OneArray Amino Allyl aRNA Amplification Kit
Contents
1. J Biotechnology 107 1 19 28 Luo L Salunga RC Guo H Bittner A Joy KC Galindo JE Xiao H Rogers KE Wan JS Jackson MR Erlander MG 1999 Gene expression profiles of laser captured adjacent neuronal subtypes Nat Med 5 1 117 122 Mujumdar RB Ernst LA Mujumdar SR Lewis CJ Waggoner AS 1993 Cyanine dye labeling reagents sulfoindocyanine succinimidyl esters Bioconjug Chem 1993 4 2 105 11 Pabon C Modrusan Z Ruvolo MV Coleman IM Daniel S Yue H Arnold LJ Jr 2001 Optimized T7 amplification system for microarray analysis Biotechniques 31 4 874 879 Polacek DC Passerini AG Shi C Francesco NM Manduchi E Grant GR Powell S Bischof H Winkler H Stoeckert CJ Jr Davies PF 2003 Fidelity of enhanced sensitivity of differential transcription profiles following linear amplification of nanogram amounts of endothelial mRNA Physiol Genomics 13 147 156 Schoor O Weinschenk T Hennenlotter J Corvin S Stenzl A Rammensee H G and Stevanovic S 2003 Moderate degradation does not preclude microarray analysis of small amounts of RNA Biotechniques 35 1192 1201 Van Gelder RN von Xastrow ME Yool A Dement DC Barchas JD Eberwine JH 1990 Amplified RNA synthesized from limited quantities of heterogeneous cDNA Proc Natl Acad Sci USA 87 1663 1667 Yue H Eastman PS Wang BB Minor J Doctolero MH Nuttall RL Stack R Becker JW Montgomery JR Vainer M Johnston R 2001 An evaluation of the performance of cDNA microarr2. These dyes are supplied in relatively large aliguots resuspend one dye vial in 88 uL of DMSO and keep in the dark at room temp for up to 1 hr until you are ready to use it Dye specifications for calculation of incorporation The specifications listed below are provided for calculation of dye incorporation into labeled aRNA described in section IV C Dye type fluorescein Alexa Fluor 546 Alexa Fluor 555 Alexa Fluor 660 Alexa Fluor 647 Oyster 556 Oyster 645 Oyster 656 Absorbance maximum 491 nm 546 nm 555 nm 663 nm 650 nm 556 nm 645 nm 656 nm Extinction coefficient 66 000 104 000 150 000 132 000 239 000 155 000 250 000 220 000 Extinction coefficient at Amax in cm M 22 OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech B References Feldman AJ Costouros NG Wang E Qian M Marincola FM Alexander HR and Libutti SK 2002 Advantages of mRNA amplification for microarray analysis Biotechniques 33 4 906 9 14 t Hoen PAC de Kort F van Ommen GJB den Dunnen JT 2003 Fluorescent Labelling of cRNA for Microarray Applications Nucleic Acids Res 31 5 e20 Kacharmina JE Crino PB Eberwine J 1999 Preparation of cDNA from single cells and subcellular regions Methods Enzymol 303 3 18 Li Y Li T Liu S Qiu M Han Z Jiang Z Li R Ying K Xie Y Mao Y 2004 Systematic comparison of the fidelity of aRNA mRNA and T RNA on gene expression profiling using cDNA microarray
3. aRNA is Not Efficiently Reverse Transcribed E Troubleshooting Dye Coupling MIISADDendbO aac etek Settee ers EF UR alae amide mene ea ee eee ee cee ee eee 22 A NHS Ester Dyes from Other Suppliers B References C OneArray Amino Allyl aRNA Amplification Kit Specifications D Quality Control Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide Manual Version 001 For research use only Not for use in diagnostic procedures By use of this product you accept the terms and conditions of all applicable Limited Label Licenses For statement s and or disclaimer s applicable to this product see below OneArray Amino Allyl aRNA Amplification Kit is manufactured for Phalanx Biotech Group Inc by Ambion Inc a wholly owned subsidiary of Applied Biosystems Literature Citation When describing a procedure for publication using this product we would appreciate that you refer to it as the OneArray Amino Allyl aRNA Amplification Kit Warranty and Liability Phalanx Biotech Group s products are intended for research use only and not intended for any other uses OneArray microarray products are designed and manufactured for research use only Buyers and users agree and understand that they are not granted the right to use OneArray products for clinical diagnostic purposes unless they obtain written approval from the appropriate government authority Phalanx Biotech Group Phalanx Biotech will not be liable for any damages arising
4. the amount of RNA to use in the procedure and how long to incubate the IVT reaction It should also address specifics that are not often included in protocols such as which tubes tube racks and incubators to use for each step in the process Finally develop a consistent work flow For example standardize stopping points in the method The idea is to standardize all of the variables discussed in this section of the Instruction Manual and carefully follow all the protocol steps in order to maximize amplification consistency among samples Tubes use non stick RNase free 0 5 mL tubes It is most convenient to conduct the OneArray Amino Allyl aRNA Amplification procedure in 0 5 mL nonstick tubes These can be thin wall PCR tubes or ordinary weight nonstick tubes 0 5 mL tubes are large enough to accommodate the cDNA Binding Buffer without having to transfer reactions to a larger tube Their small size and nonstick properties also keep the reaction components at the bottom of the tube B Prepare the Wash Buffer Add 16 mL 100 ethanol ACS grade or better to the bottle labeled Wash Buffer Mix well and mark the label to indicate that the ethanol was added 8 OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech C Reverse Transcription to Synthesize First Strand cDNA Incubators needed e 70 C thermal cycler recommended e 42 C hybridization oven or air incubator recommended 1 Mix RNA and 1 uL T7 Oligo dT Primer an
5. Primers 20 C 10 uL Control RNA 1 mg mL HeLa total RNA 20 C 875 uL Nuclease free Water any temp cDNA and aRNA purification components and dye labeling reagents Amount Component Storage 200 uL Coupling Buffer 20 C 220 uL DMSO 20 C 90 uL 4M Hydroxylamine 20 C 5 mL Nuclease free Water any tempt 20 mL Wash Buffer Add 16 mL 100 ethanol before use 4 C or room temp 3 5 mL cDNA Binding Buffer room temp 10 mL aRNA Binding Buffer room temp 12 aRNA Filter Cartridges room temp 20 aRNA Collection Tubes room temp 10 cDNA Filter Cartridges Tubes room temp 10 cDNA Elution Tubes room temp 10 Labeled aRNA Filter Cartridges Tubes room temp 10 Labeled aRNA Elution Tubes room temp Store the Nuclease free Water at 20 C 4 C or room temp tStore the Nuclease free Water at 20 C 4 C or room temp The cDNA Binding Buffer may form a precipitate if stored below room temp If a precipitate is visible redissolve it by warming the solution to 37 C for up to 10 min and vortexing vigorously Cool to room temp before use 4 OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech D Materials Not Provided with the Kit Lab equipment and supplies 100 Ethanol to prepare the Wash Buffer Additional 100 DMSO may be required depending on how the fluorescent dye is supplied Thermal cycler with adjustable temperature heated lid hybridization oven or constant temperature incubators set at 70 C 42 C 37 C and 16
6. aRNA in 9 pl Coupling Buffer Y 4 Add ll ul red to the IA and mix well a4 Y UUUU 5 5 Incubate 30 min at room temp in the dark LE amp _ _ 6 Add 4 5 ul 4M Hydroxylamine and mix 3 ay gt r UUUU 5 7 Incubate 15 min at room temp in the dark labeled aRNA k a Add 5 5 ul Nuclease free Water to bring each sample to 30 ul unused dye A Ud aan aom I Add 105 yl aRNA Binding Buffer to each sample buffer 2 Add 75 ul 100 ethanol and pipet 3 times to mi v 3 Pass samples through a Labeled aRNA Filter Cartridge s 4 Wash with 500 ul Wash Buffer i P p pws 5 labeled aRNA with 2 x 10 eated Nuclease free Water OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech Optional second round of amplification Additional amplification of an RNA sample can be achieved by subjecting the aRNA to a second round of amplification see Figure 3 The reagents and methodology used in the first and second rounds of amplification are only slightly different see section V When doing two rounds of amplification it is important to synthesize the first round aRNA with the unmodified UTP Solution because amino allyl modified aRNA transcribed with the aaUTP cannot be amplified Figure 3 Second Round Amplification aRNA from 1st round of amplification 5 uuuu _ w Reverse Transcription with Second Round Primers 5 UUU lt ss 3 AAAA FEE 5 Uu Second strand synthesis with T7 Oligo dT Primer 4 T7 TTTT 5 T7 TITT 3 3 AAAA 5 cDN
