SuperScript™ III Platinum® CellsDirect One-Step qRT
Contents
1. The following materials are provided by the user e Mammalian cell cultures in growth medium e Coulter Counter or hemacytometer e Centrifuge for pelleting cells e Incubator or thermal cycler preheated to 75 C e Trypsin for adherent cultures e 1X cold phosphate buffered saline PBS without calcium or magnesium e 0 2 ml thin walled PCR tubes or PCR plates e Ice e Pipettes The following materials are provided in the kit e Resuspension Buffer e Lysis Enhancer e MgSO 50 mM optional All steps should be performed on ice and reagents should be chilled and or thawed immediately prior to use The incubator should be preheated to 75 C Continued on next page Lysing Larger Volumes of Cells continued Lysis For adherent cell cultures follow all the steps below For cells Procedure in suspension skip Steps 1 5 and proceed to Step 6 below 1 Aspirate the media in each dish and wash each dish with an appropriate volume of 1X cold PBS e g for a 10 cm dish or a T75 flask use 10 ml PBS Aspirate the PBS 2 Addenough trypsin to cover the adherent cells in your tissue culture dish plate or flask e g for a 10 cm dish use 1 ml for a T75 flask use 3 ml 3 Incubate for 5 minutes on ice or at room temperature 4 Check for cell detachment under a microscope If cells have not detached gently tap the disk or flask to dislodge the cells or let the cells incubate longer checking them every min2. available Dual Labeled Probes Fluorescent dual labeled probe technology such as TaqMan probes requires two gene specific primers as well as a probe that hybridizes to the internal portion of the amplicon The probe sequence should be free of secondary structure and should not hybridize to itself or to primer 3 ends The optimal concentration of probe may vary between 50 and 500 nM with a recommended starting concentration of 100 nM Fluorescent Dyes This kit has been developed and optimized for use with fluorogenic primer or probe based qPCR detection technology For a CellsDirect kit with fluorescent binding dye technology we recommend the CellsDirect SYBR Green Two Step qRT PCR Kit see page vi for ordering information Continued on next page Guidelines and Recommendations One Step qRT PCR continued Detecting Genomic DNA RNaseOUT 2X Reaction Mix Note Magnesium Concentration To detect genomic DNA targets in the lysate use primers specific for your targets in the one step reaction and omit the 50 C cDNA synthesis step in the cycling program SuperScript III RT in the enzyme mix will be denatured during the 95 C PCR incubation Alternatively you can use 2 units of Platinum Tag DNA Polymerase in place of the SuperScript III RT Platinum Taq enzyme mix in the one step qRT PCR reaction RNaseOUT Recombinant Ribonuclease Inhibitor Cat No 10777 019 is included in t
3. M and Daiss J L 1994 Antibodies as thermolabile switches high temperature triggering for the polymerase chain reaction BioTechnology 12 506 509 Westfall B Sitaraman K Solus J Hughes J and Rashtchian A 1997 Focus 19 46 2010 2011 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Notes invitrogen by Life technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
4. Mix of Lysis Solution Lysis Enhancer and Resuspension Buffer may be prepared for multiple reactions Transfer 1 2 ul of cells 10 000 cells to the PCR tube well and cap or seal Vortex briefly and spin down the contents Note This mixture may be frozen and stored at 80 C until use Thaw on ice before proceeding to the next step Transfer the tube plate to an incubator water bath or thermal cycler that has been preheated to 75 C and incubate for 10 minutes After incubation spin the tube or plate briefly to collect any condensation Proceed to DNase I Digestion Optional page 12 or If you do not perform the optional DNase I digestion adjust the Mg concentration by adding 1 pl of 50 mM MgSO to each tube well Then proceed to One Step qRT PCR Guidelines and Recommendations page 13 Lysing Cells in Tissue Culture Wells Introduction This section provides a protocol for lysing cells directly in tissue culture wells e 2 in 6 well to 384 well plates For cells in larger vessels see Lysing Larger Volumes of Cells page 4 Cell Seeding For adherent cells grown in tissue culture wells seed cells so Density that 10 ul of resuspended cells will yield the desired concentration Required The following materials are provided by the user Materials e Mammalian cell cultures in growth medium e Centrifuge for pelleting cells e Incubator or thermal cycler preheated to 75 C e 1X c
5. REA end 24 BE Oi ci i a ae E R a aa e a 24 Purchaser Notificatiori ees teatri ien recien entes ea ia e E ERa Uere dedu 27 Technical See a a a a a a erra ae arera aE 29 i neiesten eser eei ara ene Sera a iiaeeeaino eR 31 iii iv Kit Contents and Storage Shipping and Kit components are shipped on dry ice and should be stored Storage at 20 C Stability can be extended by storing components at 80 C Note the following special storage conditions Catalog nos 11753 100 and 11753 500 Store tube of ROX Reference dye at 20 C in the dark Catalog nos 11754 100 and 11754 500 Store the 2X Reaction Mix containing ROX Reference dye at 20 C in the dark Kit Catalog nos 11753 100 and 11753 500 contain a separate Components tube of ROX Reference Dye Catalog nos Kit Size 11753 100 and Component 100 rxns 500 rxns 11753 500 Resuspension Buffer 10 ml 10 ml Lysis Enhancer 1ml 1ml DNase I Amplification Grade 1 U ul 500ul 2x125ml 10X DNase I Buffer 160 ul 800 ul 25 mM EDTA 400 pl 2x1ml SuperScript III RT Platinum Taq Mix with RNaseOUT Ribonuclease Inhibitor 100 pl 500 pl 2X Reaction Mix 2 x 1 25 ml 12 5 ml 50 mM MgSO 1ml 1ml DEPC treated water 2ml 12 5 ml HeLa Total RNA 10 ng ul 10 ul 10 ul ROX Reference Dye 100 pl 500 ul Kit Catalog nos 11754 100 and 11754 500 contain ROX Components Reference Dye in the 2X Reaction Mix Catalog nos Kit Size 11754 100 and Component 100 rxns 500 rxns 11754 500 Re
