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Arcturus® RiboAmp® PLUS RNA Amplification Kit User Guide, (PN

1. Reagent Maker Catalog Number CY 3 mono reactive dye Amersham PA23001 CY 5 mono reactive dye Amersham PA25001 Alexa Fluor 647 reactive Molecular A 32756 dye decapacks for Probes microarrays Alexa Fluor 555 reactive Molecular A 32757 dye decapacks for Probes microarrays Labeling reaction Re suspend 1 mg monoreactive dye in 51 uL of DMSO Teal DMSO Save unused vials in the dark at 2 6 C 1 Take 15 ug of amino allyl aRNA in 7 5 uL of nuclease free water Note Maintain sample on a cold block 25 OM aR ye Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Add 2 5 uL of Labeling Buffer Teal LB to the sample Add 10 uL of the re suspended dye into 10 uL of the sample Mix thoroughly by flicking the tube Spin down briefly Incubate at room temperature in the dark for 1 hour Proceed directly to purification of labeled aRNA 43 aRNA purification 44 Appendix A Amino allyl aRNA Labeling Protocol aRNA purification 1 Pre treat column by adding 250 ul of RNA Binding Buffer Blue RB to a new purification column Incubate the column at room temperature for 5 minutes Centrifuge at 16 000 x g for 1 minute Add 225 ul of RNA Binding Buffer to the transcript labeling reaction sample and mix thoroughly Pipette the entire sample volume into the purification column Centrifuge at 100 x g or lowest speed setting available for 2 minutes and immediately centrifuge at 10 000 x g
2. 12 1 round amplifications or 6 2 round amplifications Note This is the User Guide for the kit listed above KIT0525 RiboAmp HS PLUS 6 2 round amplifications Turbo Labeling Kits The Turbo Labeling Kits provide a proprietary non enzymatic technology for the labeling of unmodified aRNA for gene expression profiling The unmodified aRNA is labeled post amplification thereby avoiding the need to incorporate modified nucleotides The use of natural nucleotides in the amplification step results in unmodified aRNA with higher yields and longer aRNA fragments thus providing better representation of the mRNA transcript for downstream analysis KIT0608 Biotin 12 samples KIT0609 CY 3 12 samples KIT0610 CY 5 12 samples Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 55 56 Appendix E Related Reagent Kits Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chemical Safety This appendix covers General chemical safety 5 06 65 cc kc Eka l Wa see al l bbe ee cee wees 57 Safety Data Sheets varia dr Ad Web idad 58 Chemical waste safety 0 a lala aa rama ae 59 General chemical safety Chemical hazard N WARNING CHEMICAL HAZARD Before handling any chemicals refer to warning the Safety Data Sheet SDS provided by the manufacturer and observe all relevant precautions N WARNING CHEMICAL HAZARD All chemicals in the instrument including liquid in the l
3. and disposed of according to all local state provincial and or national regulations Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 59 Biological hazard safety 60 Appendix F Chemical Safety Chemical waste safety IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply General biohazard hy WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety publications index htm Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara
4. consult the SDS Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 57 Appendix F Chemical Safety Safety Data Sheets e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal Safety Data Sheets About SDSs Obtaining SDSs 58 Chemical manufacturers supply current Safety Data Sheets SDSs with shipments of hazardous chemicals to new customers They also provide SDSs with the first shipment of a hazardous chemical to a customer after an SDS has been updated SDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new SDS packaged with a hazardous chemical be sure to replace the appropriate SDS in your files The SDS for any chemical supplied by Applied Biosystems is available to you free 24 hours a day To obtain SDSs 1 2 Go to www appliedbiosystems com click Support then select SDS In the Keyword Search field enter the chemical name product name SDS part number or other information that appears in the SDS of interest S
5. for 2 minutes and immediately centrifuge at 10 000 x g for 1 minute Add 200 uL of RNA Wash Buffer Blue RW to the purification column and centrifuge at 10 000 x g for 1 minute Add 200 uL of fresh RNA Wash Buffer to the purification column and centrifuge at 16 000 x g for 2 minutes Check the column for any residual wash buffer If any wash buffer remains re centrifuge at 16 000 x g for 1 minute Discard the collection tube and flow through IMPORTANT Avoid splashing flow through in the collection tube onto the purification column If flow through waste liquid wets the outside of the purification column re centrifuge the column at 16 000 x g to remove the liquid Place the purification column into a new 0 5 mL microcentrifuge tube provided in the Kit and carefully add 30 uL of RNA Elution Buffer Blue RE directly to the center of the purification column membrane Note Gently touch the tip of the pipette to the surface of the membrane while dispensing RE to ensure maximum absorption of RE into the membrane Gently tap the purification column to distribute the buffer if necessary Incubate for one minute at room temperature Place each column tube assembly into the 2 mL support tube in the rotor with the 0 5 mL tube cap trailing the tube Centrifuge at 1000 x g for 1 minute followed immediately by 16 000 x g for 1 minute Discard the column and retain the elution containing the aRNA IMPORTANT To avoid potential breakage o
6. 1 1st Strand Enzyme Mix Red 2 Enhancer Yellow E Nuclease Mix Orange 2nd Strand Master Mix Green 1 2nd Strand Enzyme Mix Green 2 Primer A Grey A Primer B Grey B Control RNA Purple C Table 5 In Vitro Transcription IVT 1 round RA7008 Component Vial Color Vial Label IVT Buffer Blue 1 IVT Master Mix Blue 2 IVT Enzyme Mix Blue 3 DNase Mix Blue 4 Table 6 In Vitro Transcription IVT 2 round RA7009 Component Vial Color Vial Label IVT Buffer Blue 1 IVT Master Mix Blue 2 IVT Enzyme Mix Blue 3 DNase Mix Blue 4 Table 7 Amino allyl IVT RA7010 Component Vial Color Vial Label IVT Buffer Blue 1 IVT Master Mix Blue 2 IVT Enzyme Mix Blue 3 DNase Mix Blue 4 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide DNA RNA purification modules Chapter 2 Kit Components Component Vial Color Vial Label Amino allyl IVT Master Mix Teal AA Labeling Buffer Teal LB DMSO Teal DMSO Table 8 cDNA aRNA Purification RA7011 Component Vial Color Vial Label DNA Binding Buffer Red DB DNA Wash Buffer Red DW DNA Elution Buffer Red DE RNA Binding Buffer Blue RB RNA Wash Buffer Blue RW RNA Elution Buffer Blue RE 0 5 mL Microcentrifuge Tubes Purification columns with collection tubes Table 9 Amino allyl aRNA Labeling Purification RA7012 Component Vial Color Vial Label RNA Binding Buffer Blue RB RNA Wash Buffer Blue RW RNA Elution Buffer Blue RE 0 5 mL Microcentrifuge Tu
7. 2 0 and 2 6 indicates very pure aRNA Assessing input RNA sample quality This section provides instructions for assessing input RNA sample quality by quantitative Real Time PCR Although gel electrophoresis is a common approach to assessing RNA quality it is not possible to run a gel on the small quantities of input RNA used for amplification with the RiboAmp PLUS Kit Therefore you may want to assess the input RNA quality after 1 Strand Synthesis using quantitative Real Time PCR qRT PCR You can use the following guidelines To prepare Wipe all surfaces and equipment with RNase decontamination solution Use RNase free solutions and plastic ware Wear disposable gloves Perform amplification following the RiboAmp PLUS Kit protocol 1 During Round One 18 Strand Synthesis remove 2 uL of the sample 2 Pipette into a new 0 5 or 0 2 mL microcentrifuge tube Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 45 Appendix B aRNA Analysis 3 Add 8 uL of nuclease free water This is the diluted cDNA template 4 Mix the sample well Spin down and store on ice until ready to use 5 Proceed according to protocols and Instruction Manuals for the qRT PCR system utilized Use 2 uL of diluted cDNA template from step 4 Refer to the qRT PCR system manual for details concerning interpretation of data Assessing total RNA and aRNA quality This section covers assessing Total RNA and aRNA quality using the Agilent L
8. 5 RiboAmp PLUS Protocol IMPORTANT Tubes must be properly oriented in the rotor during elution To avoid potential breakage of the microcentrifuge tube cap during centrifugation insert the purification tube 0 5 mL tube assembly into a lidless 1 7 2 0 mL tube Insert this assembly into adjacent rotor holes see Figure 2 on page 52 Rest the tube cap against the tube immediately clockwise to it Place an empty lidless 1 7 2 0 mL tube into the rotor hole adjacent in the clockwise direction to the last assembly The purified aRNA is ready for use in a labeling reaction qRT PCR or for use in a second round of amplification as described in the following section of this User Guide Purified aRNA may be stored at 70 C Immediately proceed to Round Two or store the purified aRNA at 70 C overnight END OF ROUND ONE RiboAmp PLUS Round Two Protocol for performing a second round of amplification Round Two 1st strand cDNA synthesis 32 You can perform a second round of amplification to increase the yield of the amplification if necessary In second round amplification purified aRNA product from Round One is used to produce double stranded cDNA which in turn is used as template for an in vitro transcription reaction IMPORTANT Do not use the following second round amplification protocol without first performing a first round of amplification There are two significant differences between the first round and second round amp
9. Protocol Chapter contents RiboAmp PLUS Round One sin se laa eee ala te la ei 27 RiboAmp PLUS Round TWo 0 ccc cee eee eee erek dk 32 RiboAmp PLUS Round One Round One 1st IMPORTANT Read all of the notes on the previous chapter prior to beginning strand cDNA Note Do not leave reagents at room temperature Place components back onto the synthesis cold block or refreeze immediately after dispensing the reagent 1 Prepare RNA sample in a total volume of 10 11 uL in a 0 5 or 0 2 mL RNase free microcentrifuge tube and place on 4 8 C block For recommended sample input quantities see RNA input requirements and comparisons on page 11 2 Add 1 0 uL of Primer A Grey A mix well and spin down 3 Incubate at 65 C for 5 minutes then chill the samples to 4 C for at least 1 minute Hold the sample at 4 C until ready to proceed 4 Spin the contents down before proceeding to the next step 5 Thaw 1 Strand Synthesis components Red labeled Vials and place on ice Note 1 Strand Enzyme Mix Red 2 does not require thawing and can be placed directly on ice 6 Add reagents directly to the sample Note If you are performing several amplifications you will find it more convenient to prepare Complete 1 Strand Synthesis Mix according to Table 13 Table 13 Round One first strand reaction mix Complete 15t Strand Synthesis Mix 6 reaction Master Mix Component Amount Vial with 10 overage 1st