7. can lower the efficiency of reverse transcription and subsequently reduce the level of amplification An effective measure of RNA purity is the ratio of absorbance readings at 260 and 280 nm The ratio of A26 to A gt so values should fall in the range of 1 7 2 1 RNA must be suspended in high quality water or TE 10 mM Tris HCl 1 mM EDTA RNA integrity The integrity of the RNA sample or the proportion that is full length is another important component of RNA quality Reverse transcribing partially degraded mRNAs will typically generate relatively short cDNAs that potentially lack portions of the coding region RNA integrity can be evaluated by microfluidic analysis using the Agilent 2100 bioanalyzer and one of Caliper s RNA LabChip Kits Primarily full length RNA will exhibit a ratio of 28S to 18S rRNA bands that approaches 2 1 Using a bioanalyzer the RIN RNA Integrity Number can be calculated to further evaluate RNA integrity The RIN a metric developed by Agilent includes information from both the rRNA bands and outside the rRNA peaks potential degradation products to provide a fuller picture of RNA degradation states Search for RIN at the following web address for more information http www chem agilent com Denaturing agarose gel electrophoresis and nucleic acid staining can also be used to separate and visualize the major rRNA species When the RNA resolves into discrete rRNA bands i e no significant smearing below each band w
8. coupled to N hydroxysuccinimidyl ester derivatized reactive dyes NHS ester dyes such as Cy 3 and Cy5 in a simple efficient reaction see Figure 1 Once purified the labeled aRNA is suitable for use on most commercially available microarray gene expression systems Small RNA samples 0 1 100 ng can be put through two rounds of amplification if desired This strategy makes the production of microarray samples from picogram amounts of total RNA entirely possible Luo et al 1999 Benefits of RNA amplification RNA amplification was originally developed as a method to expand very small RNA samples to produce enough material for array hybridization Yue et al 2001 Several groups have conducted studies to determine whether amplification of RNA introduces bias and they report that any bias is minimal Li et al 2004 Feldman et al 2002 and Polacek et al 2003 Additionally among the benefits of amplification is a more reproducible expression profile from a wide range of RNA inputs Some researchers conclude that amplification actually improves the reliability of array results regardless of whether it is needed for sample expansion Feldman et al 2002 and Polacek et al 2003 As a result RNA amplification has become the standard method for preparing RNA samples for array analysis Kacharmina et al 1999 Pabon et al 2001 w w y E B y WAAR V yp f 0 F o myg AR Ga b y J a oH a AminoAllyl UTP n
9. from the use of its products in any manner other than their intended use or for the use of its products for clinical diagnostic purposes without written approval from the appropriate government authority The manufacture sale or importation of products from Phalanx Biotech is not permitted without the prior written consent from Phalanx Biotech Buyers and users agree and acknowledge that Phalanx Biotech is the owner and has the copyrights to the probe sequence information of the Human OneArray product and any other OneArray products Phalanx Biotech is founded on the mission to offer researchers high quality and user friendly solutions at an affordable price Your satisfaction in using our products is very important to us Therefore if any of our products is not performing to the standard we promised we are willing to replace the product or credit the product purchase price Phalanx Biotech accepts liability of ONLY the purchase price of its products and has no other liabilities Trademarks and Copyrights Human OneArray and OneArray are trademarks of Phalanx Biotech Group Inc in the United States and in other countries All trademarks and copyrights used in this manual belong to their respective owners and are the sole property of their respective owners Other Companies Trademarks Cy and CyDye are trademarks of GE Healthcare RiboGreen and Alexa Fluor are registered trademarks of Molecular Probes Inc LabChip is a registered tradem
10. in a UV Vis spectrophotometer To obtain an accurate reading at both 260 nm and at the maximum absorbance wavelength for the dye the aRNA dilution should result in an A260 between 0 1 and 1 0 If the A260 is below 0 1 reduce the dilution factor and check the A260 again 2 Measure the aRNA absorbance at 260 nm and at the absorbance max for the dye Blank the instrument with the TE used for making dilutions Measure the absorbance of each sample at 260 nm A260 and also at the maximum absorbance wavelength for the dye used in the coupling reaction Adye Specifications for other fluorescent dyes are provided in section VII A Dye specifications for calculation of incorporation Dye Absorbance maximum Extinction coefficient Cy3 550 nm 150 000 Cy5 650 nm 250 000 3 Calculate the number of dye molecules incorporated per 1000 nt Use this formula to estimate the number of dye molecules incorporated per 1000 nt of labeled aRNA dye molecules A TM sed ACMA GOUSSE n a n x _ 9010em M _ X 1000 1000 nt A260 dye extinction coefficient The expected incorporation rate is 30 60 dye molecules per 1000 nt 4 Optional Calculate the RNA concentration Use the A260 to calculate the RNA concentration if desired see section J A 1 Assessing aRNA yield by UV absorbance D Preparing Labeled aRNA for Hybridization 1 If necessary vacuum dry the labeled RNA Typically microarrays are hybridized with 25 100 uL Hybridization Solution Sinc
11. the Labeled aRNA Filter Cartridge to a Labeled aRNA Elution Tube Elute labeled aRNA with 2 x 10 uL preheated Nuclease free Water a To the center of the filter add 10 uL Nuclease free Water that is preheated to 50 60 C b Leave at room temp for 2 min and then centrifuge for 1 5 min at 10 000 X g or until the Nuclease free Water is through the filter c Repeat steps 5 a b with a second 10 uL of preheated Nuclease free Water d The aRNA will now be in the Labeled aRNA Elution Tube in 20 uL of Nuclease free Water Store purified labeled aRNA at 20 C in the dark or continue the procedure If the sample will not be used the same day store it at 209C in the dark We do not recommend storing labeled aRNA for long periods of time prepare labeled aRNA as it is needed for your experiments To continue determine the aRNA concentration using one of the methods described in section Z A aRNA Quantitation and Expected Yield The aRNA concentration must be known in order to continue to the next step fragmentation and hybridization Stopping Point The purified labeled aRNA can be stored at 20 C overnight or at 80 C for longer times if desired OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech C Spectrophotometric Analysis of Dye Incorporation 1 Determine the appropriate dilution for spectrophotometer readings Dilute labeled aRNA 1 10 in TE 10 mM Tris HCl I mM EDTA Mix well and measure the A260
12. through section II G Analysis of the positive control amplification reaction After completing the aRNA purification measure the A260 of the reaction product as described in section III A The positive control reaction should produce 70 ug of aRNA Be aware that often the positive control reaction cannot be compared to experimental reactions because many experimental amplification experiments will use less than the 1 ug of input RNA used in the positive control reaction and the aRNA yield will be proportionately lower Also the Control RNA is of exceptional guality and purity ensuring that it will amplify with extremely high efficiency Also run a 2 ug aliquot of the reaction products on a denaturing agarose gel or analyze 100 200 ng on a bioanalyzer the average size of the aRNA should be 1 kb Figure 6 shows bioanalyzer data of both amino allyl modified aRNA and unmodified aRNA produced from the Control RNA Labeling the aRNA produced in the positive control amplification reaction Use 10 ug of aRNA in a dye coupling reaction following the instructions in section IV A through IV C Evaluate the positive control reaction by determining the number of dye molecules incorporated per 1000 nt as described in section IV C Spectrophotometric Analysis of Dye Incorporation The expected incorporation rate is 30 60 dye molecules per 1000 nt 35 Ladder 30 o 25 ha S on Amino allyl aRNA a2 U 2 15 Q 10 dn o 14 19 24 29 34