6. each well The master mix should cover the cells in the well Incubate the plates on ice for up to 10 minutes During that period tap the plate periodically and check the cells under a microscope every 2 3 minutes to see whether they have detached or burst After 10 minutes gently pipet the cells up and down to dislodge the remaining attached cells Transfer 10 pl of the cell suspension to a 0 2 ml thin walled PCR tube or plate well Cap or seal the tube plate and transfer to an incubator or thermal cycler that has been preheated to 75 C Incubate for 10 minutes After incubation spin the tube or plate briefly to collect any condensation Proceed to DNase I Digestion Optional page 12 Or If you do not perform the optional DNase I digestion adjust the Mg concentration by adding 1 pl of 50 mM MgSO to each tube well Then proceed to One Step qRT PCR Guidelines and Recommendations page 13 Continued on next page Lysing Cells Obtained by LCM Introduction LCM Using Arcturus CapSure Caps Required Materials This section provides a protocol for lysing cells from frozen samples obtained by LCM The protocols in this section assume that you are using frozen LCM samples collected on Arcturus CapSure caps Two alternate lysis methods are described the cap method or the polymer peel method Gallup et al 2005 The following materials are provided by the user e Mammalian cells obtained via LCM an
7. stable baseline in multiplex reactions It is composed of a glycine conjugate of 5 carboxy X rhodamine succinimidyl ester ROX is either included in the kit in a separate tube Catalog nos 11753 100 and 11753 500 or as a component of the 2X Reaction Mix at a final concentration of 500 nM Catalog nos 11754 100 and 11754 500 TM Melting curve analysis available with LUX Fluorogenic Primers should be performed immediately after qRT PCR to identify the presence of primer dimers and analyze the specificity of the reaction Melting curve analysis can identify primer dimers by their lower annealing temperature compared to that of the amplicon The presence of primer dimers in samples containing template decreases PCR efficiency and obscures analysis and determination of cycle thresholds For multiplex applications different fluorescent reporter dyes are used to label separate primers or probes for quantification of different genes For relative expression studies using multiplex PCR the amount of primer for the reference gene e g B actin or GAPDH should be limited to avoid competition between amplification of the reference RNA and the sample gene In general the final concentration of the reference gene primer should be between 25 and 100 nM A primer titration is recommended for optimal results If additional optimization is required we first recommend increasing the MgSOsin the reaction from 3 mM to 6 mM using the ex
8. NA is degraded Add control total HeLa RNA to sample to determine if RNase is present in the first strand reaction The optional DNase I digestion can hydrolyze the RNA in the sample if the digestion time is too long Use a digestion time of 10 minutes Take appropriate cautions to prevent RNase contamination Inefficient CDNA synthesis Adjust cDNA synthesis temperature and or primer design Double the amount of reverse primer e g to 400 nM Inefficient PCR amplification Optimize PCR conditions Adjust annealing temperature as necessary Increase magnesium concentration Redesign primers Continued on next page 25 Troubleshooting continued Product detected at Template or PCR Isolate source of contamination and replace lower than carry over reagent s Use separate dedicated pipettors for expected cycle contamination reaction assembly and post PCR analysis number and or Assemble reactions except for lysate in a positive signal DNA free area Use aerosol resistant pipet tips from no template or positive displacement pipettors controls Unexpected bands Contamination by Include the optional DNase Digestion step For after genomic DNA larger samples 71 000 cells use a longer electrophoretic DNase I incubation time i e up to 10 minutes analysis Design primers that anneal to the target sequence in exons on both sides of an intron or the exon exon boundary of mRNA to diff
