e37 8 Tyagi S and Kramer F R 1996 Nature2. Biotechnology 14 303 9 Tyagi S Bratu D P and Kramer F R 1998 Nature Biotechnology 16 49 10 Kostrikis L G Tyagi S Mhlanga M M Ho D D and Kramer F R 1998 Science 279 1228 11 Holland P M Abramson R D Watson R and Gelfand D H 1991 Proc Natl Acad Sci USA 88 7276 12 Gibson U E M Heid C A and Williams P M 1996 Genome Res 6 995 13 Heid C A Stevens J Livak K J and Williams P M 1996 Genome Res 6 986 14 Nazarenko I A Bhatnagar S K and Hohman R J 1997 Nucl Acids Res 25 2516 15 Wittwer C T Herrmann M G Moss A A and Rasmussen R P 1997 Biotechniques 22 130 16 Ishiguro T Saitoh J Yawata H Yamagishi H Iwasaki S and Mitoma Y 1995 Anal Biochem 229 207 17 Chomezynski P and Sacchi N 1987 Anal Biochem 162 156 18 Chirgwin J M Przybyla A E MacDonald R J and Rutter W J 1979 Biochemistry 18 5294 19 Berger S L and Kimmel A R 1987 Methods in Enzymol 152 316 20 Gerard G F 1994 Focus 16 102 Limited Use Label License No 1 Thermostable Polymerases Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US 5 789 224 5 618 711 and 6 127 155 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal
3. Format Twelve 8 well strips come inserted in a 96 hole plate frame Push up gently on the bottom of the wells to remove each strip from the frame Strip wells are sealed with snap on strip caps Pull up gently on the end of a row of caps to unsnap them To reseal the plates for thermal cycling and storage snap the caps back into place Store any unused strips in the original resealable pouch with the desiccant pack to ensure dryness Note Only strip wells are compatible with the ABI PRISM 7900 Strip wells require the use of the MicroAmp 96 well tray retainer set ABI catalog number 403081 RNA e High quality intact RNA is essential for full length high quality cDNA synthesis and accurate mRNA quantification e RNA should be devoid of any RNase contamination and aseptic conditions should be maintained The lyophilized reaction mix includes RNaseOUT to protect against RNA degradation e To isolate total RNA we recommend the Micro to Midi Total RNA Purification System Cat no 12183 018 or TRIzol Reagent Cat Nos 15596 026 018 Oligo dT selection for poly A RNA is not necessary although incorporating this step may improve the yield of specific cDNAs e RNA may be treated with DNase I Catalog no 18068 015 to remove any contaminating genomic DNA Magnesium Concentration Magnesium is included at a final concentration of 4 mM which is appropriate for most targets ROX Reference Dye Catalog no 11728 089 incl
4. use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information about purchasing a license to use this product or the technology embedded in it for any use other than
5. 3 Invitrogen life technologies SuperScript III Platinum RTS One Step qRT PCR Kit Catalog No Size Formulation Store at room temperature 11728 081 96 well plate 11728 089 96 well plate with ROX Reference Dye 11728 097 12 x 8 strip wells plate format Description The SuperScript IN Platinum RTS One Step qRT PCR Kit provides qualified reagents in lyophilized form for the sensitive and reproducible detection and quantification of RNA in one step quantitative RT PCR qRT PCR The one step reaction mix is aliquoted into plate wells or strip wells and then lyophilized allowing for room temperature storage the addition of large sample volumes and ease of reaction setup To perform qRT PCR simply add water primers and template vortex to dissolve the pellet and proceed with the reaction This kit combines the high temperature reverse transcription capability of SuperScript II RT with the automatic hot start PCR capability provided by Platinum Taq DNA Polymerase for optimal specificity consistency and efficiency Both cDNA synthesis and qPCR are performed in a single tube using gene specific primers and RNA target s from either total RNA or mRNA The one step formulation enables highly sensitive detection from as few as 10 copies of RNA template with a broad dynamic range that supports accurate quantification of high copy mRNA at up to 1 ug of total RNA Each pellet contains SuperScript III RT Platinum Taq DNA Polyme
6. Roche LightCycler After programming your instrument follow the steps at the bottom of the page to perform the reaction LUX Primers Dual Labeled Probes Cycling Program Cycling Program Instruments using Instruments using PCR tubes plates LightCycler PCR tubes plates LightCycler 42 55 C for 10 min hold Program choice Amplification 42 55 C for 10 min hold Program choice Amplification 95 C for 2 min hold Analysis mode Quantification 95 C for 2 min hold Analysis mode Quantification 40 50 cycles of 42 55 C for 10 min hold 40 50 cycles of 42 55 C for 10 min hold 95 C 15 s 95 C for 2 min hold 95 C 15 s 95 C for 2 min hold 60 C 30 s 50 cycles of 60 C 30 s 50 cycles of 95 C 5 s 95 C 5 s 60 C 20 s single acquire 60 C 20 s single acquire Melting Curve Analysis Melting Curve Analysis Refer to instrument Program choice Melting curve Melting curve analysis is possible with some types of dual documentation Analysis mode Melting curves labeled probes but not others refer to probe documentation 95 C 0 s 55 C 15 s Use 50 C for 10 min as 95 C 0s a general starting point 40 C 0s Use 50 C for 10 min as a general starting point Protocol for Instruments Using PCR Tubes Plates Protocol for Roche LightCycler 1 Program the qPCR instrument to perform cDNA synthesis 1 Program the LightCycler to perform cDNA synthesis immediately followed by PCR amplification as specified immediate