10. cap trailing the tube Centrifuge at 1000 x g for 1 minute immediately followed by 16 000 x g for 1 minute Discard the purification column and retain the elution containing the labeled aRNA Measure the O D of the product at A260 A280 and As59 Ags59 to determine the yield and frequency of incorporation FOI by making a dilution of 1 10 5 ul sample 45 ul nuclease free water Store any remaining samples at 70 C until ready for hybridization Arcturus RiboAmp PLUS RNA Amplification Kit User Guide aRNA Analysis This appendix covers Determining RNA yieldandpurity kK KK KK KK KK KK KK KK KK 45 Assessing input RNAsamplegualiy 45 Assessing total RNAandaRNAguality KK KK KK KK KK KK 46 Analyzing aRNA by agarose gel electrophoresis 47 Determining RNA yield and purity aRNA quantitation by ultraviolet light absorbance is the simplest approach to determining amplification yield An absorbance reading at 260 nm A260 using a spectrophotometer is taken on a diluted aliquot of aRNA Typically a 1 25 to 1 50 dilution of aRNA in nuclease free water is sufficient For single stranded RNA a measurement of A269 1 0 corresponds to 40 g mL The yield can by calculated by A go dilution factor 40 ug mL RNA Measuring A go and calculating the A go A go ratio indicates the purity of the RNA sample An A 69 A280 ratio between
11. during shipment Keep thawed reagents and reaction tubes in cold blocks at 4 8 C while adding reagents to samples Prior to each incubation mix samples thoroughly by flicking the reaction tube unless noted in protocol to ensure optimal performance Spin down before proceeding Do not vortex Use a microcentrifuge to spin down all components and samples following each mixing step Clean all amplification process equipment with an RNase eliminator such as RNase AWAY Molecular Bio Products to minimize the risk of RNase contamination During enzyme and buffer dispensing keep the reaction tube with sample on ice or chilled in a 4 8 C cold block Do not freeze samples unless it is indicated in the protocol Note Although excess enzyme and reagents are provided in all vials there is insufficient volume to prepare extra reactions 1 Thaw the frozen kit components as needed and mix by flicking the tube or by inverting the tubes several times spin down and place on ice When enzyme mixtures have been removed from 20 C storage for use always keep them in a cold block or in an ice bucket at the lab bench Allow In Vitro Transcription IVT Buffer Blue labeled Vial 1 Master Mix Blue labeled Vial 2 and Enhancer Yellow labeled Vial to assume room temperature and mix by inverting or flicking the tube Spin down if necessary Dissolve all visible solids prior to use Isolate RNA from LCM samples using the PicoPure RNA I
12. for 1 minute Note To obtain 15 ug of aRNA in 7 5 ul you can dry down 15 ug of aRNA and re suspend in 7 5 ul of nuclease free water or concentrate the aRNA to 2 ug ul and use 7 5 ul of the sample Do not use re suspended dye that is over 2 days old DMSO is hygroscopic Store tightly capped e Do not allow the samples to incubate longer than 1 hour Use reagents supplied in the Labeling Purification Reagents box Discard flow through Place the column into the same collection tube Add 250 ul of RNA Wash Buffer Teal RW to the purification column and centrifuge at 10 000 x g for 1 minute Repeat Step 5 Add 250 ul of fresh RNA Wash Buffer to the column and centrifuge at 16 000 x g full speed for 2 minutes Check the purification column for any residual wash buffer If any wash buffer remains re centrifuge at 16 000 x g for 1 minute Discard the collection tube and flow through Place the purification column into a new 0 5 mL microcentrifuge tube provided in the kit and carefully add 50 ul of RNA elution Buffer Teal RE directly onto the center of the purification column membrane Gently touch the tip of the pipette to the surface of the membrane while dispensing RE to ensure maximum absorption of RE into the membrane Gently tap the purification column to distribute the buffer if necessary Incubate at room temperature for 1 minute Place the assembly into the 2 mL support tube in the rotor with the 0 5 mL tube
13. have defined the acceptable temperature ranges for our recommendations Acceptable ranges for storage are e 70 C 65 C to 80 C e 20 C 15 C to 30 C e 4 C 2 C to 8 C e Room Temperature 10 C to 30 C The RiboAmp PLUS kits have both room temperature and frozen components The room temperature components should be stored at normal room temperature The frozen components are shipped on dry ice and should be stored at 70 C until initial use After initial use we recommend 20 C to prevent unnecessary freeze thaws of the enzymes The control RNA and any RNA generated from RiboAmp PLUS RNA amplification kit should always be stored at 70 C Store the frozen reagent box at 70 C in a non frost free freezer upon receipt After the initial use of the frozen reagents storage at 20 C is recommended The reason to store the kit at 20 C instead of at 70 C is to prevent unnecessary freeze thaw of the enzymes The Control RNA vial should be stored at 70 C or below immediately upon arrival to ensure maximum stability Store the room temperature box at room temperature All reagents included with the system should be used within one year of receipt For optimal results use the reagents as soon as possible after receipt Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chapter contents Kit configurations Contents of modules Kit configurations Kit Components The table below lists the Arcturu
14. membrane 8 Gently tap the purification column to distribute the buffer if necessary Incubate for 1 minute at room temperature 9 Place the assembly into the centrifuge as shown and centrifuge at 1000 x g for 1 minute and then centrifuge at 16 000 x g for 1 minute 10 Discard the column and retain the elution containing the cDNA in the microcentrifuge tube for further processing IMPORTANT To avoid potential breakage of the microcentrifuge tube cap during centrifugation insert the purification tube 0 5 mL tube assembly into a lidless 1 7 2 0 mL tube Insert this assembly into adjacent rotor holes as illustrated Rest the tube cap against the tube immediately clockwise to it Place an empty lidless 1 7 2 0 mL tube into the rotor hole adjacent in the clockwise direction to the last assembly Note Itis safe to stop at this point in the protocol You can store the sample overnight at 209C IMPORTANT 0 5 Round Users Using Alternate IVT Labeling Procedures Stop here Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 29 Chapter 5 RiboAmp PLUS Protocol Round One In Vitro Transcription 30 0 5 Round users KIT0527 using alternative IVT labeling such as Affymetrix labeling kit 900449 stop here and proceed to Appendix C Generating Labeled aRNA Using Alternative IVT Kits Note Life Technologies recommends the Turbo Labeling microarray kit Biotin KIT0608 CY 3 KITO609 CY 5 KIT0610 for
15. recommends the Turbo Labeling microarray kit Biotin KIT0608 CY 3 KITO609 CY 5 KITO610 for higher yields and longer aRNA fragments The Turbo Labeling kit allows for a more efficient labeling process leading to a better representation of the mRNA transcript and higher present calls Round Two In vitro IVT reaction components must be thawed thoroughly mixed with all solids dissolved transcription and brought to room temperature just before use 1 Thaw IVT Buffer Blue 1 Master Mix Blue 2 and Enhancer Yellow E to room temperature and thoroughly mix to dissolve all solids 2 IVT Enzyme Mix Blue 3 does not require thawing and can be put in directly at 4 8 C Mix enzyme thoroughly by inverting several times Spin briefly 3 Add IVT components in the order listed in Table 18 Note If you are performing several amplifications you may want to prepare a Complete IVT Reaction Mix according to Table 18 and add 12 uL Complete IVT Reaction Mix to each sample Mix thoroughly by flicking the tube and spin down Table 18 Round two in vitro transcription reaction mix Complete IVT Mix 6 reaction Master Mix Component Amount Vial with 10 overage IVT Buffer 2 uL Blue 1 13 2 uL IVT Master Mix 6 uL Blue 2 39 6 uL IVT Enzyme Mix 2 uL Blue 3 13 2 uL Enhancer 2 uL Yellow E 13 2 uL Total per sample 12 uL 79 2 uL Note If you are doing amino allyl incorporation substitute Amino Allyl IVT Mas
16. stability lt ici at ele l k Heh Hele Pde edt a Peddie sa a av ale 12 Acceptablestorageranges 12 Recommended storage temperatures 12 EXPONE at cee A dete pus Lo de haste tai nee Pot teas 12 CHAPTER 2 KittComp nentS isimini derrita 13 Kit configurations saa ia oa e aya al k kin A ee ah kane dak Gh Qul K R Ee h 13 Contents of modules reen e eae neka be EE eve El A kula ei oe Hebe oe Hab E ker aya eas 14 CDNA modules ek cheats cae a dl ondo dale 14 In Vitro Transcription IVT modules 14 DNA RNA purification modules 15 CHAPTER 3 Preliminary Notes JJ k kk kk kk kak kk kk kk kk kk kk kk kk kk kak ci 17 Recommendations for RNase free technique 17 AmplifiedaRNAcontamination 17 RNA Preparation 1 0 0 18 RNA Quality 5 Serme am MM N NN MNnNnRnNnENnEnEDTREnRnHnHnHnEHnRDHnHDDJDDNA 18 RNA DD DD a HE HI ki aoe 18 RNA Storage csi si bien bien BEN 18 Additional Lab Equipment and Materials Required 19 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 3 Contents CHAPTER 4 CHAPTER 5 CHAPTER 6 APPENDIX A APPENDIX B RiboAmp PLUS Amplification 21 Over
17. temperature Note RNA Binding Buffer Red RB must be at room temperature and thoroughly mixed before use A precipitate may form during long term storage Dissolve precipitate prior to use by mixing If necessary warm the RB vial to re dissolve 1 Add 250 uL of DNA Binding Buffer Red DB to a new purification column seated in the collection tube provided Incubate for 5 minutes at room temperature Centrifuge at 16 000 x g for 1 minute Add 200 uL of DNA Binding Buffer to the 2d Strand Synthesis sample tube mix well and pipette the entire volume into the purification column To bind cDNA centrifuge at 100 x g or lowest speed setting available for 2 minutes then immediately centrifuge at 10 000 x g for 1 minute to remove flow through Add 250 uL of DNA Wash Buffer Red DW to the column and centrifuge at 16 000 x g for 2 minutes Check the purification column for any residual wash buffer If any wash buffer remains re centrifuge at 16 000 x g for 1 minute Discard the collection tube and flow through Note Avoid splashing flow through in the collection tube onto the purification column If flow through waste liquid wets the outside of the purification column re centrifuge the column at 16 000 x g to remove the liquid Place the column into the provided 0 5 mL microcentrifuge tube and carefully add 11 uL of DNA Elution Buffer Red DE onto the center of the purification column membrane Gently touch the tip of the pipette