9. RT control reaction containing 2 units of Platinum Taq DNA Polymerase see page vi for ordering information instead of the SuperScript III RT Platinum Taq Mix e For the positive control use 1 pl of the control HeLa RNA provided with the kit instead of cell lysate For the no template control omit the lysate Continued on next page One Step qRT PCR with Optional ROX continued Protocol for LUX Primers Use the following protocol with LUX Primers A standard 50 pl reaction size is provided component volumes can be scaled as desired e g scaled down to a 20 11 reaction volume for 384 well plates For smaller reactions note that using a full 1 ul of SuperScript III RT Platinum Taq Mix may increase sensitivity 1 Program your real time instrument to perform cDNA synthesis immediately followed by PCR amplification as described on page 17 Set up reactions on ice For multiple reactions prepare a master mix of common components add the appropriate volume to each tube or plate well on ice and then add the unique reaction components e lysate Preparation of a master mix is crucial in qRT PCR to reduce pipetting errors Note Be careful to thaw the 2X Reaction Mix completely before use and vortex to mix Component Single rxn SuperScript III RT Platinum Taq Mix 1ul 2X Reaction Mix 25 ul LUX labeled primer 10 uM 1ul Unlabeled primer 10 uM 1ul ROX Reference Dye optional 1 pl 0 1 ul Lysat
10. T the ABI 7300 Real Time PCR System and the ABI GeneAmp 5700 These kits are not compatible with instruments that do not use ROX or instruments that use ROX at a final concentration lower than 500 nM e g the ABI 7500 and the Stratagene Mx3000P Mx3005P and Mx40009 Continued on next page 13 Guidelines and Recommendations One Step qRT PCR continued Primers Detection Methods 14 Gene specific primers are required for one step qRT PCR LUX Fluorogenic Primers are available separately from Invitrogen see below for more information A final concentration of 200 nM per primer is effective for most reactions Doubling the amount of reverse primer to 400 nM may improve performance of certain reactions Optimal results may require a primer titration between 100 and 500 nM LUX Fluorogenic Primers LUX Primers are fluorogenic primers for qRT PCR Each LUX Primer set includes one primer labeled with single fluorophore and one corresponding unlabeled primer The labeled primer is designed with a hairpin structure that provides built in fluorescence quenching When the primer is incorporated into double stranded PCR product and extended fluorescence increases by up to 10 fold For more information visit www invitrogen com lux To design LUX Primers for specific targets visit www invitrogen com dluxdesigner Predesigned and functionally validated Certified LUX Primer Sets are also
11. and incubate in a thermal cycler preheated to 75 C for 5 minutes After incubation spin the tube briefly to collect any condensation Proceed to DNase I Digestion Optional page 12 or If you do not perform the optional DNase I digestion adjust the Mg concentration by adding 1 ul of 50 mM MgSO to the tube Then proceed to One Step qRT PCR Guidelines and Recommendations page 13 11 DNase Digestion Optional Introduction In this optional step you treat the cell lysate with DNase I to degrade the genomic DNA Materials The following materials are provided in the kit Needed e 10X DNase I Buffer e DNase I Amplification Grade e 25mMEDTA DNase 1 To each tube plate well add the following components Digestion on ice Component Amount per sample DNase I Amplification Grade 1 U pl 5 pl 10X DNase I Buffer 1 6 pl Mix by gently pipetting up and down or briefly vortexing and spin briefly to collect the contents Incubate the tube plate at 25 C or room temperature for 5 minutes Note A longer incubation time up to 10 minutes may be used for larger samples gt 5 000 cells However incubation times exceeding 10 minutes can greatly reduce yield Spin briefly and add 4 pl of 25 mM EDTA to each tube plate well on ice Mix by gently pipetting up and down and spin briefly to collect the contents Incubate at 70 C for 10 minutes Spin briefly and place the tube or plate on ice before pr
12. ct genomic DNA targets Continued on next page Introduction continued SuperScript Ill RT Platinum Taq DNA Polymerase Control RNA SuperScript III Reverse Transcriptase RT is an engineered version of M MLV RT with reduced RNase H activity and increased thermal stability Gerard et al 1986 Kotewicz et al 1985 The enzyme can synthesize cDNA at a temperature range of 42 60 C providing increased specificity higher yields of cDNA and more full length product than other reverse transcriptases Because SuperScript III RT is not inhibited significantly by ribosomal and transfer RNA it can effectively synthesize cDNA directly from total RNA Platinum Taq DNA is a recombinant Tag DNA polymerase complexed with a proprietary antibody that inhibits polymerase activity at ambient temperatures Chou et al 1992 Sharkey et al 1994 Westfall et al 1997 Full polymerase activity is restored after the denaturation step in PCR providing an automatic hot start for increased amplification efficiency sensitivity and yield HeLa Total RNA is included in the kit for use as a positive control The concentration of HeLa Total RNA provided 10 ng pl is equivalent to 1 000 cells Methods General Guidelines for Lysing Cells Introduction Cell Types and Density T Important This section provides general guidelines for lysing cells You may perform an optional DNase I digestion to remov