7. aqMan probes and molecular beacons 6 16 We do not recommend using this kit with dsDNA binding dyes such as SYBR Green I The final volume of each reaction including primers template and water is 25 ul Cat No Product Format No of Reactions Instrument Recommendation 11728 081 96 well plate 96 ABI PRISM 7000 7300 7700 7500 Bio Rad iCycler Stratagene Mx3000P and Mx4000 MJ Opticon 11728 089 96 well plate with ROX Reference Dye 96 ABI PRISM 7000 7300 7700 7500 Stratagene Mx3000P and Mx4000 MJ Opticon 2 11728 097 12 x 8 strip wells plate format 96 Roche LightCycler Corbett Rotor Gene Cepheid Smart Cycler 25 ul reaction volume including primers template and water Quality Control The product is tested functionally by real time quantitative analysis using total HeLa RNA as template Kinetic analysis must demonstrate a linear dose response with decreasing target concentration and detection of B actin mRNA in 1 pg of total HeLa RNA Related Products Product Amount Catalog no Micro to Midi Total RNA Purification System 50 rxns 12183 018 TRIzol Reagent 100 ml 15596 026 200 ml 15596 018 DNase I Amplification Grade 100 units 18068 015 Custom Primers to order visit www invitrogen com Part no 11728 pps Rev date 04 September 2010 This product is distributed for laboratory research use only CAUTION Not for diagnostic use The safety and efficacy of this product in diagnostic or o
8. for research use please contact Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 or e mail outlicensing lifetech com Limited Use Label License No 14 Platinum products Licensed to Life Technologies Corporation under U S Patent Nos 5 338 671 5 587 287 and foreign equivalents for use in research only Limited Use Label License No 24 Use of TaqMan in 5 nuclease assays The use of TaqMan fluorogenic probes in 5 nuclease assays is covered by U S Patent No 5 538 848 owned by Applied Biosystems Purchase of the product does not provide a license to use this patented technology A license to practice this technology must be obtained from Applied Biosystems 850 Lincoln Center Drive Forest City California 94404 or from Roche Molecular Systems Inc 1145 Atlantic Avenue Alameda California 94501 2004 2010 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use
9. ld be free of secondary structure and should not hybridize to itself or to primer 3 ends e For multiplex applications the concentration of each probe may need to be adjusted independently to obtain optimal fluorescent signals The amount of probe for the reference gene such as B actin or GAPDH should be limited as described above for primers Reaction Setup and Conditions e Efficient CDNA synthesis can be accomplished in a 5 30 min incubation at 45 60 C Optimal temperature varies for different primers and templates A good starting point is 50 C for 10 minutes For problematic templates or to increase the specificity of cDNA priming increase the cDNA synthesis temperature to 55 C e SuperScript II RT is inactivated the RNA cDNA hybrid is denatured and Platinum Taq DNA polymerase is activated during the 2 min incubation at 95 C e The annealing temperature should be 0 10 C below the melting temperature of the primers used e The extension time of 30 seconds for instruments that use PCR tubes plates is appropriate for the short amplicons that are typically used in qPCR Page 3 Quantitative One Step RT PCR Protocol The tables below show the cycling conditions for one step qRT PCR using either LUX Primers or dual labeled probes Separate cycling programs and protocols are provided for instruments that use PCR tubes plates e g ABI PRISM Bio Rad iCycler Stratagene Mx4000 Cepheid Smart Cycler and the