18. 46 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Appendix B aRNA Analysis Analyzing aRNA by agarose gel electrophoresis Analyzing aRNA using agarose gel electrophoresis is one method to visualize the RNA profile and relative quantity after amplification You can use standard protocols for agarose gel electrophoresis The following protocol uses commercially available reagents Required materials These materials are required for this procedure Suggested protocol 1 1 25 Agarose Portrait Gel or 1 25 Agarose Medium Gel EmbiTec cat GE 6010 or GE 6030 10X RNA MOPS Running Buffer EmbiTec cat EC 1020 2X Gel Loading Buffer various RNA Ladder various SYBR Gold Nucleic Acid Gel Stain Molecular Probes cat S 11494 or Ethidium Bromide Stain Nuclease free Water Determine the concentration of the aRNA by UV absorbance with a spectrophotometer Dilute the aRNA sample s with nuclease free water Each gel well can be loaded with 1 3 ug of aRNA Prepare aRNA gel sample by mixing 6 uL of diluted aRNA with 6 uL of 2X Gel Loading Buffer Incubate for 3 5 minutes at 65 C Cool on ice Prepare 1X RNA MOPS Running Buffer and fill gel electrophoresis unit Place agarose gel into the unit Load 12 uL of sample per well of the agarose gel Include RNA Ladder in one or more lanes Electrophorese at 5 7 volts per centimeter for 30 minutes Stain the gel with SYBR Gold Nucleic Acid Gel Stain for 30 minutes
19. A amplified through linear amplification to labeled unamplified RNA should be designed to compare differential gene expression using RNA samples that are processed using identical methods Performance specifications One round of amplification using a recommended input quantity of Control RNA supplied with the RibobAmp PLUS RNA Kit typically yields about 30 50 ug of aRNA Master Mix quantity 10 The RiboAmp PLUS RNA Kit is designed with the assumption that you will use the mix when you need three or more samples and that you will not use it for two or fewer samples The kits have been calculated to have a 10 overage for three samples If you exceed 10 overage for master mixes this may result in insufficient material to complete all six reactions A suggested master mix size for six samples is included when appropriate Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chapter 1 Product Introduction RNA input requirements and comparisons The two tables in this section provide the total RNA input requirements for RiboAmp PLUS RNA Amplification Table 1 and the differences between the RiboAmp PLUS RNA Amplification Kit and the RiboAmp HS PLUS RNA Amplification Kit Table 2 Table 1 Total RNA input requirements for RiboAmp PLUS DNA Amplification RiboAmp PLUS Input Amounts Minimum Recommended RiboAmp PLUS 1 round 500 ng 2 Ug 10 pg RiboAmp PLUS 2 round 5 ng 10 ng 40 ng RiboAmp HS PLUS 2 r
20. A provided in the RiboAmp PLUS Kit to verify kit functionality Starting RNA sample quality has been compromised The greatest factor affecting amplification efficiency is the integrity of the RNA used in the RiboAmp PLUS amplification process Suspend RNA in nuclease free water prior to amplification Avoid using organic solvents such as phenol in RNA isolation procedures Trace amounts of organic solvents that carry over into amplification reactions will impair the amplification process If input RNA is from cells obtained by LCM use specialized LCM sample preparation protocols designed to preserve RNA Arcturus HistoGene and PicoPure Kits are optimized to preserve the integrity of RNA and maximize recovery You can use Quantitative Real Time PCR of 1 Strand Synthesis product to verify the quality and quantity of the RNA input There is no RNA in the input sample Run a control RNA sample with a known quantity of RNA to ensure that amplification is successful Reagent concentrations in reaction mixtures are incorrect due to inadequate thawing or mixing Ensure all reagents are completely thawed mixed and all solids dissolved prior to use Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 39 Chapter 6 Troubleshooting 40 Cause Suggestion Reagent concentrations in the reaction mixtures are incorrect due to inadequate reaction volume collection in the reaction tub
21. AB iis stem Arcturus RiboAmp PLUS RNA Amplification Kit User Guide For Research Use Only Not intended for any animal or human therapeutic or diagnostic use Information in this document is subject to change without notice APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE TO THE FULLEST EXTENT ALLOWED BY LAW IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners RNase AWAY is a registered trademark of Molecular Bio Products Inc LabChip is a trademark of Caliper Life Sciences Inc CY is a registered trademark of GE Healthcare The products in this User Guide may be covered by one or more Limited Use Label License s Please refer to the respective product documentation or the Applied Biosystems website under www appliedbiosystems com for the comprehensive license information By use of these products the purchaser accepts t
22. Arcturus RiboAmp HS 12672 00 Provides instructions for using the Arcturus PLUS Kit User Guide Rev E RiboAmp HS PLUS Kit Arcturus Paradise PLUS 12872 00 Provides instructions for using the Arcturus Reagent System Kit User Rev D Paradise PLUS Reagent System Kit Guide Arcturus Paradise Whole 14360 00 Provides instructions for using the Arcturus Transcript RT WT RT Rev C Paradise PLUS WT RT Kit Reagent System Kit User Guide Arcturus Turbo Labeling 14827 00 Provides instructions for using the Arcturus Kit User Guide Rev B Turbo Labeling Kit with Biotin Cy93 and Cy 5 dyes Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 61 Documentation and Support How to obtain support How to obtain support 62 For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems web site you can Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents SDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Part Number 12293 00ARC Rev B 02 2011 gt applied A h
23. Hold the sample at 4 8 C until ready to proceed Spin down the contents and place on 4 8 C block before proceeding to the next step Thaw the 2 4 Strand Master Mix Green 1 at 4 8 C cold block Thoroughly mix and spin 24 Strand Master Mix Note 2nd Strand Enzyme Mix Green 2 does not require thawing Mix enzyme thoroughly by inverting several times spin briefly and place at 48 C Add 28d Strand Synthesis components separately in the order listed in Table 17 Note If you are performing several amplifications you may want to prepare a Complete 274 Strand Synthesis Mix based on Table 17 and add 30 uL Complete 24 Strand Synthesis Mix to each sample Mix thoroughly by flicking the tube and spin down Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 33 Chapter 5 RiboAmp PLUS Protocol Table 17 Round Two 2d strand cDNA synthesis reaction mix Round Two cDNA purification 34 Complete 2nd Strand Synthesis Mix 6 reaction Master Mix Component Amount pL Vial with 10 overage pL 2nd Strand Master Mix 29 uL Green 1 191 4 ul 2nd Strand Enzyme Mix Tul Green 2 6 6 ul Total per sample 30 uL 198 uL 8 Incubate the sample s as follows e 37 C 15 minutes e 70 C 5 minutes e 4 8 C Hold until ready to proceed up to a maximum of 30 minutes IMPORTANT Place components back onto the cold block or refreeze immediately after dispensing the reagent Do not leave reagents at room
24. Strand Master Mix 7 uL Red 1 46 2 uL 1st Strand Enzyme Mix 2 uL Red 2 13 2 uL Total per sample 9 uL _ 59 4 uL 7 Incubate at 42 C for 45 minutes then chill the sample to 4 8 C for at least 1 minute Do not hold samples at this step for a prolonged period of time Keep samples at 4 8 C until next incubation Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 27 Chapter 5 RiboAmp PLUS Protocol 8 Optional You can remove a 2 0 uL sample at this point in the protocol to assess the integrity of the starting mRNA by qRT PCR This may reduce your final yield 9 Thoroughly mix and spin down Nuclease Mix orange Place on ice 10 Add 2 0 uL of Nuclease Mix to the sample and mix thoroughly by flicking the tube and then spin down 11 Incubate the sample at 37 C for 30 minutes followed by 95 C for 5 minutes 12 Chill the sample to 4 8 C for at least 1 minute Note It is safe to stop at this point in the protocol You can store the sample overnight at 20 C Round One 2nd 1 Place sample on 4 8 C block and allow to thaw if frozen at 4 8 C strand cDNA 2 Thaw Primer B Gray B mix thoroughly and spin down synthesis 3 Add 1 0 uL of Primer B Mix thoroughly by flicking the tube and spin down 4 Incubate sample at 95 C for 2 minutes then chill and maintain the sample at 4 8C for at least 2 minutes 5 Thaw 21d Strand Master Mix at 4 8 C cold block Green 1 Mix thoroughly and s
25. T 2 2 In vitro transcription 3 4 5 aRNA Purification 0 5 0 5 Total 5 5 7 Thermal cyclers provide a convenient and reproducible method of incubating reactions according to specified temperatures and times in the RiboAmp PLUS Kits protocol A thermal cycler program for use with the RiboAmp PLUS Kit appears below The program is not intended for automatic progression from one time and temperature set to another The program lists a 4 8 C hold after each incubation or incubation cycle when it is necessary to remove the reactions from the thermal cycler to add reagents After the addition of reagents place the sample back into the thermal cycler and resume the program IMPORTANT Using a thermal cycler with a heated lid is important The heated lid ensures proper temperature distribution within the reaction tube and prevents evaporative condensation that alters the reaction mixture concentrations The 4 89C steps in the thermal cycler program allow for buffer and reagent addition and mixing steps at certain points during the amplification process and are not intended for indefinite hold unless noted The thermal cycler should be calibrated to the manufacturer s specifications Table 11 RiboAmp PLUS Thermal Cycler Program Round One oc Time 1st Strand 65 C 5 minutes Synthesis 490 hold 42 C 45 minutes 49C hold 37 C 20 minutes 95 C 5 minutes 49C hold Arcturus RiboAmp PLUS RNA Amplificat
26. ab on a Chip system The Agilent Lab on a Chip system provides a fast and effective approach to assessing the integrity of an aRNA sample The system requires very small quantity of sample Refer to the Agilent 2100 Bioanalyzer and RNA LabChip Kit Instruction Manuals for details Required This equipment and these materials are required for this procedure equipment and Agilent 2100 Bioanalyzer System Agilent materials e RNA 6000 Nano Assay Kit Agilent e Ice or cold block 4 8 C e Spectrophotometer Before you begin refer to the instruction manual for the RNA 6000 Nano Assay Kit Prepare necessary reagents and supplies as required by the kit To prepare Wipe all surfaces and equipment with RNase decontamination solution Use RNase free solutions and plastic ware Wear disposable gloves Su gg ested protocol 1 Determine the concentration of the aRNA generated through RiboAmp PLUS by UV spectrophotometry 2 Based on the optical density reading prepare a dilution of the sample to a concentration of 200 300 ng uL Note Store the sample on ice or in a cold block until ready to load on to the RNA chip 3 Follow the RNA 6000 Nano Assay Kit protocol loading 1 uL of the prepared sample dilution from step 2 For details of data interpretation refer to the bioanalyzer instruction manual The aRNA appears on the bioanalyzer as a single broad peak The size of the aRNA ranges in length from 200 to 2000 bases