13. to RCFs over 16 000 x g because the force could cause mechanical damage and or may deposit glass filter fiber in the eluate Preheat Nuclease free Water to 50 60 C Before beginning the aRNA purification preheat the 10 mL bottle of Nuclease free Water to 50 60 C for at least 10 min Assemble aRNA Filter Cartridges and tubes For each sample place an aRNA Filter Cartridge into an aRNA Collection Tube and set aside for use in step 3 1 Add 350 uL aRNA Binding Buffer to each sample Check to make sure that each IVT reaction was brought to 100 uL with Nuclease free Water Add 350 uL of aRNA Binding Buffer to each aRNA sample Proceed to the next step immediately 2 Add 250 uL 100 ethanol and pipet 3 times to mix Add 250 uL of ACS grade 10090 ethanol to each aRNA sample and mix by pipetting the mixture up and down 3 times Do NOT vortex to mix and do NOT centrifuge Proceed immediafely to the next step as soon as you have mixed the ethanol into each sample Any delay in proceeding could result in loss of aRNA because once the ethanol is added the aRNA will be in a semiprecipitated state 3 Pass samples through an aRNA Filter Cartridge s a Pipet each sample mixture from step 2 onto the center of the filter in the aRNA Filter Cartridge b Centrifuge for 1 min at 10 000 X g Continue until the mixture has passed through the filter c Discard the flow through and replace the aRNA Filter Cartridge back into the aRNA Collection Tube 4
14. uL T7 Oligo dT Primer to cDNA sample b Mix well by gently vortexing then centrifuge briefly 5 sec to collect the sample at the bottom of the tube 2 Incubate for 10 min at 70 C then place on ice a Incubate 10 min at 70 C in a thermal cycler b Place the reaction on ice briefly before adding the remaining second strand cDNA synthesis reagents 3 Add 74 uL Second Strand Master Mix to each sample a On ice prepare a Second Strand Master Mix by adding the following reagents in the order listed below Assemble master mix for all the samples in the experiment including 5 overage to cover pipetting error Assemble the Second Strand Master Mix on ice in the order shown Second Strand Master Mix for a single 100 uL reaction Amount Component 58 uL Nuclease Free Water 10 uL 10X Second Strand Buffer 4 uL dNTP Mix 2 ML DNA Polymerase b Mix well by gently vortexing Centrifuge briefly 5 sec to collect the Second Strand Master Mix at the bottom of the tube and place on ice c Transfer 74 uL of Second Strand Master Mix to each sample Mix thoroughly by pipetting up and down 2 3 times then flicking the tube 3 times and centrifuge briefly to collect the reaction in the bottom of the tube d Place the tubes in a pre cooled 16 C thermal cycler It is important to cool the thermal cycler block to 16 C before placing the reaction tubes subjecting the reactions to temperatures gt 16 C could compromise aRNA yield 4 Incuba
15. 0 mL aRNA Binding Buffer room temp 20 aRNA Filter Cartridges room temp 40 aRNA Collection Tubes room temp 20 cDNA Filter Cartridges Tubes room temp 20 cDNA Elution Tubes room temp 20 Labeled aRNA Filter Cartridges Tubes room temp 20 Labeled aRNA Elution Tubes room temp ttStore the Nuclease free Water at 20 C 4 C or room temp Store the Nuclease free Water at 20 C 4 C or room temp The cDNA Binding Buffer may form a precipitate if stored below room temp If a precipitate is visible redissolve it by warming the solution to 37 C for up to 10 min and vortexing vigorously Cool to room temp before use 24 OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech To obtain Material Safety Data Sheets e Email your request to info phalanxbiotech com Specify the catalog or part number s of the product s and we will e mail the associated MSDSs unless you specify a preference for fax delivery For customers without access to the internet or fax our technical service department can fulfill MSDS requests placed by telephone or postal mail D Quality Control Functional testing The Control RNA is used in a OneArray Amino Allyl aRNA Amplification reaction following the instructions in section VLA The aRNA yield is assessed by measuring the Azs on the NanoDrop ND1000A spectrophotometer The median size of the aRNA is assessed using the mRNA smear assay on the Agilent 2100 bioanalyzer CyDye is used to label 10 ug
16. 3 Cy5 N hydroxysuccinimide ester J TE a A wen wy m y S P 0 P 0 P 0 Nn ge ey dle Cy5 UTP i Figure 1 Amino Allyl Labeling Reaction B Procedure Overview The OneArray Amino Allyl aRNA amplification procedure is depicted in Figure 2 e Reverse Transcription to Synthesize First Strand cDNA is primed with the T7 Oligo dT Primer to synthesize cDNA containing a T7 promoter sequence e Second Strand cDNA Synthesis converts the single stranded cDNA into a double stranded DNA dsDNA template for transcription The reaction employs DNA Polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA e cDNA Purification removes RNA primers enzymes and salts that would inhibit in vitro transcription e In Vitro Transcription to Synthesize Amino Allyl Modified aRNA with aaUTP generates multiple copies of amino allyl modified aRNA from the double stranded cDNA templates this is the amplification step e aRNA Purification removes unincorporated NTPs salts enzymes and inorganic phosphate to improve the stability of the aRNA and to facilitate NHS ester coupling or subsequent enzymatic reactions e aRNA Dye Coupling Reaction takes place between the amino allyl modified UTP residues on the aRNA and amine reactive Cy3 or Cy5 dyes Dye Labeled aRNA Purification removes free dye and exchanges the buffer with Nuclease free Water Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit U
17. 39 44 49 54 59 64 69 74 79 84 Time seconds Figure 6 Bioanalyzer Electropherogram of aa UTP Modified aRNA and Unmodified aRNA The same double stranded cDNA was used as a template for in vitro transcription reactions containing either 5090 aaUTP amino allyl aRNA or 100 UTP unmodified aRNA Each reaction was run on an Agilent bioanalyzer using the RNA 6000 Marker and Nano LabChip Kit The average size and yield of aRNA is very similar regardless of whether 5090 aaUTP was included in the amplification reaction B Factors that Affect Both the Positive Control and Experimental Samples If the positive control reaction yield or amplification product size does not meet expectations consider the following possible causes and troubleshooting suggestions These suggestions also apply to problems with amplification of experimental RNA a Incubation temperature s were incorrect The incubation temperatures are critical for effective RNA amplification Check the temperatures of all incubators used in the procedure with a calibrated thermometer If a thermal cycler is used for incubation check the accuracy of the adjustable temperature lid If the lid temperature cannot be adjusted to match the desired reaction temperature use the lid with the heat turned off or do not use the lid to cover the reaction vessel s b Condensation formed in the tube during the reaction incubation s Condensation occurs when the cap of the reaction vessel is co
18. 5 uL of aRNA Binding Buffer to each aRNA sample Proceed to the next step immediately Add 75 uL 100 ethanol and pipet 3 times to mix Add 75 uL of ACS grade 100 ethanol to each labeled aRNA sample and mix by pipetting the mixture up and down 3 times Do NOT vortex and do NOT centrifuge Proceed immediately to the next step as soon as you have mixed the ethanol into each sample Any delay in proceeding could result in loss of aRNA because once the ethanol is added the aRNA will be in a semiprecipitated state Pass samples through a Labeled aRNA Filter Cartridge s a Pipet each sample mixture onto the center of the filter in the Labeled aRNA Filter Cartridge b Centrifuge for 1 min at 10 000 X g Continue until the mixture has passed through the filter c Discard the flow through and replace the Labeled aRNA Filter Cartridge back into the Labeled aRNA Collection Tube Note The filter in the Labeled aRNA Filter Cartridge may acguire the color of the fluorescent dye during the purification This is from the labeled aRNA binding to the filter Most of the color should disappear when the purified aRNA is eluted in step 5 Wash with 500 pL Wash Buffer a Apply 500 uL Wash Buffer to each Labeled aRNA Filter Cartridge b Centrifuge for 1 min at 10 000 X g or until all the Wash Buffer passes through the filter o Discard the flow through and centrifuge for an additional 1 min to remove trace amounts of Wash Buffer d Transfer