13. d immobilized on polymer film lining an Arcturus CapSure cap e Centrifuge for pelleting cells e Heat block preheated to 75 C for the cap method e 1 7 ml Eppendorf microcentrifuge tube for the cap method e Thermal cycler or incubator preheated to 50 C and 75 C for the polymer peel method e RNase and DNase free forceps for the polymer peel method e 02 ml thin walled PCR tubes e Pipettes The following materials are provided in the kit e Resuspension Buffer e Lysis Enhancer e MgSO 50 mM optional For collection of very small samples using LCM i e 100 cells or less glycogen is commonly suggested This kit is compatible with samples collected with glycogen Continued on next page Lysing Cells Obtained by LCM continued Cap Lysis Method 10 The following method lyses cells obtained by LCM directly on the Arcturus CapSure cap 1 Fora single reaction prepare 11 ul of Lysis Solution by adding 1 ul of Lysis Enhancer to 10 ul of Resuspension Buffer Note A Master Mix may be prepared for multiple reactions 2 Invert the Arcturus CapSure cap and carefully pipet 11 ul of Lysis Solution onto the immobilized cells Be careful to place the drop directly on the sample surface tension should keep this volume as a drop over the sample 3 Carefully fit a microcentrifuge tube upside down onto the cap Be careful not to disturb the surface tension of the Lysis Solution over the cell sam
14. e genomic DNA from the lysate prior to qRT PCR see page 12 However if your gene specific primers are well designed i e spanning an exon intron exon junction to avoid amplifying genomic DNA you may skip this step for convenience Alternatively you may use this kit to detect genomic DNA targets in the lysate omitting DNase I digestion The SuperScript One Step qRT PCR Kit has been optimized for small cell samples ranging from 1 to 10 000 cells This kit is compatible with several different mammalian cell lines including HeLa COS 7 293 Jurkat CV1 and primary cells including stem cells and neural cells Cells may be grown under a variety of conditions and treatments and any type of culture vessel can be used This kit is not intended for whole blood or macrophages e We recommend using a maximum of 10 000 cells per reaction Higher numbers of cells may inhibit one step qRT PCR and result in reduced yields and or truncated product e Make sure that all solutions and equipment that come in contact with the cells are sterile Always use proper sterile technique and work in a laminar flow hood when handling cells Lysing Larger Volumes of Cells Introduction Required Materials A Note This section provides a protocol for lysing larger volumes of cells i e cells in larger plates and flasks For smaller samples in tissue culture wells see Lysing Cells in Tissue Culture Wells starting on page 7
15. e 2 20 ul DEPC treated water to 50 ul See the table on page 18 for the amount concentration of ROX to use for your specific instrument Cap or seal the reaction tube PCR plate Centrifuge briefly to make sure that all components are at the bottom of the tube plate Place reactions in a preheated real time instrument programmed as described in step 1 Collect data and analyze results Continued on next page 19 One Step qRT PCR with Optional ROX continued Protocol for TaqMan Probes 20 Use the following protocol with TaqMan Probes A standard 50 pl reaction size is provided component volumes can be scaled as desired e g scaled down to a 20 ul reaction volume for 384 well plates For smaller reactions note that using a full 1 ul of SuperScript III RT Platinum Taq Mix may increase sensitivity 1 Program your real time instrument to perform cDNA synthesis immediately followed by PCR amplification as described on page 17 Set up reactions on ice For multiple reactions prepare a master mix of common components add the appropriate volume to each tube or plate well on ice and then add the unique reaction components e lysate Preparation of a master mix is crucial in qRT PCR to reduce pipetting errors Note Be careful to thaw the 2X Reaction Mix completely before use and vortex to mix Component Single rxn SuperScript III RT Platinum Taq Mix 1ul 2X Reaction Mix 25 ul Fo
16. eaction size is provided component volumes can be scaled as desired e g scaled down to a 20 11 reaction volume for 384 well plates For smaller reactions note that using a full 1 ul of SuperScript III RT Platinum Tag Mix may increase sensitivity 1 Program your real time instrument to perform cDNA synthesis immediately followed by PCR amplification as described on page 17 Set up reactions on ice For multiple reactions prepare a master mix of common components add the appropriate volume to each tube or plate well on ice and then add the unique reaction components e g lysate Note Preparation of a master mix is crucial in qRT PCR to reduce pipetting errors Component Single rxn SuperScript III RT Platinum Taq Mix 1ul 2X Reaction Mix with ROX 25 ul Forward primer 10 uM 1ul Reverse primer 10 uM 1ul Fluorogenic probe 1gul Lysate 2 20 ul DEPC treated water to 50 ul Cap or seal the reaction tube PCR plate Centrifuge briefly to make sure that all components are at the bottom of the tube plate Place reactions in a preheated real time instrument programmed as described in step 1 Collect data and analyze results 23 Appendix Troubleshooting Problem Possible Cause Suggested Solution Cells in tissue culture wells do not detach burst Incubation temperature of lysis reaction is too low Incubate lysis reaction at room temperature instead of on ice No amplification curve appea