27. bes Purification columns with collection tubes Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 15 Chapter 2 Kit Components 16 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Preliminary Notes Chapter contents RecommendationsforRNase freetechnigue 17 Amplified aRNA contamination eee eee KK KK KK KK KK eens 17 RNA Preparation 41 300444 w k n W anak ee ORR Oe Rae ale ate 18 Recommendations for RNase free technique RNase contamination will cause experimental failure Minimize RNase contamination by adhering to the following recommendations throughout your experiment Wear disposable gloves and change them frequently e After putting on gloves avoid touching surfaces that may introduce RNases onto the glove surface Do not use reagents not supplied in the RiboAmp PLUS Kit or recommended for use within this guide Substitutions of reagents or kit components may adversely affect yields or introduce RNases Use only new sterile RNase free pipette tips and microcentrifuge tubes e Clean work surfaces with commercially available RNase decontamination solutions prior to performing reactions Amplified aRNA contamination Stray amplified aRNA in the work area can contaminate precious samples if the work area is routinely used for performing amplifications To ensure a work area free of amplified aRNA e Irradiate the work area hood wit
28. cells The PicoPure RNA Kit comes with optimized buffers MiraCol Purification Columns and an easy to use protocol to maximize recovery of high quality total cellular RNA ready for amplification with the RiboAmp Plus RNA Amplification Kits KIT0204 40 isolations PicoPure DNA Extraction Kit The PicoPure DNA Extraction Kit is optimized to maximize the recovery of genomic DNA from 10 or more cells captured by LCM The kit comes with reagents and protocols tested to ensure complete extraction of DNA from LCM samples prepared with any standard tissue preparation procedure DNA prepared using the kit is PCR ready and needs no additional purification to perform amplification KIT0103 150 HS cap extractions 30 Macro cap extractions or 10 tissue scrapes Paradise PLUS FFPE Kits The Paradise PLUS Reagent System is designed to enable gene expression studies using formalin fixed paraffin embedded FFPE tissue samples Components include sample preparation and staining reagents RNA extraction and isolation reagents RNA amplification reagents and a comprehensive user guide KIT0312 12 samples KIT0312B 12 samples with biotin labeling KIT0312C 12 samples with CY 3 labeling KIT0312D 12 samples with CY 5 labeling Paradise PLUS FFPE WT RT Kit 54 The Paradise PLUS Whole Transcript Reverse Transcription WT RT Reagent System Kit enables QRT PCR using formalin fixed paraffin embedded FFPE tissue samples The kit was
29. cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Documentation and Support Related documentation The following documents are available for the Arcturus LCM System and related products Document Fart Description number Instruments Arcturus LCM System 0112 0153 Provides a detailed set of instructions for using User Guide Rev C the Arcturus Instrument Arcturus Troubleshooting 4458770 Provides tips and recommendations for Guide Rev A handling problems encountered while using the Arcturus Instrument Reagents Arcturus HistoGene 12294 00 Provides instructions for using the Arcturus Frozen Section Staining Kit Rev C HistoGene Frozen Section Staining Kit User Guide Arcturus HistoGene 12653 00 Provides instructions for using the Arcturus Immunofluorescence Rev C HistoGene Immunofluorescence Staining Kit Staining Kit User Guide Arcturus PicoPure DNA 12637 00 Provides instructions for using the Arcturus Extraction Kit User Guide Rev D PicoPure DNA Extraction Kit Arcturus PicoPure RNA 12682 Provides instructions for using the Arcturus Isolation Kit User Guide OOARC PicoPure RNA Isolation Kit Rev A
30. container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Handle chemical wastes in a fume hood e After emptying a waste container seal it with the cap provided e Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental and health regulations Waste disposal If potentially hazardous waste is generated when you operate the instrument you must Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory e Ensure the health and safety of all personnel in your laboratory e Ensure that the instrument waste is stored transferred transported
31. d Synthesis components in the order listed in Table 16 on page 33 Note If you are performing several amplifications you may wish to prepare a Complete 15t Strand Synthesis Mix based on the table below and add 9 0 uL Complete 15 Strand Synthesis Mix to each sample Mix thoroughly by flicking the tube and spin down Do not vortex Table 16 Round Two 1S strand cDNA synthesis reaction mix Complete 1 4 Strand Synthesis Mix 6 reaction Master Mix Component Amount Vial with 10 overage 1st Strand Master Mix 7 uL Red 1 46 2 uL 1st Strand Enzyme Mix 2 uL Red 2 13 2 uL Total per sample 9 uL _ 59 4 uL 8 Incubate the sample s at 25 C for 10 minutes then at 37 C for 45 minutes 9 Chill the sample s to 4 8 C for at least 1 minute Note Tt is safe to stop at this point in the protocol You can store the sample overnight at 20 C IMPORTANT Place the components back onto the cold block or refreeze immediately after dispensing the reagent Do not leave reagents at room temperature for any extended period of time Round Two 2nd 1 strand cDNA 2 synthesis 7 Place sample on a 4 8 C block and allow to thaw if frozen at 4 8 C Thaw Primer A Gray A thoroughly mix spin down and place on a 4 8 C block Add 1 0 uL of Primer A to the sample Mix thoroughly by flicking the tube and spin down Incubate the sample at 95 C for 2 minutes then cool sample to 4 8 C for at least 1 minute
32. d symbol and hazard type appear by a chemical name or instrument hazard see the Safety appendix at the end of the manual for the complete alert on the chemical Four safety alert words appear in this user documentation at points in the document where you need to be aware of relevant hazards Each alert word implies a particular level of observation or action as defined below IMPORTANT Indicates information that is necessary for accurate chemistry kit use or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices ha WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury n DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANTs each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 7 About This Guide Safety information 8 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Product Introduction Chapter contents Backgroundinformation KK KK KK KK KK KK KK KK KK nee 9 Performance SpedcificatlonS 352 0 ay ke kek di
33. developed specifically to overcome obstacles often associated with formalin fixed tissue such as chemical modification and RNA fragmentation The kit provides RNA isolation and reverse transcription reagents optimized for use with archived FFPE samples at small sample input amounts and delivers unparalleled yield fidelity and representation The kit was designed with exon spanning primers at varying distances from the 3 end of the transcript and allows the study of splice variants in archived or degraded samples The Paradise WT RT system also allows the use of gene specific primers for reverse transcription to suit specific assay requirements KIT0315 12 Samples Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Appendix E Related Reagent Kits RiboAmp PLUS RNA Amplification Kits The RiboAmp PLUS RNA Amplification Kit enables the production of microgram quantities of antisense RNA aRNA from as little as picogram amounts of total cellular RNA Amplified RNA produced using the kit is suitable for labeling and use on expression microarrays The kit achieves 1 000 to 3 000 fold amplifications in one round of amplification and up to 1 000 000 fold in two rounds The kits include microarray labeling options for biotin fluorescent dyes and amino allyls Kits are available in two sensitivity options RiboAmp Plus 5 40 ng input and a high sensitivity version RiboAmp HS Plus 0 1 to 5 ng input KIT0521 RiboAmp PLUS
34. e Reagent concentrations in reaction mixtures are incorrect due to evaporative condensation onto the wall of the reaction tube during incubation Thoroughly thaw and mix all reagents prior to dispensing Ensure all reagents are dispensed at proper volumes Briefly spin down the reaction mix prior to incubation to ensure all reagents are collected in the reaction volume and the reaction mix has the proper concentrations of reagents Briefly spin down the sample following incubation steps to maintain proper volumes and concentrations of reagents and ensure that all nucleic acid templates are mixed with reaction components Use a thermal cycler with a heated lid Incubation temperatures are incorrect Verify the accuracy of all incubation temperatures If you are using a thermal cycler make sure that the programmed temperatures read correctly and the instrument has been calibrated to establish and maintain accurate temperature settings RNA yield is diminished during column purification Verify centrifugal force used during nucleic acid purification Improper binding washing and elution centrifugal forces can decrease the recovery of nucleic acid from the purification column Microcentrifuges should be calibrated to deliver the correct centrifugal force Message content is low within the total RNA being used in your study Check amplification efficiency using control RNA Use higher RNA inputs to compensate fo
35. e working with the tabletop microcentrifuge Eppendorf 5415 Table 22 Centrifugal forces for selected rotations Rotations Per Minute Centrifugal Force 14 000 rpm 13 000 g 12 000 rpm 10 000 g 10 000 rpm 7000 g 8000 rpm 4500 g 5500 rpm 2200 g 5000 rpm 2000 g Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 51 Appendix D Centrifuge Information Centrifuge rotation The graphic below shows the centrifuge IMPORTANT IMPORTANT To avoid potential breakage of the microcentrifuge tube cap during centrifugation insert the purification tube 0 5 mL tube assembly into a lidless 1 7 2 0 mL tube Insert this assembly into adjacent rotor holes as illustrated below Rest the tube cap against the tube immediately clockwise to it Place an empty lidless 1 7 2 0 mL tube into the rotor hole adjacent in the clockwise direction to the last assembly Figure 2 Centrifuge drawing 52 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Related Reagent Kits This appendix covers HistoGene LCM FrozenSectionStainingkKit 53 HistoGene LCM ImmunofluorescenceStainingKit 53 PicoPure RNArlsolationKit KK KK KK KK KK KK KK KK KK kk 54 PicoPure DNA ExtractionKit KEK KK KK KK KK KK KK KK KK 54 Paradise PLUS FFPE Keira ii KK kk 54 Paradise PLUSFFPEWTRTKit 54 RiboAmp PLUSRNAAm