19. A Coupling Buffer mixture Mix well by vortexing gently 5 Incubate 30 min at room temp in the dark This 30 min incubation at room temp allows the dye coupling reaction to occur To keep the samples in the dark simply put the tubes in a closed drawer 6 Add 4 5 pL 4M Hydroxylamine and mix To quench the reaction add 4 5 uL 4M Hydroxylamine and mix well by vortexing gently 7 Incubate 15 min at room temp in the dark Incubate the reaction in the dark at room temp for 15 min During this incubation the large molar excess of hydroxylamine will guench the amine reactive groups on the unreacted dye molecules 8 Add 5 5 uL Nuclease free Water to bring each sample to 30 uL Add 5 5 uL Nuclease free Water to each sample to bring the volume to 30 uL Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide 15 Dye Labeled aRNA Purification This purification removes excess dye from the labeled aRNA Important All centrifugations in this purification procedure should be done at 10 000 x g typically 10 000 rpm at room temp aRNA Filter Cartridges should not be subjected to RCFs over 16 000 x g because the force could cause mechanical damage and or may deposit glass filter fiber in the eluate Preheat Nuclease free Water to 50 60 C 1 4 16 Before beginning the labeled aRNA purification preheat the 10 mL bottle of Nuclease free Water to 50 60 C for at least 10 min Add 105 uL aRNA Binding Buffer to each sample Add 10
20. A purification amino allyl 4 in vitro transcription with aaUTP amplification modified aRNA Suse ES 5 UUUU Fe o r 5 UUUU Fe y 3 ce aRNA purification Coupling to NHS ester dye Dye quenching labeled aRNA purification dye labeled aRNA 5 UUU Tt _ _ t3 Sue r x x Suu y3 The OneArray Amino Allyl advantage The first strand cDNA synthesis reaction employs an engineered reverse transcriptase to ensure that every cDNA bears a T7 promoter at its 5 end and that even very limited amounts of mRNA are fully converted to full length cDNA The second strand cDNA synthesis reaction is designed for the efficient synthesis of full length double stranded cDNAs and the complete conversion of single stranded cDNA into double stranded transcription templates The cDNA purification procedure not only removes enzymes salts and unincorporated dNTPs but also efficiently removes RNA from the cDNA sample This eliminates the heating or enzymatic digestion step that is commonly used in other procedures to degrade RNA especially ribosomal RNA The IVT reaction optimized to ensure efficient transcription of limited amounts of template DNA as well as synthesis of long transcripts The NTPs are provided in three separate vials Providing the UTP and the aaUTP separately provides flexibility to make unlabeled aRNA or to incorporate aaUTP during either the first or second round of amplification Unlike most modified nucleotides the inclusio
21. C See Incubator recommendations for more information Vacuum centrifuge concentrator Vortex mixer Microcentrifuge Non stick RNase free 0 5 mL microfuge tubes Optional RNA controls for microarrays analysis such as Array Control RNA Spikes from Ambion Cat AM1780 Optional materials and equipment for RNA analysis Spectrophotometer such as the NanoDrop ND 1000A UV Vis Spectrophotometer With the NanoDrop Spectrophotometer the user simply pipets 1 5 2 uL of sample onto the measurement pedestal no dilutions or cuvettes are necessary The NanoDrop Spectrophotometer performs all UV Vis spectrophotometric analyses carried out by traditional spectrophotometers Optional Reagents and apparatus for preparation and electrophoresis of agarose gels Optional RiboGreen RNA Ouantitation Assay and Kit Molecular Probes Inc Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide 5 ll aRNA Amplification Protocol A Important Parameters for Successful Amplification Input RNA quantity and IVT reaction incubation time Consider both the amount of sample RNA you have and the amount of aRNA needed for your analysis when planning OneArray Amino Allyl aRNA Amplification experiments These factors will influence how much input RNA to use whether one or two rounds of amplification should be done and how long to incubate the IVT reaction Accurate quantitation For experiments where the aRNA yield from different samples will be co
22. OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech Group OneArray Amino Allyl aRNA Amplification Kit User Guide k MINTS i ica cata is a gh dia a aT FFON Shon a aca SYTN ARE aw he af aan side E A Product Description and Background B Procedure Overview C Materials Provided with the Kit and Storage Conditions D Materials Not Provided with the Kit Il aRNA Amplification Protocol Y FF FY tees A Important Parameters for Successful Amplification B Prepare the Wash Buffer C Reverse Transcription to Synthesize First Strand cDNA D Second Strand cDNA Synthesis E cDNA Purification F In Vitro Transcription to Synthesize Amino Allyl Modified aRNA G aRNA Purification Ill Assessing aRNA Yield and Quality A aRNA Quantitation and Expected Yield B Analysis of aRNA Size IV Dye Coupling and Labeled aRNA Cleanup YYF eee 15 A aRNA Dye Coupling Reaction B Dye Labeled aRNA Purification C Spectrophotometric Analysis of Dye Incorporation D Preparing Labeled aRNA for Hybridization V Optional Second Round Amplification 00 ccc FF ua 18 A Synthesis of First Strand cDNA Second Round B Synthesis of Second Strand cDNA Second Round VI Troubleshooting sei oe hd bbe Be one Cn a ees DG Du aah oo EA 20 A Positive Control Reaction B Factors that Affect Both the Positive Control and Experimental Samples C Troubleshooting Low Yield and Small Average aRNA Size D
23. Reverse Transcription Master Mix to each RNA sample Mix thoroughly by pipetting up and down 2 3 times then flicking the tube 3 4 times and centrifuge briefly to collect the reaction in the bottom of the tube d Place the samples in a 42 C incubator 4 Incubate for 2 hr at 42 C Incubate reactions for 2 hr at 42 C hybridization oven or air incubator is recommended After the incubation centrifuge briefly 5 sec to collect the reaction at the bottom of the tube Place the tubes on ice and immediately proceed to second strand cDNA synthesis below D Second Strand cDNA Synthesis Incubator needed e 16 C thermal cycler recommended 1 Add 80 uL Second Strand Master Mix to each sample a On ice prepare a Second Strand Master Mix in a nuclease free tube in the order listed below Assemble enough to synthesize second strand cDNA from all the samples in the experiment including 5 overage to cover pipetting error Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide 9 Assemble the Second Strand Master Mix on ice in the order shown Second Strand Master Mix for a single 100 uL reaction Amount Component 63 uL Nuclease free Water 10 uL 10X Second Strand Buffer 4 uL dNTP Mix 2 uL DNA Polymerase 1 uL RNase H b Mix well by gently vortexing Centrifuge briefly 5 sec to collect the Second Strand Master Mix at the bottom of the tube and place on ice c Transfer 80 uL of Second Strand Master Mix to each
24. Wash with 650 pL Wash Buffer Apply 650 uL Wash Buffer to each aRNA Filter Cartridge 2 oz Centrifuge for 1 min at 10 000 X g or until all the Wash Buffer is through the filter c Discard the flow through and spin the aRNA Filter Cartridge for an additional 1 min to remove trace amounts of Wash Buffer d Transfer Filter Cartridge s to a fresh aRNA Collection Tube 5 Elute aRNA with 100 uL preheated Nuclease free Water a To the center of the filter add 100 uL Nuclease free Water preheated to 50 60 C b Leave at room temp for 2 min and then centrifuge for 1 5 min at 10 000 X g or until the Nuclease free Water is through the 12 OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech filter c The aRNA will now be in the aRNA Collection Tube in 100 uL of Nuclease free Water d Store purified aRNA at 20 C or determine its concentration using one of the methods described in section J A aRNA Quantitation and Expected Yield The aRNA concentration must be known in order to continue to the dye coupling reaction Stopping Point The purified aRNA can be stored at 20 C overnight or at 80 C for longer times if desired Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide 13 III Assessing aRNA Yield and Quality A aRNA Quantitation and Expected Yield 1 Assessing aRNA yield by UV absorbance The concentration of an aRNA solution can be determined by measuring its absorbance at 260