17. ent For instrument specific protocols tips and troubleshooting including information about LUX Primers visit www invitrogen com qpcr For additional information about probes consult your instrument documentation and or your probe technology documentation 24 Continued on next page Troubleshooting continued Problem Possible Cause Suggested Solution Poor sensitivity Not enough template RNA Increase the number of cells used Scaled down reaction volume e g 20 ul does not include enough enzyme Use 1 ul of SuperScript III RT Platinum Taq Mix even in smaller reaction volumes Signals are present in no template controls and or multiple peaks are present in the melting curve graph Template or reagents are contaminated by nucleic acids DNA cDNA Use melting curve analysis if possible and or run the PCR products on a 4 agarose gel after the reaction to identify contaminants Include the optional DNase I digestion step Primer dimers or other primer artifacts are present Use melting curve analysis to identify primer dimers by their lower melting temperature if possible We recommend using validated pre designed primer sets or designing primers or primer probe combinations using dedicated software programs or primer databases Check the purity of your primers by gel electrophoresis Product detected at higher than expected cycle number R
18. erentiate between amplification of CDNA and potential contaminating genomic DNA To test if products were derived from DNA prepare a negative RT control Nonspecific Vary the annealing conditions annealing of qPCR Optimize magnesium concentration for each pHinere template and primer combination 26 Purchaser Notification Limited Use Label License No 358 Re search Use Only Trademarks The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 PRISM and GeneAmp are registered trademarks of Applera Corporation CapSure is a registered trademark of Arcturus Inc TaqMan is a registered trademark of Roche Molecular Systems Inc iCycler Mx3000P Mx3005 Mx4000 Rotor Gene DNA Engine Opticon Ch
19. he SuperScript III RT Platinum Taq Mix to safeguard against degradation of target RNA due to ribonuclease contamination 2X Reaction Mix consists of a proprietary buffer system MgSO dNTPs and stabilizers all at optimized concentrations Catalog nos 11754 100 and 11754 500 also include ROX Reference Dye in the 2X Reaction Mix Be careful to thaw the 2X Reaction Mix completely before use and vortex briefly to mix Incomplete thawing may result in a salt concentration that is too low which may reduce the efficiency of the qRT PCR reaction The 2X Reaction Mix provided with each kit supplies a final magnesium concentration of 3 mM This works well for most targets however the optimal concentration may range from 3 to 6 mM If necessary use the separate tube of 50 mM magnesium sulfate to increase the magnesium concentration Use the following table to determine the amount of MgSO to add to achieve the specified concentration in a 50 ul PCR with 25 pl of 2X Reaction Mix Volume of 50 mM MgSO per 50 ul Rxn Final MgSO Conc iol 4 0 mM 2 pl 5 0 mM 3 pl 6 0 mM Decrease the amount of water in the reaction accordingly Continued on next page 15 Guidelines and Recommendations One Step qRT PCR continued ROX Reference Dye Melting Curve Analysis Multiplexing 16 ROX Reference Dye is used to adjust for non PCR related fluctuations in fluorescence between qPCR reactions and provides a
20. invitrogen by technologies CellsDirect One Step qRT PCR Kits For one step real time quantitative RT PCR from cell lysate Catalog Nos 11753 100 11753 500 11754 100 11754 500 Rev Date 25 May 2011 Manual part no 25 0870 MAN0000536 ii Table of Contents Kit Contents and Storage c ccccceccessesseseesessesssseseesesssssesesesssssssseseeseesessesusseeseeseassauesseseansaneseeseeses v Additional Products ia E EE a E EES E RRA vi Tint UH OD a icto oe ei e m etna e e e aca EERE E edens 1 Methods ic a etse dac EMI d 3 General Guidelines for Lysing Cells c ccccscssessesessesteseesessessesneseeseessssesteseeseaseenesneseesseneasesneaes 3 Lysing Larger Volumes of Cells sss tenente entente 4 Lysing Cells in Tissue Culture Wells sssssssssseeseeneneeneetenettent tenete 7 Lysing Cells Obtained by LCM sssssssssseseseeeeeenennene tenete trennen 9 DNase I Digestion Optional c ccccesssssesessesesesseseeseeseesssseseeseesssneseeseesessesnsseeseessensseseeseeneenes 12 Guidelines and Recommendations One Step qRT PCR sss 13 Cycling Programs One Step qRT PCR ccscssessessesessesesseseeseesesesteseeseesesseaneseeseesssneaneseeseenesses 17 One Step qRT PCR with Optional ROX c cceccscssesessesesteseeseeseseseeseeseesessesnesseseaseaneseeseeseaneanes 18 One Step qRT PCR with ROX in the 2X Reaction Mix 21 Appendix e ee LL UU
21. ng the CellsDirect One Step qRT PCR Kit you lyse the cells and add the complete lysate directly to a one step qRT PCR reaction with minimal handling and sample loss You can add DNase I to the lysate as an optional step to eliminate genomic DNA prior to qRT PCR Alternatively you can analyze genomic DNA targets by omitting the DNase T step and using qPCR primers designed for your genomic sequence of interest The CellsDirect One Step qRT PCR Kit has been optimized for small cell samples ranging from 10 000 cells down to a single cell This kit is compatible with fluorogenic primer technology such as LUX Primers or fluorogenic probe based technology such as TaqMan probes The kit is also compatible with high throughput applications and frozen samples obtained by Laser Capture Microdissection LCM This kit offers the following advantages e Eliminates time consuming purification procedures that contribute to sample loss and PCR inhibition e Compatible with a wide range of mammalian cell types grown under different treatment conditions e Total lysate volume may be used depending on the capacity of your real time instrument increasing sensitivity and allowing for detection of rare transcripts e Convenient one step enzyme mix includes both SuperScript III Reverse Transcriptase and Platinum Taq DNA Polymerase e Compatible with high throughput applications and frozen samples obtained by LCM e Can also be used to dete