10. ly followed by PCR amplification as specified above Optimal temperatures and incubation times may vary above Optimal temperatures and incubation times may vary for different target sequences see Reaction Setup and for different target sequences see Reaction Setup and Conditions previous page Conditions previous page 2 Add primers template and sterile distilled water to each well 2 Set the fluorescence on the LightCycler to the appropriate to a final volume of 25 ul as follows channel for the reporter dye used e g F1 channel for FAM e Amount of template 1 pg to 1 ug total RNA 3 Add primers template and sterile distilled water to each well e Recommended final concentration of primers 200 nM each to a final volume of 25 pl as follows e g 0 5 ul of a 10 uM stock e Amount of template 1 pg to 1 ug total RNA e Ifyou are using TaqMan probes recommended final e Recommended final concentration of primers 500 nM each concentration of probe 100 nM e g 0 25 ul of a 10 uM stock e g 1 25 ul of a 10 uM stock 3 Seal the plates strip wells see page 2 for sealing instructions e Ifyou are using TaqMan probes recommended final and vortex for 5 10 seconds Note Vortexing is crucial to concentration of probe 250 nM e g 0 625 ul of a 10 uM ensure complete dissolution of the pellet Centrifuge briefly stock to collect the contents es 4 Seal the plates strip wells see page 2 for sealing instructions 4 If your
11. ording to standard PCR guidelines We recommend using primer design software such as OligoPerfect Designer www invitrogen com oligoperfect LUX Primers must be designed using LUX Designer www invitrogen com lux e Primers should be specific for the target sequences be free of internal secondary structure and should avoid complementation at 3 end within each primer primer pair or hybridization probe sequence except as required for hairpin primers such as LUX For best results the amplicon size should be limited to 80 200 bp e Design primers that anneal to exons on both sides of an intron or within the exon exon boundary of the mRNA to allow differentiation between amplification of cDNA and potential contaminating genomic DNA e Optimal results may require a primer titration between 100 and 500 nM A final concentration of 200 nM per primer is effective for most reactions e For multiplex applications limit the amount of primer for the reference gene such as B actin or GAPDH to avoid competition between amplification of the reference gene and sample gene In general the final concentration of the reference gene primer should be between 25 and 100 nM However a primer titration is recommended for optimal results Dual Labeled Probes e The optimal concentration of probe may vary between 50 and 800 nM See the protocol on the next page for a recommended starting concentration e The probe sequence shou
12. qPCR instrument cannot accommodate the plates or and vortex for 5 10 seconds Note Vortexing is crucial to strip wells transfer each 25 ul reaction to a tube appropriate ensure complete dissolution of the pellet Centrifuge briefly for use with your qPCR instrument to collect the contents 5 Place reactions in a thermal cycler programmed as described 5 Unseal uncap the wells and transfer 20 ul of each reaction to above Collect and analyze the results each LightCycler capillary tube Cap the capillary tube 6 Centrifuge tubes at 700 x g for 5 seconds 7 Place reaction tubes in the rotor of the LightCycler and run the program Collect and analyze the results ABI PRISM and GeneAmp are registered trademarks of Life Technologies Corporation TaqMan is a registered trademark of Roche Molecular Systems Inc iCycler Mx4000 Mx3000P Rotor Gene Opticon and Smart Cycler are trademarks or registered trademarks of their respective companies TRIzol is a registered trademark of Molecular Research Center Inc LightCycler is a registered trademark of Idaho Technologies Inc Troubleshooting Guide Page 4 Problem Possible Cause Probable Solution No amplification product Relative fluorescent signal lt background or no template control Problem with reporter dye or instrument settings cDNA synthesis temperature too high low priming efficiency RT or cDNA primer blocked by secondary st
13. rase a proprietary buffer system Mg dNTPs RNaseOUT Recombinant Ribonuclease Inhibitor and stabilizers The final concentration of dNTPs is 200 nM each and the final concentration of Mg is 4 mM Catalog no 11728 089 also includes ROX Reference Dye in each pellet to normalize the fluorescent signal between reactions for instruments that are compatible with this option SuperScript III Reverse Transcriptase is a version of M MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability 1 2 The enzyme can be used to synthesize cDNA at a temperature range of 45 60 C providing increased specificity higher yields of cDNA and more full length product than other reverse transcriptases Because SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA it can be used to synthesize cDNA from total RNA Platinum Taq DNA polymerase is recombinant Tag DNA polymerase complexed with a proprietary antibody that blocks polymerase activity at ambient temperatures 3 4 5 Activity is restored after the denaturation step in PCR cycling at 94 C providing an automatic hot start in PCR for increased sensitivity specificity and yield The SuperScript III Platinum RTS One Step qRT PCR Kit provides optimal performance for both fluorogenic primer based detection technology such as Invitrogen s LUX Primers and fluorogenic hybridization probe based detection methods such as T