25. an provide a fast and accurate size distribution profile of aRNA samples but aRNA yield should be determined by UV absorbance or RiboGreen analysis To analyze aRNA size using a bioanalyzer follow the manufacturer s instructions for running the assay using purified aRNA from step II G 5 Expected aRNA size Agilent bioanalyzer analysis The expected aRNA profile is a distribution of sizes from 250 5500 nt with most of the aRNA between 1000 1500 nt Figure 6 To compare bioanalyzer profiles of different aRNA samples be sure to load equal mass amounts to get an accurate comparison When comparing unmodified aRNA with amino allyl modified aRNA you will notice that amino allyl aRNA migrates at a slightly higher molecular weight than unmodified aRNA Denaturing agarose gel analysis Amplified aRNA should appear as a smear from 250 to 5000 nt The average size of amino allyl aRNA should be approximately 1400 nt the average size of unmodified aRNA should be 1150 nt 14 OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech IV Dye Coupling and Labeled aRNA Cleanup Any label moiety should be capable of coupling to the amino allyl modified aRNA generated with this kit For a partial list of commercially available NHS ester dyes and instructions for preparing them for use with this kit see section VII A NHS Ester Dyes from Other Suppliers The choice of dye will depend on your preference and on the microarray scanning equipment a
26. ark of Caliper Technologies Corp NanoDrop is a registered trademark of NanoDrop Technologies Inc Oyster is a trademark of DenovoBiolabels GmbH Copyright 2008 by Phalanx Biotech Group Inc All Rights Reserved OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech I Introduction A Product Description and Background The OneArray Amino Allyl aRNA Amplification Kit is based on the RNA amplification protocol developed in the Eberwine laboratory Van Gelder et al 1990 The procedure consists of reverse transcription with an oligo dT primer bearing a T7 promoter using a reverse transcriptase RT engineered to produce higher yields of first strand cDNA than wild type enzymes The RT enzyme catalyzes the synthesis of virtually full length cDNA which is the best way to ensure production of reproducible microarray samples The cDNA then undergoes second strand synthesis and clean up to become a template for in vitro transcription IVT with T7 RNA polymerase To maximize aRNA yield IVT technology is used to generate hundreds to thousands of antisense RNA copies of each mRNA in a sample In this Instruction Manual the antisense amplified RNA is referred to as aRNA it is also commonly called CRNA The IVT is configured to incorporate the modified nucleotide 5 3 aminoallyl UTP aaUTP into the aRNA during in vitro transcription aaUTP contains a reactive primary amino group on the C5 position of uracil that can be chemically
27. ata are subjected to rigorous statistical analysis Schoor et al 2003 Reaction incubation times should be precise and consistent The incubation times for most of the enzymatic reactions in the protocol were optimized in conjunction with the kit reagents to ensure the maximum yield of nucleic acid product in each step adhere to them closely An exception is the IVT reaction where a range of 4 14 hr incubation time is acceptable step II F 2 Refer to Figure 4 to help determine what incubation time to use Keep this IVT incubation time uniform if aRNA yield from different samples will be compared or if you want to have equal amplification of different samples Although differences in IVT incubation time among samples has had very little if any effect on array results in our hands we still recommend using a uniform IVT incubation time for the most reproducible amplification and array analysis Master mixes We strongly recommend preparing master mixes for the OneArray Amino Allyl aRNA Amplification procedure This approach reduces the effects of pipetting error saves time and improves reproducibility Using master mixes is especially important when aRNA yield from different samples will be compared Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide 7 Thorough mixing is very important for reproducibility Below are specific instructions for mixing kit reagents master mixes and individual reactions For maximum repro
28. ays for detecting changes in global mRNA expression Nucleic Acids Res 29 8 E41 1 Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide 23 C OneArray Amino Allyl aRNA Amplification Kit Specifications OneArray Amino Allyl aRNA Amplification Kit Components The OneArray Amino Allyl aRNA Amplification Kit includes reagents for single round amplification of 20 samples or double round amplification of 10 samples Properly stored kits are guaranteed for 6 months from receipt cDNA synthesis and in vitro transcription Amount Component Storage 60 uL T7 Oligo dT Primer 20 C 22 uL ArrayScript 20 C 22 uL RNase Inhibitor 20 C 42 uL 10X First Strand Buffer 20 C 170 uL dNTP Mix 20 C 210 uL 10X Second Strand Buffer 20 C 42 uL DNA Polymerase 20 C 22 uL RNase H 20 C 84 uL T7 Enzyme Mix 20 C 84 uL T7 10X Reaction Buffer 20 C 95 uL UTP Solution 50 mM 20 C 64 uL aaUTP 50 mM 5 3 amino allyl UTP 20 C 255 uL ATP CTP GTP Mix 25 mM each nucleotide 20 C 40 uL Second Round Primers 20 C 10 uL Control RNA 1 mg mL HeLa total RNA 20 C 1 75 mL Nuclease free Water any temptt cDNA and aRNA purification components and dye labeling reagents Amount Component Storage 400 uL Coupling Buffer 20 C 440 uL DMSO 20 C 180 pL 4M Hydroxylamine 20 C 10 mL _ Nuclease free Water any temp t 40 mL Wash Buffer Add 32 mL 100 ethanol before use 4 C or room temp 7 mL cDNA Binding Buffer room temp 2
29. d aRNA from the first round of amplification into a sterile RNase free microfuge tube With very small RNA samples we have dried the entire first round amplification reaction and used it as starting material for the second round amplification Important The volume of the aRNA must be 10 uL If necessary concentrate the aRNA in a vacuum centrifuge Do not dry the aRNA completely as this could impede reverse transcription b Add 2 uL of Second Round Primers c Add Nuclease free Water to bring the volume to 12 uL vortex briefly to mix and centrifuge briefly to collect the reaction in the bottom of the tube Incubate 10 min at 70 C a Incubate for 10 minutes at 70 C in a thermal cycler b Remove the RNA samples from the 70 C incubation and centrifuge briefly 5 sec to collect the reaction at the bottom of the tube Place the reaction on ice briefly before starting step 3 Add 8 uL of Reverse Transcription Master Mix and place at 42 C a At room temp prepare Reverse Transcription Master Mix in a nuclease free tube Assemble enough master mix for all of the samples in the experiment including 5 overage to cover pipetting error Assemble the Reverse Transcription Master Mix in the order shown Reverse Transcription Master Mix for a single 20 uL reaction Amount Component 2 uL 10X First Strand Buffer 4 uL dNTP Mix 1 uL RNase Inhibitor 1 uL Reverse Transcriptase b Mix well by gently vortexing Centrifuge briefly 5 sec t
30. d adjust volume to 12 uL a Place a maximum volume of 10 uL of total RNA 1000 ng recommended or poly A selected RNA typically 10 100 ng into a nonstick sterile RNase free 0 5 mL tube RNA must be in high guality water or TE b Add 1 uL of T7 Oligo dT Primer c Add Nuclease free Water to a final volume of 12 uL vortex briefly to mix then centrifuge to collect the mixture at the bottom of the tube Note If your experiment will include RNA Spikes e g Ambion s ArrayControl Kit add them to samples at this step 2 Incubate 10 min at 70 C then place on ice a Incubate 10 min at 70 C in a thermal cycler b Centrifuge samples briefly 5 sec to collect them at the bottom of the tube Place the mixtures on ice 3 Add 8 uL of Reverse Transcription Master Mix and place at 42 C a At room temp prepare Reverse Transcription Master Mix in a nuclease free tube Assemble enough to synthesize first strand cDNA from all the RNA samples in the experiment including 5 overage to cover pipetting error At room temp assemble the Reverse Transcription Master Mix in the order shown Reverse Transcription Master Mix for a single 20 uL reaction Amount Component 2 uL 10X First Strand Buffer 4 uL dNTP Mix 1 uL RNase Inhibitor 1 ML Reverse Transcriptase b Mix well by gently vortexing Centrifuge briefly 5 sec to collect the Reverse Transcription Master Mix at the bottom of the tube and place on ice c Transfer 8 uL of