22. oceeding to the next section 12 Guidelines and Recommendations One Step qRT PCR Introduction Important Amount of Starting Material Instrument Compatibility This section provides guidelines for setting up your one step qRT PCR reaction Since PCR is a powerful technique capable of amplifying trace amounts of DNA all appropriate precautions should be taken to avoid cross contamination The amount of lysate you can use in the qRT PCR reaction depends on your real time instrument and the total reaction volume it can accommodate For a 50 ul total reaction volume up to 20 pl of lysate may be used For a 20 ul total reaction volume up to 10 pl of lysate may be used Catalog nos 11753 100 and 11753 500 These kits can be used with a variety of real time instruments including but not limited to the ABI PRISM 7000 7700 and 7900HT the ABI 7300 and 7500 Real Time PCR Systems the ABI GeneAmp 5700 the Bio Rad iCycler the Stratagene Mx3000P Mx3005P and Mx4000 the Corbett Research Rotor Gene the MJ Research DNA Engine Opticon Opticon 2 and Chromo 4 Real Time Detector and the Cepheid Smart Cycler Optimal cycling conditions will vary with different instruments Catalog nos 11754 100 and 11754 500 These kits include ROX Reference Dye in the 2X Reaction Mix at a final concentration of 500 nM This concentration is compatible with the ABI PRISM 7000 7700 and 7900H
23. old phosphate buffered saline PBS without calcium or magnesium e 0 2 ml thin walled PCR tubes or PCR plates e Ice e Pipettes The following materials are provided in the kit e Resuspension Buffer e Lysis Enhancer e MgSO 50 mM optional Preparing For multiple reactions prepare a Master Mix of Lysis Master Mix of Solution Resuspension Buffer Lysis Enhancer in a ratio of Lysis 10 1 See the chart below for the minimum volume of Lysis Solution Solution required to cover the cells in each type of tissue culture well Larger volumes may be used if desired Tissue Culture Volume per Well Plate Resuspension Buffer Lysis Enhancer Total 6 well 400 nl 40 pl 440 ul 12 well 200 pl 20 pl 220 pl 24 well 100 ul 10 ul 110 ul 48 well 50 ul 5 pl 55 pl 96 well 20 pl 2 pl 22 ul 384 well 10 ul 1ul 11 ul Continued on next page Lysing Cells in Tissue Culture Wells Continued Q Important Direct Lysis of Cells in Tissue Culture Wells If you are processing many samples additional Resuspension Buffer and Lysis Enhancer may be required You can order additional CellsDirect Resuspension Buffer and Lysis Enhancer from Invitrogen see page vi For adherent cells grown in tissue culture wells perform the following lysis procedure Aspirate the medium in each well and wash each well with 1X cold PBS without magnesium calcium Aspirate the PBS Add the Lysis Solution master mix see table on previous page to
24. on times may vary for different target sequences Select Fast Mode on the Thermal Profile tab 50 C for 5 minutes hold 95 C for 2 minutes hold 40 50 cycles of 95 C 3 seconds 60 C 30 seconds For LUX Primers only Perform melting curve analysis Refer to your specific instrument documentation 17 One Step qRT PCR with Optional ROX Introduction This section provides general reaction setup and protocol instructions for kits with ROX supplied as a separate tube Catalog nos 11753 100 and 11753 500 The use of ROX Reference Dye is optional The following Note instruments do not use ROX Bio Rad iCycler Corbett Research Rotor Gene MJ Research DNA Engine Opticon Opticon 2 and Chromo 4 Real Time Detector and Cepheid Smart Cycler Amount of ROX Reference Dye is supplied at a 25 uM concentration Use ROX to Use the following table to determine the amount of ROX to use with your particular instrument Amount of ROX per Final ROX Instrument f 50 pl reaction Concentration ABI 7000 7300 7700 and 7900HT 1 0 pl 500 nM ABI 7500 Stratagene Mx3000 Mx3005P and Mx4000 0 1 pl aoa To accurately pipet 0 1 ul per reaction we recommend diluting ROX 1 10 immediately before use and use 1 ul of the dilution Control Reactions 18 For the reactions in this section set up the following controls e To test for genomic DNA contamination prepare a negative
25. ple 4 With the tube still inverted on the cap place the cap and tube assembly directly on a heat block pre heated to 75 C and incubate for 15 minutes 5 After incubation vortex the inverted cap and tube assembly briefly and then turn the tube right side up and spin down briefly to collect the contents to the bottom of the tube Discard the cap 6 Proceed to DNase I Digestion Optional page 12 or If you do not perform the optional DNase I digestion adjust the Mg concentration by adding 1 pl of 50 mM MgSO to the tube Then proceed to One Step qRT PCR Guidelines and Recommendations page 13 Continued on next page Lysing Cells Obtained by LCM continued Polymer Peel Lysis Method The following method involves peeling off the polymer film with the attached cells from the Arcturus CapSure cap and then lysing the cells in a tube 1 Using clean forceps that are RNase and DNase free carefully peel off the polymer film sticker with the attached cells from the cap Place the polymer film at the bottom of a 0 2 ml thin walled PCR tube and add 10 pl of Resuspension Buffer and 1 pl of Lysis Enhancer a Master Mix may be prepared for multiple reactions Incubate the tube in a thermal cycler or incubator at 50 C for 10 minutes Vortex the tube and spin down briefly to collect the polymer and other debris at the bottom of the tube Transfer the aqueous solution to a new 0 2 ml thin walled PCR tube Cap the tube