36. ed aRNA when coupled with the Arcturus Turbo Labeling Kit which is then ready for microarray experiments The kit contains e A complete set of reaction reagents Nucleic acid purification columns e A control RNA sample e This user guide Reagents and materials are supplied for 12 one round amplifications or 6 two round amplifications The kit provides premixed enzymes and buffers to save time and increase ease of handling The protocol is streamlined to enable fast processing while generating reproducible results Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 9 Chapter 1 Product Introduction aRNA product size The kit generates aRNA product that is shorter than the starting mRNA template The bulk of the aRNA product is 250 1800 bases in length after one round of amplification and slightly shorter under 200 to over 600 bases after a second round Messenger RNA makes up an estimated 1 5 of total cellular RNA One round of amplification typically yields up to 1000 fold amplification of the mRNA while two rounds may yield up to one million fold amplification of the mRNA Amplified aRNA produced using the RiboAmp PLUS Kit is ready for subsequent labeling and probing of CDNA microarrays Note RNA amplification begins at the 3 end of the substrate molecule therefore amplified RNA product should not be used to prepare full length cDNA libraries IMPORTANT Microarray experiments that compare labeled RN
37. elect the language of your choice then click Search Find the document of interest right click the document title then select any of the following Open To view the document e Print Target To print the document e Save Target As To download a PDF version of the document to a destination that you choose Note For the SDSs of chemicals not distributed by Applied Biosystems contact the chemical manufacturer Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Appendix F Chemical Safety Chemical waste safety Chemical waste safety Chemical waste N CAUTION HAZARDOUS WASTE Refer to Safety Data Sheets and local hazards regulations for handling and disposal n WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical waste To minimize the hazards of chemical waste safety guidelines Read and understand the Safety Data Sheets SDSs provided by the manufacturers of the chemicals in the waste
38. equired equipment Equipment and Thermal cycler with heated lid Materials Required Microcentrifuge for 1 5 mL and 0 5 mL tubes Eppendorf 5414D or similar e 0 5 10 uL pipettor e 20 uL pipettor 200 uL pipettor 1000 uL pipettor e Ice bath or cold block 4 C Vortex mixer optional These are the required materials 0 5 mL or 0 2 mL RNase free microcentrifuge tubes e 2mL lidless tube for centrifuge PGC Scientific Cat 16 8101 06 Nuclease free pipette tips Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 19 Chapter 3 Preliminary Notes 20 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide RiboAmp PLUS Amplification Chapter contents Overviewoftheamplificationprocess 21 Necessary preliminaries 000 24 Overview of the amplification process The Arcturus RiboAmp PLUS RNA Amplification i Kit is optimized to amplify nanogram amounts of Figure 1 Rih Amp li starting RNA The RiboAmp PLUS Kit utilizes up to RNA Amplification Process two rounds of a five step process for linear amplification of the mRNA fraction of total cellular RNA These five steps are listed below and shown in Mees Callas EMA Figure 1 E o 1 15t strand synthesis reaction that yields CDNA specific Ist Strand The RiboAmp Amplification Process 1st Prime incorporating a T7 promoter sequence ES cDNA 2 29d strand synthesis reaction utilizing exo
39. erform RNA transcript labeling according to the protocol of the IVT labeling kit using the sample from step 2 above as the cDNA template Adjust the final volume of the cDNA sample as necessary Antisense RNA purification 1 Add 250 uL of RNA Binding Buffer Blue RB to a new purification column and incubate for 5 minutes at room temperature Centrifuge at 16 000 x g for 1 minute Add 200 uL of RNA Binding Buffer to the Transcript Labeling Reaction sample and mix thoroughly Pipet the entire sample volume into the purification column Centrifuge at 100 x g or lowest speed setting available for 2 minutes and immediately centrifuge at 10 000 x g for 1 minute Add 200 uL of RNA Wash Buffer Blue RW to the purification column and centrifuge at 10 000 x g for 1 minute Note RNA Binding Buffer Blue RB must be at room temperature and thoroughly mixed before use A precipitate may form during long term storage Dissolve precipitate by mixing If necessary warm the RB vial to redissolve Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 49 50 Appendix C Generating Labeled aRNA Using Alternative IVT Kits Add 200 uL of fresh RNA Wash Buffer to the column and centrifuge at 16 000 x g for 2 minutes Check the purification column for any residual wash buffer If any wash buffer remains re centrifuge at 16 000 x g for 1 minute Discard the collection tube and flow through Place the purification column into a new 0 5 mL m
40. f the microcentrifuge tube cap during centrifugation insert the purification tube 0 5 mL tube assembly into a lidless 1 7 2 0 mL tube Insert this assembly into adjacent rotor holes Rest the tube cap against the tube immediately clockwise to it Place an empty lidless 1 7 2 0 mL tube into the rotor hole adjacent in the clockwise direction to the last assembly 11 Measure the O D of the product at A269 and Aygo Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chapter 5 RiboAmp PLUS Protocol 12 Analyze the aRNA using the Agilent Bioanalyzer or by gel electrophoresis The purified aRNA is ready for use in a labeling reaction with the Turbo Labeling kit for microarrays You can store purified aRNA at 70 C Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 37 Chapter 5 RiboAmp PLUS Protocol 38 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chapter contents Troubleshootin Amplificationyieldispoor 0 WENA kk AA Low molecular weight product appears on a gel Amplification yield is poor The table below lists possible reasons why the amplification yield is poor and provides suggestions for correcting the problem Table 19 Suggestions for correcting low amplification yield g Cause Suggestion Starting RNA sample quality varies If you observe low yields with different RNA samples run an amplification control using the Control RN
41. g long term storage Dissolve precipitate prior to use by mixing If necessary warm the DB vial to redissolve 1 Add 250 uL of DNA Binding Buffer Red DB to a DNA RNA Purification Column seated in the collection tube provided Hold for 5 minutes at room temperature 2 Centrifuge at 16 000 x g for 1 minute 3 Add 200 uL of DNA Binding Buffer to the 21d Strand Synthesis sample tube mix well and pipet the entire volume into the purification column 4 To bind cDNA to column centrifuge at 100 x g for 2 minutes or lowest speed setting available immediately followed by a centrifugation at 10 000 x g for 1 minute to remove flow through 5 Add 250 uL of DNA Wash Buffer Red DW to the column and centrifuge at 16 000 x g for 2 minutes Check the purification column for any residual wash buffer If any wash buffer remains re centrifuge at 16 000 x g for 1 minute 6 Discard the flow through and collection tube IMPORTANT Avoid splashing flow through in the collection tube onto the column If flow through waste liquid wets the outside of the purification column re centrifuge the column at 16 000 x g to remove liquid 7 Place the column into the provided 0 5 mL microcentrifuge tube and carefully add 11 uL of DNA Elution Buffer Red DE onto the center of the purification column membrane Gently touch the tip of the pipette to the surface of the membrane while dispensing the elution buffer to ensure maximum absorption of DE into the
42. genous Tome primers that yields double stranded cDNA pas 2nd 2nd Strand 3 cDNA purification using specially designed Primer y enes MiraCol Purification Columns eee HO 4 In vitro transcription IVT utilizing T7 RNA TITITITI polymerase yields antisense RNA aRNA Zax Purification 5 aRNA isolation with the MiraCol Purification wu Columns ei an The RiboAmp PLUS Kit allows for up to two rounds Amplified aRNA of amplification for each sample In vitro transcription EVE A SES can be performed overnight with the proper thermal oes ome po cycler programming The entire process can be EN Sees completed by the morning of the third day and the ne product is then ready for labeling and microarray hybridization or qRT PCR Mame beli or 2nd round amplification Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 21 Chapter 4 RiboAmp PLUS Amplification Time requirements Thermal cycler programming 22 The table below presents typical time requirements for completion of the protocol Times reflect total handling and reaction times of each step Note that although there are safe stopping points for pausing the amplification process the times presented reflect a continuous uninterrupted process Table 10 Time Required for RiboAmp PLUS Steps 15t Round hours 2nd Round hours 1st Strand Synthesis 1 1 2nd Strand Synthesis 0 5 0 5 cDNA Purification 0 5 0 5 Total before IV
43. h UV overnight every three to four days e Clean surfaces with commercially available decontamination solutions Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 17 Chapter 3 Preliminary Notes RNA Preparation RNA Quality RNA Input RNA Storage 18 The success of amplification using the RiboAmp PLUS RNA Amplification Kit depends on the quality of the source RNA Integrity is affected by exposure to internal and external sources of RNases Avoiding RNA degradation due to intracellular RNases is often the most critical step in isolating good quality RNA Isolation from cell cultures should be performed immediately after harvesting the cell to avoid RNase activity Quiescent cells such as those in tissue samples require immediate freezing in embedding media to inactivate RNases Subsequent processing of those samples requires methods that preserve RNA integrity We recommend the use of the PicoPure RNA Isolation Kit for isolating RNA from tissue or cell samples Cells captured by LCM will yield high quality RNA when appropriate protocols are applied such as those used in the Arcturus HistoGene LCM Frozen Section Staining Kit the HistoGene LCM Immunofluorescence Staining Kit and PicoPure RNA Isolation Kit We strongly recommend performing quality assessment of the tissue prior to amplification Tissue that has degraded RNA prior to freezing will not yield good results If the quality of the source tissue is unknow