14. research No right under any other patent claim no right to perform any patented method and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is conveyed expressly by implication or by estoppel This product is for research use only Diagnostic uses under Roche patents require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Limited Use Label License No 5 Invitrogen Technology The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to
15. ructure RNA has been damaged or degraded RNase contamination Reporter dye not functional Run the sample on a gel to determine if the PCR product was generated If the appropriate bands are seen see Reporter dye not functional under Possible Cause or check the instrument settings Lower incubation temperature Raise incubation temperature Redesign primer s Replace RNA if necessary Maintain aseptic conditions add RNase inhibitor Validate fluorescent primer or probe design and presence of fluorophore and or quencher Redesign and or resynthesize if necessary Poor sensitivity Not enough starting template RNA Increase the concentration of template RNA use 10 ng to 1 ug of total RNA Product detected at higher than expected cycle number RNA has been damaged or degraded RNase contamination RT inhibitors are present in RNA Inefficient CDNA synthesis Inefficient PCR amplification Replace RNA if necessary Maintain aseptic conditions add RNase inhibitor Remove inhibitors in the RNA preparation by an additional 70 ethanol wash Inhibitors of RT include SDS EDTA guanidium salts formamide sodium phosphate and spermidine 19 20 Adjust cDNA synthesis temperature and or primer design Optimize PCR conditions Adjust annealing temperature as necessary Increase magnesium concentration Redesign primers Product detected at lower than expected cycle number Too much sample added to reactions
16. ther clinical uses has not been established For technical questions about this product call the Invitrogen TECH LINESM 800 955 6288 Form No 18057N Printed on Recycled Paper Recommendations and Guidelines Plate Formats SuperScript Platinum RTS One Step qRT PCR reaction mix comes lyophilized in a 96 well microtiter plate or 12 x 8 strip wells in plate format 96 Well Plate The microtiter plate comes sealed To open peel away the foil seal to expose the wells you want to use and add template and primer Then reseal the wells for thermal cycling using the heat seal tape provided in each kit To seal wells for thermal cycling and subsequent storage 1 Peel away the plastic backing to expose the sticky side of the heat seal tape 2 Position the tape sticky side down on the plate so that all wells are covered Press down gently and evenly to seal Important Do not use the heat seal tape provided in the kit to cover wells containing unreconstituted pellets because the pellets will stick to the tape Wells containing pellets should remain covered with the original foil seal The plate can be cut into four sections of 24 wells each 8 wells x 3 rows Each plate section can then be run in a separate reaction To separate carefully cut the foil seal and tabs connecting each section Store any unused foil covered wells in the original resealable pouch with the desiccant pack to ensure dryness 12 x8 Strip Wells in Plate
17. udes ROX Reference Dye in the pellet to normalize the fluorescent reporter signal in qPCR for instruments that are compatible with this option Note that the use of ROX Reference Dye is not supported on the iCycler Rotor Gene Opticon and LightCycler platforms ROX Reference Dye can be used to adjust for non PCR related fluctuations in fluorescence between reactions and provides a stable baseline in multiplex reactions It is composed of a glycine conjugate of 5 carboxy X rhodamine succinimidy ester 25 uM in 20 mM Tris HC pH 8 4 0 1 mM EDTA and 0 01 Tween 20 Page 2 Instrument Settings This kit can be used with a variety of qPCR instruments including but not limited to the ABI PRISM 7000 7300 7500 7700 7900 and GeneAmp 5700 the Bio Rad iCycler the Stratagene Mx3000P and Mx4000 the Corbett Research Rotor Gene the MJ Research DNA Engine Opticon and Opticon 2 the Cepheid Smart Cycler and the Roche LightCycler Please refer to your instrument user manual for operating instructions Optimal cycling conditions will vary with different machines The protocols on the following page have been optimized for the ABI PRISM 7700 and the Roche LightCycler Primers e Gene specific primers GSP are required We do not recommend using oligo dT or random primers which may generate nonspecific products in the one step procedure and reduce the amount of product e Primers should be designed acc
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