31. ducibility and aRNA yield follow these instructions closely Mix each kit component after thawing Mix enzyme solutions by gently flicking the tube a few times before adding them to reactions Thaw frozen reagents completely at room temperature i e primers nucleotides and 10X buffers then mix thoroughly by vortexing and keep on ice before use Mix master mixes by gentle vortexing After assembling master mixes gently vortex to make a homogenous mixture without inactivating the enzyme s Mix individual reactions by pipetting and flicking the tube After adding master mixes or other reagents to individual reactions pipet up and down 2 3 times to rinse reagents from the pipet tip Then flick the tube with your finger 3 4 times to mix thoroughly and finish by centrifuging briefly to collect the reaction at the bottom of the tube Incubator recommendations We recommend a calibrated hybridization oven thermal cycler or other constant temperature air incubator for most enzymatic reaction incubations We do not recommend using ordinary laboratory heat blocks or water baths for any OneArray Amino Allyl aRNA Amplification reaction incubations To avoid any potential influence on the reaction temperature from the tube holder let tube holders equilibrate in the incubator for sufficient time or use a tube holder that does not touch the sides and bottoms of the tubes for example a floating tube support For the 16 C second strand synth
32. e the cDNA Filter Cartridge in the wash tube Important Make sure that the ethanol has been added to the bottle of Wash Buffer before using it in this Step 3 Wash with 500 pL Wash Buffer o b c d Apply 500 uL Wash Buffer to each cDNA Filter Cartridge Centrifuge for 1 min at 10 000 X g or until all the Wash Buffer is through the filter Discard the flow through and spin the cDNA Filter Cartridge for an additional minute to remove trace amounts of Wash Buffer Transfer cDNA Filter Cartridge to a cDNA Elution Tube 4 Elute cDNA with 2 x 9 uL 50 55 C Nuclease free Water It is important to use Nuclease free Water that is at 50 559C for the cDNA elution Colder water will be less efficient at eluting the F cDNA and hotter water 58 C may result in reduced aRNA yield Apply 9 uL of Nuclease free Water preheated to 50 55 C to the center of the filter in the cDNA Filter Cartridge Leave at room temperature for 2 min and then centrifuge for 1 5 min at 10 000 x g or until all the Nuclease free Water is through the filter Elute with a second 9 uL of preheated Nuclease free Water The double stranded cDNA will now be in the eluate 14 uL Proceed directly to section F next or place the cDNA at 20 C Stopping Point The purified cDNA can be stored overnight at 20 C at this point if desired In Vitro Transcription to Synthesize Amino Allyl Modified aRNA Incubator needed 37 C hybridization ove
33. e the labeled aRNA is eluted in only 20 uL it may or may not need to be concentrated in order to use it for microarray hybridization If it is necessary to concentrate the labeled aRNA we recommend vacuum drying in the dark until the volume is reduced to 1 10 uL cover the lid of the vacuum drier with aluminum foil if necessary to keep out ambient light Bring the volume of the aRNA to 10 uL with either hybridization buffer or Nuclease free Water and calculate the aRNA concentration based on the amount of RNA coupled to dye or the A260 reading 2 Fragment labeled RNA for hybridization to oligonucleotide microarrays For microarrays printed with oligonucleotides the labeled aRNA must be fragmented for proper hybridization Any RNA hydrolysis method that produces RNA fragments in the 60 200 nucleotide size range can be used Follow the procedure associated with the fragmentation method used 3 Dilute the labeled aRNA into the hybridization solution aRNA amount The amount of aRNA to use for hybridization will depend on your microarray type and will have to be optimized for maximum sensitivity and minimal background Important Using the coverslip method and the volumes described in the HOA or MOA User Guide we recommend hybridizing with at least 2 ug of labeled aRNA per microarray Choice of hybridization solution We recommend using the Hybridization Solution provided with your Human or Mouse OneArray microarray See the HOA or MOA Use
34. ently Reverse Transcribed 1 The cDNA procedure relies on oligo dT priming The aRNA has a poly U tract near the 5 end but lacks a poly A tract at its 3 end Thus any reverse transcription procedures that rely on oligo dT primers will not effectively convert aRNA to cDNA Try using gene specific or random primers 2 The filter in the aRNA Filter Cartridge was not completely dried after the wash steps If the aRNA contains ethanol carried over from the Wash Buffer it can inhibit reverse transcription Make sure that the filter is completely dry at step II G 4 c just before eluting the aRNA 3 Absorbance readings are inaccurate Confirm that your spectrophotometer is accurate by measuring the absorbance of an RNA or DNA sample of known concentration Alternatively assess the quantity of aRNA by a different method such as fractionating on an agarose gel adjacent to an RNA sample whose concentration is known and comparing the ethidium bromide staining or using a sensitive RNA dye like RiboGreen E Troubleshooting Dye Coupling NHS ester dye coupling is a simple and robust chemical reaction but unreacted NHS ester dye solutions are susceptible to hydrolysis and photo bleaching Dye esters that have been exposed to water or stored improperly may lose much of their reactivity Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide 21 VII Appendix A NHS Ester Dyes from Other Suppliers Amine reactive fluorescent dye suppl
35. esis reaction incubation step ll D 2 we recommend using a thermal cycler Ideally these reactions should be incubated in a calibrated thermal cycler with a lid temperature that matches the block temperature If your machine does not have this feature and the lid temperature is static 100 C use it with the lid heat turned off or do not close the heated lid Otherwise heat from the lid will raise the temperature of the solution in the tube compromising the reaction The procedure is very sensitive to temperature therefore use incubators that have been professionally calibrated according the manufacturer s recommended schedule Variable or inaccurate incubation temperatures can limit aRNA synthesis Preheat incubators so that the correct temperature has stabilized before reactions are placed in the incubator It is also very important that condensation does not form in the reaction tubes during any of the incubations Condensation changes the composition of reaction mixtures and can greatly reduce yield Maintaining consistency Procedural consistency is very important for amplification experiments Consider implementing a detailed procedural plan that will be used by everyone in the lab to maintain consistency This type of plan will minimize variation due to subtle procedural differences that can influence RNA amplification and may complicate gene expression studies The plan should include basic information such as the method of RNA isolation
36. iers Mono reactive NHS esters of any label moiety should be capable of coupling to the amino allyl modified aRNA generated with this kit Common dyes are listed below e Amersham Biosciences CyDye Post Labeling Reactive Dye Pack Cat RPN 5661 e Molecular Probes Alexa Fluor Succinimidyl Esters Alexa Fluor 546 Cat A 20002 Alexa Fluor 555 Cat A 20009 Alexa Fluor 647 Cat A 20006 Alexa Fluor 660 Cat A 20007 e Pierce Biotechnology NHS Fluorescein Product 46100 e Denovo Biolabels GmbH Oyster Dyes Oyster 556 Cat OY 556 1 N 1x0 2 Oyster 645 Cat HOY 645 2 N 1x0 2 Oyster 656 Cat OY 656 1 N 1x0 2 Preparation of dye for coupling Prepare dye immediately before starting the dye coupling procedure the table below shows recommendations for solubilizing several commercially available NHS ester dyes Store any unused solubilized dye in the dark at 80 C Note that most dye manufacturers do not recommend storing solubilized dyes for more than 1 month Dye Type Amersham Biosciences CyDye Post Labelling Reactive Dyes Denovo Biolabels GmbH Oyster Dyes Molecular Probes Alexa Fluor Succinimidyl Esters Preparation Instructions These dyes are supplied in single use guantities Resuspend one vial with 11 uL of DMSO and keep in the dark at room temp for up to 1 hr until you are ready to use it Resuspend one dye vial in 55 yL of DMSO and keep in the dark at room temp for up to 1 hr until you are ready to use it