44. he terms and conditions of all applicable Limited Use Label Licenses These products are sold for research use only and are not intended for human or animal diagnostic or therapeutic uses unless otherwise specifically indicated in the applicable product documentation or the respective Limited Use Label License s For information on obtaining additional rights please contact outlicensingfdlifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 2011 Life Technologies Corporation All rights reserved 12293 00ARC Rev B 02 2011 Contents About This Guide 2 m0 kk kak cient a KK KK kk kk eee 7 Purpose of this quide ci n se A kad GY eA es 7 Prerequisitesa rito dd E ay And Je aes ER he NS ala ds a e lerle 7 Safety informations 52 3 21 xware iy a la i RE A eter a n Wa ace n a 7 CHAPTER 1 Productintroduction RR RR RR RR eee 9 Backgroundinformation kk kk 9 illa VIG LASS yale ND Ne a DD DD A ARDEN 9 Kitcontenis ts A Pa A DA Ea E PEDE va T on ae e de E di e ME 9 ARNA productssize Zayend tenet ce WADA ed img imla Ar NAN AS A bag 10 Performance specifications tte kk kk kk kk kk kk 10 Master Mixguantity kk kk kk kk kK kk kk kk kk kk kk kk kk kk 10 RNA input reguirementsandcomparisons 11 Storage and
45. higher yields and longer aRNA fragments The Turbo Labeling kit allows for a more efficient labeling process leading to a better representation of the mRNA transcript and higher present calls Thaw IVT Buffer Blue 1 Master Mix Blue 2 and Enhancer Yellow E to room temperature and thoroughly mix to dissolve all solids IVT Enzyme Mix Blue 3 does not require thawing and can be put in directly at 4 8 C 2 Mix enzyme thoroughly by inverting several times Spin briefly Note IVT reaction components must be thawed thoroughly mixed with all solids dissolved and brought to room temperature just before use 3 Add IVT components in the order listed in the following table Note If you are performing several amplifications you may want to prepare a Complete IVT Reaction Mix according to this table and add 12 uL Complete IVT Reaction Mix to each sample Mix thoroughly by flicking the tube and then spin down Table 15 Round One in vitro transcription reaction mix Complete 2d Strand Synthesis Mix 6 reaction Master Mix Component Amount Vial with 10 overage HL IVT Buffer 2 uL Blue 1 13 2 uL IVT Master Mix 6 uL Blue 2 39 6 ul IVT Enzyme Mix 2 uL Blue 3 13 2 uL Enhancer 2 uL Yellow E 13 2 uL Total per sample 12 uL 79 2 uL 4 Incubate at 42 C for 3 hours Optional You can use four hour incubation for additional aRNA yield Chill the sample s to 4 8 C Note At this point i
46. icrocentrifuge tube provided in the kit and carefully add 30 uL of RNA Elution Buffer Blue RE directly onto the center of the purification column membrane Note Gently touch the tip of the pipette to the surface of the membrane while dispensing RE to ensure maximum absorption of RE into the membrane Gently tap the purification column to distribute the buffer if necessary Incubate at room temperature for 1 minute Place the assembly into the 2 mL support tube in the rotor with the 0 5 mL tube cap trailing the tube Centrifuge at 1000 x g for 1 minute and immediately centrifuge at 16 000 x g for 1 minute Discard the purification column and retain the elution containing the labeled aRNA Measure the O D of the product at A269 and Aygq to determine the yield of labeled aRNA Perform electrophoretic analysis if necessary Proceed to protocols for microarray hybridization IMPORTANT Avoid splashing flow through in the collection tube onto the column If flow through waste liquid wets the outside of the purification column re centrifuge the column at 16 000 x g to remove liquid Note Tubes must be properly oriented in the rotor during elution Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Centrifuge Information This appendix covers Centrifugal force Centrifuge rotation Centrifugal force The table below shows corresponding centrifugal forces g for selected rotations per minute rpm when you ar
47. iesystems by Lefe technologies Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 760 603 7200 www lifetechnologies com Technical Resources and Support For the latest technical resources and support information for all locations please refer to our Web site at www appliedbiosystems com
48. ines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument N WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles N WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of breaking or shattering Reagent and waste bottles can crack and leak Each waste bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles Chemical safety To minimize the hazards of chemicals guidelines Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About SDSs on page 58 e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines
49. ion Kit User Guide Chapter 4 RiboAmp PLUS Amplification 2nd Strand 95 C 2 minutes Synthesis 496 hold 25 C 5 minutes 37 C 10 minutes 70 C 5 minutes 49C hold IVT 42 C 3 hours optional 4 hours 4 C hold optional overnight hold 37 C 15 minutes 49C hold Table 12 RiboAmp PLUS Thermal Cycler Program Round Two oc Time 1st Strand 65 C 5 minutes Synthesis 49 hold 25 C 10 minutes 37 C 45 minutes 4 C hold 2nd Strand 95 C 2 minutes Synthesis 49 hold 37 C 15 minutes 70 C 5 minutes 4 C hold IVT 42 C 4 6 hours 49C hold optional overnight hold 37 C 15 minutes 4 C hold IMPORTANT Do not allow incubation times and temperatures to deviate from the protocol Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 23 Chapter 4 RiboAmp PLUS Amplification Necessary preliminaries Protocol notes Preparing samples and reagents 24 Before you begin Round One of the thermal cycling review the recommendations and instructions provided here When adding reagent to samples or master mixes pipette mixtures up and down several times to ensure complete transfer of reagent from the pipette tip Prior to the first use of an enzyme gently mix do not vortex and briefly microcentrifuge the vial to ensure that all enzyme is mixed and collected at the bottom of the vial Enzyme may collect on the vial wall or cap
50. kel Ne rude el a ene Ate Da meee 58 Obtaining SDSS 424 21 ete de ee Et Lt e ta Ae al 58 Chemical waste safety KK kK KK eee kk ka 59 Chemical waste hazards 0000s cc kK KK KK kK KK KK KK KK KK tte KK kk kk 59 Chemical waste safety guidelines 59 Waste disposalkt tastiest Sainte ili dk da da ea as tte cl hs 59 Biological Hazard Safety pee eee pe pi sml ei 60 Documentation and Support 61 Related documentation KK KK KK kk kk kk kK kk KK kk kK kk kk 61 u leya to Ob tain SUpport Ayka deca 62 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 5 Contents 6 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide About This Guide Purpose of this guide This user guide is for use with Arcturus RiboAmp PLUS RNA Amplification Kit You can view and download this user guide from www appliedbiosystems com Prerequisites This guide is intended for those who use the RiboAmp PLUS RNA Kit Life Technologies is not liable for damage or injury that results from use of this manual by unauthorized or untrained parties Instructions in this guide use conventions and terminology that assume a working knowledge of the Microsoft Windows operating system the Internet and Internet based browsers Safety information For general safety information see this section When a hazar
51. lification protocols Since Primer A is a component of 2nd Strand Synthesis and Primer B is a component of 15 Strand Synthesis reaction temperatures and incubation intervals are different The second round amplification protocol does not make use of 1 Strand Nuclease Mix Note The aRNA product produced after the second round of amplification is somewhat shorter than that formed from one round Typically the bulk of the aRNA visualized through gel electrophoresis will range from under 200 to over 600 bases 1 Thaw samples from round one at 4 8 C if necessary Place samples on a 4 89C block 2 Thaw Primer B Grey B thoroughly mix spin down and place on a 4 89C block 3 Into eluted aRNA product from Round One add 1 0 uL of Primer B mix thoroughly by flicking the tube and spin down 4 Incubate the microcentrifuge tube at 65 C for 5 minutes then chill the samples to 4 8 C for 1 minute 5 Spin down the contents and place on 4 8 C block before proceeding to next step 6 Place 1 Strand Synthesis components Red 1 and Red 2 at 4 8 C 15t Strand Master Mix must be thawed thoroughly mixed with all solids dissolved and maintained at 4 8 C until used Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chapter 5 RiboAmp PLUS Protocol Note 1 Strand Enzyme Mix does not require thawing and can be placed directly at 4 8 C Mix enzyme thoroughly by inverting several times Spin briefly Add 1 Stran
52. n Ensure that all pipettes are properly calibrated to dispense correct volumes Concentrations of Primer 1 Primer 2 Primer 3 or 15t Strand Nuclease Mix are incorrect due to inadequate mixing or reaction volume collection inside the reaction tube Thoroughly mix and spin down the sample after adding the primers or 1 Strand Nuclease Mix into the reaction mix and prior to incubation This ensures the correct concentration of primers or nuclease in each respective reaction mix Input RNA was not isolated using the PicoPure RNA Isolation Kit and no nucleic acid carrier was added Low molecular weight material may result from lack of RNA and carrier Using the PicoPure RNA Isolation Kit is recommended to prepare samples that contain carrier Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 41 Chapter 6 Troubleshooting 42 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Amino allyl aRNA Labeling Protocol This appendix covers Appropriate reagen S xa ot ees bi Deletes 43 Labeling reaction B A db dedi mean a ate 43 ARNA purification se i 4 445341543 22444531444 des eed oe eee bee eee dads 44 Appropriate reagents This protocol is intended for use with amino allyl modified aRNA that was generated using the optional Amino Allyl IVT components of RA7010 and RA7012 The table below lists appropriate reagents Table 21 Reagents appropriate for Amino allyl aRNA labeling