26. romo 4 and Smart Cycler are trademarks or registered trademarks of their respective companies 27 Technical Service World Wide Visit the Invitrogen Web site at www invitrogen com for Web Technical resources including manuals vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete technical service contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Tel Toll Free 1 800 955 Paisley PA4 9RF UK Minato ku Tokyo 108 0022 44 0 141 814 6100 Tel 81 3 5730 6509 6288 Tech Fax 44 0 141 814 Fax 1 760 602 6500 FARS uae Dnus 6117 E mail E mail E E mail tech_service invitrogen com jpinfo invitrogen com eurotech invitrogen com SDS Safety Data Sheets SDSs are available at www invitrogen com sds Certificate of The Certificate of Analysis provides detailed quality control and Analysis product qualification information for each product Certificates of Analysis are available on our website Go to www invi
27. rs on the qPCR graph There is no PCR product Run the PCR product on a gel to determine whether PCR worked Then proceed to the troubleshooting steps below No PCR product is evident either in the qPCR graph or on a gel Procedural error Confirm that all steps were followed Use the control RNA to verify the efficiency of the reaction see the next page on troubleshooting with the Control RNA RNA is degraded Add control total HeLa RNA to sample to determine if RNase is present in the first strand reaction The optional DNase I digestion can hydrolyze the RNA in the sample if the digestion time is too long Use a digestion time of 10 minutes Take appropriate cautions to prevent RNase contamination Fluorescent probe not functional Validate probe design and presence of fluorophore and quencher Treat TaqMan Probe with DNase and check for increase in fluorescence Redesign and or resynthesize probe if necessary Target mRNA contains strong transcriptional pauses Maintain an elevated temperature after the annealing step Redesign the primers Increase the temperature of cDNA synthesis up to 60 C PCR product is evident in the gel but not on the qPCR graph qPCR instrument settings are incorrect Confirm that you are using the correct instrument settings dye selection reference dye filters acquisition points etc Problems with your specific qPCR instrum
28. rward primer 10 uM 1ul Reverse primer 10 uM 1gul Fluorogenic probe lul ROX Reference Dye optional 1 pl 0 1 ul Lysate 2 20 ul DEPC treated water to 50 ul See the table on page 18 for the amount concentration of ROX to use for your specific instrument Cap or seal the reaction tube PCR plate Centrifuge briefly to make sure that all components are at the bottom of the tube plate Place reactions in a preheated real time instrument programmed as described in step 1 Collect data and analyze results One Step qRT PCR with ROX in the 2X Reaction Mix Introduction This section provides general reaction setup and protocol instructions for kits with ROX included in the 2X Reaction Mix Catalog nos 11754 100 and 11754 500 These kits include ROX Reference Dye in the 2X Reaction Mix Note at a final concentration of 500 nM For information about instrument compatibility see page 13 Control For the reactions in this section set up the following controls Reactions e To test for genomic DNA contamination prepare a negative RT control reaction containing 2 units of Platinum Tag DNA Polymerase see page vi for ordering information instead of the SuperScript III RT Platinum Taq Mix e For the positive control use 1 ul of the control HeLa RNA provided with the kit instead of cell lysate For the no template control omit the lysate Continued on next page 21 One Step qRT PCR with ROX in the 2X Reaction Mi
29. suspension Buffer 10 ml 10 ml Lysis Enhancer 1ml 1ml DNase I Amplification Grade 1 U ul 500pl 2x125ml 10X DNase I Buffer 160 pl 800 ul 25 mM EDTA 400 pl 2x1ml SuperScript III RT Platinum Tag Mix with RNaseOUT Ribonuclease Inhibitor 100 pl 500 ul 2X Reaction Mix with ROX 2 x 1 25 ml 12 5 ml 50 mM MgSO 1ml 1ml DEPC treated water 2ml 12 5 ml HeLa Total RNA 10 ng ul 10 ul 10 ul Continued on next page Vv Additional Products Product LUX Custom Primers CellsDirect Two Step qRT PCR Kit CellsDirect SYBR Green Two Step qRT PCR Kit CellsDirect cDNA Synthesis System TM CellsDirect Resuspension and Lysis Buffer Platinum Taq DNA Polymerase Size Catalog No To order visit www invitrogen com 100 rxns 500 rxns 100 rxns 500 rxns 25 rxns 100 rxns 10 ml Resuspension Buffer and 1 ml Lysis Enhancer 100 rxns 250 rxns 500 rxns 11737 030 11737 038 11738 060 11738 068 18080 200 18080 300 11739 010 10966 018 10966 020 10966 034 vi Introduction System Overview Advantages of the Kit The CellsDirect One Step qRT PCR Kit is an optimized kit for the detection and quantification of RNA or DNA directly from mammalian cell lysate without a separate purification step In traditional real time quantitative RT PCR qRT PCR you first isolate RNA from cells in a time consuming procedure that can lead to loss of material Usi