37. ignificant amounts of contaminating DNA protein phenol ethanol or salts are reverse transcribed poorly and subsequently generate less aRNA than pure RNA samples Phenol extract and ethanol precipitate your RNA or use a column based purification kit to further purify your RNA before reverse transcription 2 Lower than expected input RNA concentration Take another A260 reading of your RNA sample or try using more RNA in the aRNA amplification procedure 3 RNA integrity is compromised RNA that is partially degraded generates cDNA that is relatively short This will reduce the average size of the aRNA population and subsequently reduce the yield of aRNA You can assess the integrity of an RNA sample by determining the size of the 18S and 28S rRNA bands and the relative abundance of 28S to 18S rRNA See section J A RNA integrity for more information 4 The mRNA content of your total RNA sample is lower than expected Different RNA samples contain different amounts of mRNA In healthy cells mRNA constitutes 1 390 of total cellular RNA The actual amount of mRNA depends on the cell type and the physiological state of the sample When calculating the amount of amplification the starting mass of mRNA in a total RNA prep should always be considered within a range of 10 30 ng per ug of total RNA assuming good RNA quality Most total RNA samples can be amplified up to 1000 fold producing 10 30 ug of aRNA from 1 ug of total RNA D aRNA is Not Effici
38. ith the 28S rRNA band appearing approximately twice as intense as the 18S rRNA band then the mRNA in the sample is likely to be mostly full length The primary drawback to gel electrophoresis is that it reguires microgram amounts of RNA 28S Intact Total RNA Typical Total RNA Prep 7 Lower Integrity RNA Prep ssaes ssa g Fluorescence Fluorescence ee 8 Fluorescence 0 o f t t t f t t t t t 1 rit 279 3 3 a 19 m 29 4 1 14 54 Time seconds Time seconds Time seconds Figure 5 Bioanalyzer Images of Total RNA Preparations These electropherograms from the Agilent 2100 bioanalyzer show RNA samples with decreasing integrity that are all of sufficient guality to use as input for the OneArray Amino Allyl aRNA Amplification Kit The trace labeled Intact Total RNA represents the ideal for a bioanalyzer trace of total RNA Notice how the ribosomal RNA peaks are at a ratio of about 2 28S 18S in this sample this represents ultrahigh quality RNA in terms of integrity Most RNA samples will more closely resemble the center trace where there are nearly egual amounts of 28S and 18S rRNA The trace on the right shows a fairly typical human RNA prep with rRNA peaks that are lower than in the other two traces and where lower molecular weight degradation products become apparent RNA samples with suboptimal integrity can yield meaningful array analysis results if the d
39. mpared it is essential to accurately quantify the input RNA used in the OneArray Amino Allyl aRNA Amplification procedure We recommend the NanoDrop 1000A Spectrophotometer for rapid accurate quantitation of nucleic acids however any reliable RNA quantitation method such as traditional spectrophotometry or RiboGreen can be used Recommended minimum and maximum amounts of input RNA Table 1 shows the mass of total RNA that can be used in the OneArray Amino Allyl aRNA Amplification procedure Alternatively 10 100 ng of poly A selected RNA can be used in the procedure The RNA volume must be 10 uL Table 1 Amount of Total RNA to Use in OneArray Amino Allyl aRNA Amplification Amplification Recommended Minimum Maximum single round 1000 ng 100 ng 5000 ng two rounds 100 ng 0 1 ng 100 ng Determining input RNA amount and IVT reaction incubation time The procedure can accommodate a wide range of input RNA amounts but for reproducible and comparable results use a fixed amount of input RNA for all experiments Tailor both the amount of input RNA and the amplification procedure to produce the amount of aRNA needed for your microarray hybridizations Figure 4 shows aRNA yields from different amounts of Control RNA amplified with increasing IVT incubation times Typically 0 2 5 ug of cyanine dye labeled aRNA is used for hybridization but the exact amount will depend on the array platform used Typically 100 ng of total RNA input is the lower limit fo
40. n of aaUTP in an IVT reaction has only a minor effect on the reaction efficiency and yield Hoen et al 2003 Additionally since the incorporation of aaUTP by IVT will be virtually identical in different samples and since the dye coupling reaction is efficient and reproducible labeled samples will not have the biases that can result from direct incorporation of modified nucleotides by in vitro transcription Simple rapid aRNA purification procedures are used to prepare the aRNA for dye coupling and to remove free dye molecules after the coupling reaction in preparation for array hybridization Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide 3 C Materials Provided with the Kit and Storage Conditions OneArray Amino Allyl aRNA Amplification Kit components The OneArray Amino Allyl aRNA Amplification Kit includes reagents for single round amplification of 20 samples or two round amplification of 10 samples cDNA synthesis and IVT Amount Component Storage 30 uL T7 Oligo dT Primer 20 C 11 uL Reverse Transcriptase 20 C 11 uL RNase Inhibitor 20 C 21 uL 10X First Strand Buffer 20 C 85 uL dNTP Mix 20 C 105 uL 10X Second Strand Buffer 20 C 21 uL DNA Polymerase 20 C 11 uL RNase H 20 C 42 uL T7 Enzyme Mix 20 C 42 uL T7 10X Reaction Buffer 20 C 48 uL UTP Solution 50 mM 20 C 50 uL aaUTP 50 mM 5 3 amino allyl UTP 20 C 128 uL ATP CTP GTP Mix 25 mM each nucleotide 20 C 20 uL Second Round
41. n or air incubator recommended 1 Add 26 uL of IVT Master Mix to each sample and mix Because the amino allyl UTP nucleotide aaUTP does not contain a bulky modification it is possible to replace all of the UTP with aaUTP in the IVT reaction However we recommend using a 1 1 ratio of UTP to aaUTP i e 50 for most samples Using a higher ratio of aaUTP will produce aRNA that can be coupled with more dye molecules which could lead to brighter signal on microarrays but which may cause a decrease in the signal to noise ratio The kit contains 50 mM aaUTP for 20 IVT reactions at the 1 1 ratio recommended Important If two rounds of amplification will be done this first round transcription should be unmodified containing only UTP no aaUTP At room temp prepare an IVT Master Mix by adding the following reagents to a nuclease free microcentrifuge tube in the order listed below Assemble enough for all the samples in the experiment including 5 overage to cover pipetting error Assemble the IVT Master Mix at room temp in the order shown IVT Master Mix for a single 40 uL reaction Amino allyl Unmodified Component 3 uL a aaUTP 50 mM 12 uL 12 uL ATP CTP GTP Mix 25 mM 3 uL 6 uL UTP Solution 50 mM 4 uL 4 uL T7 10X Reaction Buffer 4 uL 4 uL T7 Enzyme Mix b Mix well by gently vortexing Centrifuge briefly 5 sec to collect the IVT Master Mix at the bottom of the tube and place on d Ice Transfer 26 uL of IVT Mas
42. nm We recommend using the NanoDrop 1000A Spectrophotometer for its convenience No dilutions or cuvettes are needed just measure 1 5 uL of the aRNA sample directly Alternatively the aRNA concentration can be determined by diluting an aliquot of the preparation in TE 10 mM Tris HCl pH 8 1 mM EDTA and reading the absorbance in a traditional spectrophotometer at 260 nm Find the concentration in g mL by multiplying the A260 by the dilution factor and the extinction coefficient 1 A260 40 ug RNA mL A260 X dilution factor X 40 g RNA ml 2 Assessing aRNA yield with RiboGreen If a fluorometer or a fluorescence microplate reader is available Molecular Probes RiboGreen fluorescence based assay for RNA quantitation is a convenient and sensitive way to measure RNA concentration Follow the manufacturer s instructions for using RiboGreen Note Cy5 labeled aRNA cannot be accurately quantitated using RiboGreen 3 Expected yield The aRNA yield will depend on the amount and quality of poly A RNA in the input total RNA Since the proportion of poly A RNA in total RNA is affected by influences such as health of the organism and the organ from which it is isolated aRNA yield from equal amounts of total RNA may vary considerably B Analysis of aRNA Size The size distribution of aRNA can be evaluated using an Agilent 2100 bioanalyzer with Caliper s LabChip technology or by conventional denaturing agarose gel analysis The bioanalyzer c