53. n then performing a quality assessment of the tissue block prior to spending the time and expense of Laser Capture and amplification is imperative A protocol for quality assessment is included in Appendix B aRNA Analysis in this document Using isolated total cellular RNA rather than mRNA for amplification is recommended to reduce the loss of valuable transcripts during mRNA isolation from extremely small samples Isolating RNA with the PicoPure RNA Isolation Kit is also highly recommended Furthermore input RNA should be DNase treated to eliminate genomic DNA contamination The RNA must be provided in RNase free water without the presence of organic solvents salts or contaminating cellular material IMPORTANT The RiboAmp PLUS RNA Amplification kits are not designed for use with formalin fixed material For Formalin Fixed materials please use the Arcturus Paradise PLUS Reagents System RNA intended for use with the RiboAmp PLUS Kits should be used immediately after isolation or stored at 70 C until use The control RNA provided with each RiboAmp PLUS Kit should be stored at 70 C immediately upon arrival Avoid multiple freeze thaw cycles Amplified aRNA produced using the RiboAmp PLUS Kit should be used for labeling reactions as soon as possible Alternatively the aRNA may be stored at 70 C Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chapter 3 Preliminary Notes Additional Lab This is the r
54. n the protocol you may hold the reaction mixture at 4 8 C in the thermal cycler overnight 5 Move the samples directly to a 4 8 C block 6 Add 1 uL DNase Mix Blue 4 Mix thoroughly and spin down Note DNase Mix must be thoroughly mixed and held at 4 C until used 7 Incubate at 37 C for 15 minutes 8 Chill the sample s to 4 8 C Proceed immediately to aRNA purification Note RNA may be adversely affected if not purified immediately after DNase treatment Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chapter 5 RiboAmp PLUS Protocol Round One aRNA Note RNA Binding Buffer Blue RB must be at room temperature and thoroughly Purification mixed before use A precipitate may form during long term storage Dissolve precipitate prior to use by mixing If necessary warm the RB vial to re dissolve 1 Add 250 uL of RNA Binding Buffer Blue RB to a new purification column and incubate for 5 minutes at room temperature 2 Centrifuge at 16 000 x g for 1 minute 3 Add 120 uL of RNA Binding Buffer to the IVT reaction sample and mix thoroughly 4 Pipet the entire volume into the purification column 5 To bind aRNA centrifuge at 100 x g or lowest speed setting available for 2 minutes immediately followed by a centrifugation at 10 000 x g for 1 minute to remove flow through 6 Add 200 uL of RNA Wash Buffer Blue RW to the purification column and centrifuge at 10 000 x g for 1 minute 7 Add 200
55. nin sk eee 10 Master Mix quantity i n3154 48 2 252 ELA 2 2440454 AA send 10 RNA input reguirementsandcomparisons 00000000 11 Storage and stabil 4 iy e ts u zeka e n y k zn 12 Background information High yields Kit contents The Arcturus RiboAmp PLUS RNA Amplification Kit enables you to produce microgram quantities of antisense RNA aRNA from nanogram quantities of total cellular RNA Small sample collection methods such as needle aspiration and Laser Capture Microdissection LCM limit starting quantities of RNA available for amplification Microarray hybridization experiments require microgram quantities of probe per array to enable detection and most studies entail multiple experiments The RiboAmp PLUS RNA Kit achieves high yields of aRNA with a patented linear amplification process using double stranded cDNA as template ina T7 RNA polymerase catalyzed amplification This amplification process yields highly reproducible results through an optimized system of reagents purification devices and protocols The resulting aRNA is suitable for use in RT PCR quantitative Real Time PCR qRT PCR and microarray hybridization experiments The Arcturus RiboAmp PLUS RNA Kit can generate different forms of aRNA e Unlabeled aRNA ready for reverse transcription and quantitative Real Time PCR qRT PCR e Amino allyl incorporated aRNA ready to be labeled for microarray analysis e Label
56. ontents APPENDIX C Generating Labeled aRNA Using Alternative IVT Kits 49 Overview of substitution proceSS 00 0 cece KK KK KK ete kk kk kk kk ka 49 Antisense RNA purification 49 APPENDIX D o Centrifugelnformatlon 2 aaa 51 Gentolu allorce a e nee es Ain e ii em ere os o ii Gr A alae 51 Centrituge rotation ie ai bell ee co aa e o eo o do u say ile 52 APPENDIX E Related Reagent Kits gt 12 4 a kk kk kk a kaka kk kk kk 53 HistoGene LCMFrozenSectionStainingKit 53 HistoGene LCM Immunofluorescence StainingKit 53 PicoPure RNA Isolation Kit 54 PicoPure DNA Extraction Kilos iS a it WE EZ 54 Paradise PLUS REPE Kits ar Mandal eee ts hie ee 54 Paradise PLUS FFPE WT RT Kit WW kk kk kk kK kK KK cece KK KK eee KK KK eee KK kk 54 RiboAmp PLUS RNA Amplification Kits 0 0 00 00 0 cc ccc KK KK KK KK KK KK KK KK eas 55 Turbo Labeling Kits el a n N la Sate ke e Qi es AW 55 APPENDIX F Chemical Safety sa sk ak kal ka dl kc al eae ara a lad ed es 57 General chemical satety 5 xway Wn 2k ge eed od Bee ee da 57 Chemical hazard warning M K kk kk kk kK cece KK KK KK KK KK K KK KK etnies 57 Chemicalsafetyguidelines 57 Safety Data Sheets esc MM A eed E HHHH rere 58 ADOUE SDSS a tere Mat eee a
57. or according to the protocol supplied with the reagent Alternatively stain with Ethidium Bromide 0 5 1 0 ug mL Visualize the gel on a UV transilluminator The size of the aRNA ranges from 200 to 2000 bases in length Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 47 48 Appendix B aRNA Analysis Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Generating Labeled aRNA Using Alternative IVT Kits This appendix covers Overview of substitutionprocess KK KK KK KK KK K 49 Antisense RNA purification KK KK KK KK KK KK KK KK KK KK KK KK KK KI 49 Overview of substitution process This appendix explains how to use the RiboAmp PLUS Kits with alternative IVT labeling such as Affymetrix labeling kit 900449 to yield suitable RNA sample for hybridizing to GeneChip Probe Arrays You substitute these kit reagents and protocol during the second IVT reaction of the RiboAmp PLUS Kit protocol You subsequently purify the Labeled aRNA with the RiboAmp PLUS Kit and MiraCol Purification Columns as described below 1 Perform Round One of amplification according to the RiboAmp PLUS Amplification Kit protocol starting from the recommended input for the kit Note Due to IVT efficiency we do not recommend using the minimum input amounts with an alternative labeling kit Perform Round Two of amplification through cDNA Purification according to the Kit protocol P
58. ound 100 pg 500 pg 5 ng Input required to obtain sufficient aRNA yields for microarray hybridizations If your application is anything other than microarray please contact us for input recommendations Minimum input will yield gt 15 ug of aRNA Recommended input will yield gt 30 ug of aRNA The RiboAmp HS PLUS kit has been validated down to 100 Ps However new users should start with the control RNA provided with the kit until they become comfortable with the use of the kit Note Do not overload amplification reactions Overloading high sensitivity kits may deplete key components during the reaction and may lead to no amplification Table 2 Differences between the RiboAmp HS PLUS RNA Kit and RiboAmp Plus RNA kit RiboAmp HS PLUS RNA Kit RiboAmp PLUS RNA Kit Higher sensitivity Faster processing 2 rounds of amplification 1 or 2 rounds of amplification Recommend 500 pg 5 ng total RNA input Recommend 5 40 ng total RNA input for 2 rounds of amplification Requires 2 5 days Requires 13 hours total for 2 rounds Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 11 Chapter 1 Product Introduction Storage and stability Acceptable storage ranges Recommended storage temperatures Expiration 12 Life Technologies makes recommendations for storage temperatures throughout this document Realizing that not every laboratory has a freezers set at these temperatures we
59. oundlwo 2ndstrandcDNAsynthesis 33 Roundiwo cDNApurification 34 Roundilwo lnvitrotranscription 35 Roundiwo aRNApurification 36 Troubleshooting gt css mii en Bl de ak de l ka 39 Amplification yield is poor 39 Lowmolecularweightproductappearsonagel 41 Amino allyl aRNA Labeling Protocol 43 Appropriate reagents kk kk kk kk kk kk kk kk kk kk kk k 43 Pa beling Econ taie e ein a a PP eJrWUUN DE 43 ARNA ni a O aaa apak a k gn 44 ARNA Analysis cosida raras Aa E 45 Determining RNA yield and purity kk kk kk kk kk kk kK KK kK KK KK KK kK kK KK kk kK kK kk kk 45 AssessinginpputRNAsamplegualiiy 45 Assessing total RNA and aRNA quality WW kk kk kk kk kk KK KK KK KK KK KK KK KK KK KK eens 46 Required equipment and materials 46 Suggested protocols icc a daia a WEZ atid ee OD dd di cd 46 Analyzing aRNA by agarose gel electrophoresis 47 Required materials 47 Suggested protocol ct ya kerda e ONA K e N iia AS 47 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide C
60. pin 294 Strand Master Mix 6 Mix enzyme thoroughly by inverting several times spin briefly and then place at 4 8 C Note 2nd Strand Enzyme Mix Green 2 does not require thawing 7 Add 28d Strand Synthesis components separately in the order listed in the following table If you are performing several amplifications you may want to prepare a Complete 2 4 Strand Synthesis Mix based on Table 14 and add 30 uL Complete 2nd Strand Synthesis Mix to each sample Mix thoroughly by flicking the tube and spin down Store at 4 C until needed Table 14 Round One second strand reaction mix Complete 274 Strand Synthesis Mix 6 reaction Master Mix Component Amount pL Vial with 10 overage UL 2nd Strand Master Mix 29 uL Green 1 191 4 uL 2nd Strand Enzyme Mix 1 uL Green 2 6 6 uL Total per sample 30 uL _ 198 uL IMPORTANT Place components back onto the cold block or refreeze immediately after dispensing the reagent Do not leave reagents at room temperature for any extended period of time 8 Incubate the sample as follows e 25 C 5 minutes 37 C 10 minutes 28 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chapter 5 RiboAmp PLUS Protocol e 70 C 5 minutes e 4 8 C Hold until ready to proceed up to a maximum of 30 minutes Round One cDNA Note DNA Binding Buffer Red DB must be at room temperature and mixed purification thoroughly by shaking before use A precipitate may form durin
61. r lower message content Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chapter 6 Troubleshooting Low molecular weight product appears on a gel Occasionally a predominant band below the expected aRNA smear will appear on a gel This band will lead to improper estimation of yield and may result in high backgrounds on microarrays The RiboAmp PLUS Kit components are formulated and tested to avoid the synthesis of this material but if it does occur refer to Table 20 The table below lists possible reasons why low molecular weight material is present and provides suggestions for correcting the problem Table 20 Suggestions for correcting low molecular weight in gel Cause Suggestion Quality of the starting RNA is inadequate Poor RNA quality can lead to the formation of the reaction artifact visible as a low molecular weight band Check the quality of your input RNA One approach is to utilize the Agilent Lab on a Chip System with an RNA LabChip Kit For additional recommendations to check for the quality of the input RNA contact Technical Support at 1 800 831 6844 Concentrations of Primer 1 Primer 2 Primer 3 or 15t Strand Nuclease Mix are incorrect due to inadequate thawing or dispensing Thaw and thoroughly mix each reagent vial prior to dispensing If incompletely thawed and mixed the concentrations of these reagents may not be dispensed at optimal concentrations for the reactio