30. tra MgSO provided in the kit Then we recommend doubling the amount of Platinum Tag DNA Polymerase to 0 06 U per pl of reaction volume or 3 U per 50 ul reaction Add Platinum Taq DNA polymerase stand alone enzyme see page vi for ordering information to double the amount of enzyme Cycling Programs One Step qRT PCR Introduction Standard Cycling Program Fast Cycling Program for the ABI 7500 in Fast Mode This section provides general cycling programs for one step qRT PCR on ABI real time instruments For more instrument specific programs go to www invitrogen com qpcr To detect genomic DNA targets omit the 50 C cDNA synthesis step SuperScript III RT will be denatured in the 2 minute 95 C PCR incubation Program your real time instrument to perform cDNA synthesis immediately followed by PCR amplification as shown below Optimal temperatures and incubation times may vary for different target sequences 50 C for 15 minutes hold cDNA synthesis temperature may range from 42 60 C time may range from 5 20 minutes 95 C for 2 minutes hold 40 50 cycles of 95 C 15 seconds 60 C 30 45 seconds 60 seconds for the 7900HT For LUX Primers only Perform melting curve analysis Refer to your specific instrument documentation Program the ABI 7500 with Fast Mode capability to perform cDNA synthesis immediately followed by PCR amplification as shown below Optimal temperatures and incubati
31. trogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Continued on next page 28 Technical Service Continued Limited Warranty Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 10076 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If yo
32. u discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 29 30 References Chou Q Russel M Birch D Raymond J and Bloch W 1992 Prevention of pre PCR mis priming and primer dimerization improves low copy number amplifications Nucl Acids Res 20 1717 Gallup J Kawashima K Lucero G and Ackerman M 2005 New quick method for isolating RNA from laser captured cells stained by immunofluorescent immunohistochemistry RNA suitable for direct use in fluorogenic TaqMan one step real time RT PCR Biological Procedures Online 7 70 92 Gerard G F D Alessio J M Kotewicz M L and Noon M C 1986 Influence on stability in Escherichia coli of the carboxy terminal structure of cloned Moloney murine leukemia virus reverse transcriptase DNA 5 271 279 Kotewicz M L D Alessio J M Driftmier K M Blodgett K P and Gerard G F 1985 Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli Gere 35 249 258 Sharkey D J Scalice E R Christy K G Atwood S
33. ute under a microscope 5 When all the cells have detached add serum containing medium to a final volume equaling the volume of PBS used in the Step 1 for 6 and 12 well plates add a 1X 2X volume of medium Note that the medium must contain serum to inactivate the trypsin 6 Pipetthe cells gently up and down to mix and then transfer the cell suspension to a centrifuge tube 7 Spinthecells at 200 x 2 for 5 minutes to pellet or spin at the recommended speed and time appropriate for your cell line 8 Aspirate the medium and wash the cell pellet with 5 10 ml of 1X cold PBS 9 Spin the cells at 200 x g for 5 minutes to pellet 10 Aspirate the PBS and resuspend the pellet in 500 pl to 1 ml of 1X cold PBS Mix the cell solution gently Protocol continued on next page Continued on next page Lysing Larger Volumes of Cells continued Lysis Procedure continued Protocol continued from previous page A 12 13 14 15 16 17 Collect a small aliquot 10 pl to verify that the cells are at the desired concentration Determine cell density electronically using a Coulter Counter or manually using a hemacytometer chamber The cell density should be 10 000 cells ul If necessary adjust the density using cold PBS Count the cells again to verify cell concentration To a 0 2 ml thin walled PCR tube or plate well on ice add 1 pl of Lysis Enhancer and 10 pl of Resuspension Buffer Note A Master
34. x continued Protocol for LUX Primers 22 Use the following protocol with LUX Primers A standard 50 pl reaction size is provided component volumes can be scaled as desired e g scaled down to a 20 ul reaction volume for 384 well plates For smaller reactions note that using a full 1 ul of SuperScript III RT Platinum Tag Mix may increase sensitivity 1 Program your real time instrument to perform cDNA synthesis immediately followed by PCR amplification as described on page 17 Set up reactions on ice For multiple reactions prepare a master mix of common components add the appropriate volume to each tube or plate well on ice and then add the unique reaction components e g lysate Note Preparation of a master mix is crucial in qRT PCR to reduce pipetting errors Component Single rxn SuperScript III RT Platinum Taq Mix 1ul 2X Reaction Mix with ROX 25 ul LUX labeled primer 10 uM 1nul Unlabeled primer 10 uM Tul Lysate 2 20 pl DEPC treated water to 50 ul Cap or seal the reaction tube PCR plate Centrifuge briefly to make sure that all components are at the bottom of the tube plate Place reactions in a preheated real time instrument programmed as described in step 1 Collect data and analyze results Continued on next page One Step qRT PCR with ROX in the 2X Reaction Mix continued Protocol for TaqMan Probes Use the following protocol with TaqMan Probes A standard 50 pl r
Download Pdf Manuals
Related Search