62. s RiboAmp PLUS RNA Amplification Kit configurations The catalog numbers are provided These kits can be ordered online Table 3 RiboAmp PLUS RNA Amplification Kit Configurations Catalo al Amplifi ll Description 9 of cDNA p IVT Purifica Labeling Number cation Samples tion RiboAmp PLUS KITO521 6 1x 1x 1x RA7016 RA7011 RA7009 RiboAmp PLUS with KITO511 B 12 2x 2x 2x 1x Biotin Labeling RA7016 RA7011 RA7009 KITO608 RiboAmp PLUS with KITO511 C 12 2x 2x 2x 1x CY 3 Labeling RA7016 RA7011 RA7009 KITO609 RiboAmp PLUS with KITO511 D 12 2x 2x 2x 1x CY 5 Labeling RA7016 RA7011 RA7009 KITO610 RiboAmp PLUS KITO521aa 6 1x 1x 1x 1x Amino Allyl RA7016 RA7011 RA7010 RA7012 RiboAmp Plus bulk KITO501 24 4x 4x 4x RA7016 RA7011 RA7009 RiboAmp PLUS KIT0501aa 24 4x 4x 4x 4x Amino Allyl bulk RA7016 RA7011 RA7010 RA7012 RiboAmp PLUS 1 5 KITO526 6 1x 1x 1x Rounds RA7016 RA7011 RA7008 RiboAmp PLUS 0 5 KITO527 12 4x 4x Rounds bulk RA7016 RA7011 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 13 Chapter 2 Kit Components Contents of modules cDNA module In Vitro Transcription IVT modules 14 The tables in this section list the contents of the various modules Table 4 RiboAmp PLUS RA7016 Component Vial Color Vial Label 1st Strand Master Mix Red
63. solation Kit KIT0204 for best results Note Avoid using organic solvents in RNA isolation procedures Trace amounts of organic solvents that carry over into amplification reactions will impair the RiboAmp PLUS Kit amplification process First DNase treat the RNA prior to putting it into the RiboAmp PLUS Kits to eliminate possible genomic DNA interference in the amplification process You must then remove the DNase enzyme and buffers prior to putting the RNA into the RiboAmp PLUS Kits protocol Note You can incorporate DNase treatment directly into the protocol of the PicoPure RNA Isolation Kit or other purification column based approaches Note Using nucleic acid carrier is not necessary Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chapter 4 RiboAmp PLUS Amplification Usingspincolumns Spin columns and 0 5 mL microcentrifuge tubes are provided for nucleic acid elution for nucleic acid Improper orientation of tubes during centrifugation may result in cap breakage or elution sample loss To use the column tube assembly correctly 1 Insert a spin column into the 0 5 mL tube aligning the two cap hinges as illustrated 2 Load Elution Buffer onto the column and incubate as directed 3 Place the column tube assembly into a 2 mL lidless support tube PGC Scientific Catalog 16 8101 06 in the centrifuge rotor alternately retain and reuse the 2 mL lidless collection tubes provided Note Some varie
64. ter Mix teal AA here 4 Incubate at 42 C for 4 hours Optional If you want additional yield you can incubate the IVT reaction for up to six hours Chill the sample s to 4 8 C Note It is safe to stop at this point in the protocol You may hold the reaction mixture at 48 C in the thermal cycler overnight Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 35 Chapter 5 RiboAmp PLUS Protocol Round Two aRNA purification 36 Move the samples directly to a 4 8 C block Add 1 uL DNase Mix Blue 4 Mix thoroughly and spin down Incubate at 37 C for 15 minutes Chill the sample s to 4 8 C Proceed immediately to aRNA purification Note DNase Mix must be thoroughly mixed and held at 4 C until used RNA may be adversely affected if not purified immediately after DNase 1 10 Add 250 uL of RNA Binding Buffer Blue RB to a new purification column seated in the collection tube provided Incubate for 5 minutes at room temperature Centrifuge at 16 000 x g for 1 minute Note RNA Binding Buffer Blue RB must be at room temperature and thoroughly mixed before use A precipitate may form during long term storage Dissolve precipitate prior to use by mixing If necessary warm the RB vial to re dissolve Add 120 uL of RNA Binding Buffer to the IVT reaction sample and mix thoroughly Pipet the entire volume into the purification column To bind aRNA centrifuge at 100 x g or lowest speed setting available
65. ties of 2 mL tubes will not provide enough support Contact Life Technologies Technical Support for other alternatives Call 1 800 831 6844 4 Skip one rotor position between assemblies and position assemblies with the 0 5 mL tube cap trailing the tube during centrifugation as shown in Appendix D Centrifuge Information 5 Check for a mark on the centrifuge indicating rotation direction 6 Centrifuge as directed in the protocol Using the control A control RNA sample Purple is provided along with each kit to be used as a control templates template to verify amplification efficacy Use 10 uL of this RNA for control amplifications For RiboAmp PLUS 10 uL of control RNA contains 2 ug 200 ng uL of total RNA This is enough RNA to be used as a control for one round amplifications For use as a 2 round control you may adjust input to lower amounts 1 4 ng uL When diluting control RNA use 10 ng uL Poly I to perform dilutions Avoiding Due to the sensitivity of the RiboAmp PLUS Kits it is very important to prevent contamination RNA DNA and nuclease contamination Clean work surfaces before and after each use Perform all dispensing in a work hood that has been irradiated with UV to remove contaminants from previous amplification experiments Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 25 Chapter 4 RiboAmp PLUS Amplification 26 Arcturus RiboAmp PLUS RNA Amplification Kit User Guide RiboAmp PLUS
66. to the surface of the membrane while dispensing DE to ensure maximum absorption of DE into the membrane Gently tap the purification column to distribute the buffer if necessaty Incubate for one minute at room temperature Place each column tube assembly into the 2 mL support tube in the rotor with the 0 5 mL tube cap trailing the tube Centrifuge at 1000 x g for 1 minute and then immediately centrifuge by 16 000 x g for 1 minute Discard the column and retain the elution containing the cDNA Arcturus RiboAmp PLUS RNA Amplification Kit User Guide Chapter 5 RiboAmp PLUS Protocol IMPORTANT To avoid potential breakage of the microcentrifuge tube cap during centrifugation insert the purification tube 0 5 mL tube assembly into a lidless 1 7 2 0 mL tube Insert this assembly into adjacent rotor holes as illustrated in Appendix D Centrifuge Information Rest the tube cap against the tube immediately clockwise to it Place an empty lidless 1 7 2 0 mL tube into the rotor hole adjacent in the clockwise direction to the last assembly Note Itis safe to stop at this point in the protocol You can store the sample overnight at 209C IMPORTANT 1 5 Round Users Using Alternate IVT Labeling Procedures Stop Here 1 5 Round users KIT0526 using alternative IVT labeling such as Affymetrix labeling kit 900449 stop here and proceed to Appendix C Generating Labeled aRNA Using Alternative IVT Kits Note Life Technologies
67. uL of fresh RNA Wash Buffer to the purification column and centrifuge at 16 000 x g for 2 minutes 8 Check the purification column for any residual wash buffer If any wash buffer remains re centrifuge at 16 000 x g for 1 minute 9 Discard the collection tube and flow through IMPORTANT Avoid splashing flow through in the collection tube onto the purification column If flow through waste liquid wets the outside of the purification column re centrifuge the column at 16 000 x g to remove the liquid 10 Place the purification column into a new 0 5 mL microcentrifuge tube provided in the kit and carefully add RNA Elution Buffer Blue RE directly to the center of the purification column membrane Add one of the following volumes e 30 uL if stopping with one round of amplification or e 12 uLif going on to a second round 11 Gently touch the tip of the pipette to the surface of the membrane while dispensing RE to ensure maximum absorption of RE into the membrane Gently tap the purification column to distribute the buffer if necessary 12 Incubate at room temperature for 1 minute 13 Place each column tube assembly into the centrifuge rotor with the 0 5 mL tube cap trailing the tube 14 Centrifuge at 1000 x g for 1 minute and immediately centrifuge at 16 000 x g for 1 minute Discard the purification column and retain the elution containing the aRNA Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 31 Chapter
68. view of the amplification process 21 Time requirements 1 1 0 22 Thermal cycler programming iesnas ben cece eee eee kan dul u dk a TAYA k 22 Necessary preliminaries M M kk kk kk kk kk kk kk kk kk kk kK kk kk kk kK kk kk kk kk kk kk kk kk k 24 Protocol notes isco cee Wa yaka eee Kawana Wa Wl Le ba Wa ka kk ba ve Ge E 24 Preparing samples and reagents 24 Using spin columns for nucleic acid elution 25 singthecontroltemplates 25 Avoidingcontamination 25 RiboAmp PLUS Protocol kk kk kk a 27 RiboAmp PLUS Round One 27 Round One 1st strand cDNA synthesis 27 Round ne 2ndstrandcDNAsynthesis 28 Round ne cDNApurification 29 RoundOne inVitrolranscription 30 Round ne aRNAPurification 31 RiboAmp PLUS Round TWO 32 Protocol for performing a second round of amplification 32 Round Two 1st strand cDNA synthesis 32 R
69. yplificationKits 55 Turbo Labeling Kits u kk sikii ke yeis emel kk biri gli 55 Note Only the most frequently used kits are listed here Additional kit configurations are available depending on individual research needs For more information go to www appliedbiosystems com HistoGene LCM Frozen Section Staining Kit The HistoGene LCM Frozen Section Staining Kit is used to process tissue sections for LCM in order to maximize the quality and yield of RNA from the LCM cells The kit comes with all dehydration and staining reagents disposable staining jars specially treated slides and a detailed protocol and troubleshooting guide KIT0401 72 slides HistoGene LCM Immunofluorescence Staining Kit The HistoGene LCM Immunofluorescence Staining Kit is designed to enable retrieval of high quality RNA from immunofluorescently stained frozen tissue It enables convenient and reliable staining dehydration and LCM of tissue sections The kit s protocols are streamlined and optimized for efficient LCM capture while maintaining RNA quality for downstream applications that require intact RNA such as microarray analysis and RTPCR KIT0420 32 slides Arcturus RiboAmp PLUS RNA Amplification Kit User Guide 53 Appendix E Related Reagent Kits PicoPure RNA Isolation Kit The PicoPure RNA Isolation Kit is used for the extraction and isolation of total RNA from small samples particularly LCM

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