43. o collect the Reverse Transcription Master Mix at the bottom of the tube and place on ice c Transfer 8 uL of Reverse Transcription Master Mix to each sample Mix thoroughly by pipetting up and down 2 3 times then flicking the tube 3 4 times and centrifuge briefly to collect the reaction in the bottom of the tube d Place the tubes in a 42 C incubator Incubate 2 hr at 42 C Incubate reactions for 2 hr at 42 C hybridization oven or air incubator recommended After the incubation centrifuge briefly 5 sec to collect the reaction at the bottom of the tube OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech 5 Add 1 pL RNase H and incubate 30 min at 37 C RNase H specifically degrades the aRNA leaving only the cDNA as template for second strand synthesis This helps assure that the second strand synthesis reaction will be primed exclusively by the T7 Oligo dT Primer a Add 1 uL RNase H to each reaction Mix thoroughly by pipetting up and down 2 3 times then flicking the tube 3 4 times and centrifuge briefly to collect the reaction in the bottom of the tube b Incubate for 30 min at 37 C in a hybridization oven or air incubator After the incubation proceed directly to Second Strand Synthesis below B Synthesis of Second Strand cDNA Second Round Incubator needed e 70C thermal cycler recommended e 16 C thermal cycler recommended 1 Add 5 uL T7 Oligo dT Primer to each sample a Add 5
44. of the amino allyl aRNA following the instructions in section IV A through IV C Nuclease testing Relevant kit components are tested in the following nuclease assays RNase activity Meets or exceeds specification when a sample is incubated with 25 ng labeled RNA and analyzed by PAGE Nonspecific endonuclease activity Meets or exceeds specification when a sample is incubated with 300 ng supercoiled plasmid DNA and analyzed by agarose gel electrophoresis Exonuclease activity Meets or exceeds specification when a sample is incubated with 40 ng labeled Sau3A fragments of pUC19 and analyzed by PAGE Protease testing Meets or exceeds specification when a sample is incubated with 1 ug protease substrate and analyzed by fluorescence Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide 25 Phalanx Biotech Group OneArray Amino Allyl aRNA Amplification Kit User Guide 2008 Phalanx Biotech All rights reserved
45. oler e g room temperature than the bottom of the tube As little as 1 2 uL of condensate in an IVT reaction tube throws off the concentrations of the nucleotides and magnesium which are crucial for good yield 20 OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech If you see condensation spin the tube briefly and mix the reaction gently Move the tube s to an incubator where condensation does not occur or is minimized c Nuclease contaminated tubes tips or equipment Using pipettes tubes or equipment that are contaminated with nucleases can cleave the RNA or DNA being generated at each step in the procedure This will reduce the size of the aRNA products and decrease aRNA yield d Absorbance readings were inaccurate Confirm that your spectrophotometer is accurate by measuring the absorbance of an RNA or DNA sample of known concentration Alternatively assess the aRNA concentration by fractionating on an agarose gel adjacent to an RNA sample whose concentration is known Comparing the ethidium bromide staining of the aRNA and control samples can approximate the concentration of the aRNA C Troubleshooting Low Yield and Small Average aRNA Size Consider the following troubleshooting suggestions if the positive control reaction produced the expected results but amplification of your experimental samples results in less or smaller average lt 500 nt aRNA than expected 1 Impure RNA samples RNA samples with s
46. r Guide for details on how to prepare the Hybridization Solution for use Extinction coefficient at Amax in cm M Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide 17 V Optional Second Round Amplification If one cycle of amplification does not yield the amount of aRNA necessary for your experiments a second round of amplification can be conducted to generate additional aRNA see Figure 3 for an overview of second round amplification In order to conduct two rounds of amplification the first round of amplification must contain only unmodified UTP amino allyl modified aRNA cannot undergo a second round of amplification The procedure is similar to the first round of amplification and the reaction products are equivalent but different primers are used for first and second strand synthesis and the reaction setup is slightly different Second round amplification products are typically shorter than first round amplification products but we have not seen that this has adverse effects on array hybridization results if all samples are prepared using two rounds of amplification A Synthesis of First Strand cDNA Second Round Incubators needed 1 18 e 70 C thermal cycler recommended e 42 C hybridization oven or air incubator recommended e 37 C hybridization oven or air incubator recommended Mix 2 ug aRNA with 2 pL Second Round Primers and adjust the volume to 12 uL a Place up to 2 ug of purifie
47. r synthesizing 10 ug of aRNA using a 4 hr IVT reaction If your total RNA input will be 100 ng or less we advise conducting a preliminary amplification from a representative sample to determine how much aRNA you can expect from your experimental samples If the preliminary experiment does not produce enough aRNA for two array hybridizations repeat the experiment using an IVT incubation as long as 14 hr and or consider using two rounds of amplification Yield ug 50 100 500 1000 Total RNA Input ng Figure 4 aRNA Yield vs IVT Incubation Time and Total RNA Input The indicated amounts of Control RNA HeLa total RNA were amplified for the IVT incubation times shown Although longer IVT incubation times produced more aRNA the minimum amount of aRNA needed for Human or Mouse OneArray hybridization was obtained with a 4 hour incubation time with 500 ng input RNA Note that this is empirical data obtained using the Control RNA provided with the kit aRNA yield from experimental samples may be considerably different 6 OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech RNA purity The quality of the RNA is the single most important factor affecting how efficiently an RNA sample will be amplified using the OneArray Amino Allyl aRNA Amplification Kit RNA samples should be free of contaminating proteins DNA and other cellular material as well as phenol ethanol and salts associated with RNA isolation procedures Impurities
48. room temp cDNA Filter Cartridges should not be subjected to RCFs over 16 000 x g because the force could cause mechanical damage and or may deposit glass filter fiber in the eluate Preheat Nuclease free Water to 50 55 C Before beginning the cDNA purification preheat the 10 mL bottle of Nuclease free Water to 50 55 C for at least 10 min Important Preheat the Nuclease free Water to a maximum of 55 C temperatures above 58 C can partially denature the cDNA compromising final aRNA yield 1 Add 250 uL cDNA Binding Buffer to each sample Important Check the cDNA Binding Buffer for precipitation before using it If a precipitate is visible redissolve it by warming the solution to 37 C for up to 10 min and vortexing vigorously Cool to room temp before use Add 250 uL of cDNA Binding Buffer to each sample and mix thoroughly by pipetting up and down 2 3 times then flicking the tube 3 4 times Follow up with a guick spin to collect the reaction in the bottom of the tube Proceed guickly to the next step 2 Pass the mixture through a cDNA Filter Cartridge Check that the cDNA Filter Cartridge is firmly seated in its wash tube supplied a Pipet the cDNA sample cDNA Binding Buffer from step 1 onto the center of the cDNA Filter Cartridge b Centrifuge for 1 min at 10 000 x g or until the mixture is through the filter 10 OneArray Amino Allyl aRNA Amplification Kit User Guide Phalanx Biotech c Discard the flow through and replac
49. sample Mix thoroughly by pipetting up and down 2 3 times then flicking the tube 3 times and centrifuge briefly to collect the reaction in the bottom of the tube d Place the tubes in a 16 C thermal cycler It is important to cool the thermal cycler block to 16 C before adding the reaction tubes because subjecting the reactions to temperatures gt 16 C will compromise aRNA yield 2 Incubate for 2 hr at 16 C Incubate 2 hr in a 16 C thermal cycler If the lid temperature cannot be adjusted to match the 16 C block temperature cover the reactions with the heated lid turned off or if the lid cannot be turned off do not cover the tubes with it Do not use a water bath or a heat block in a 4 C refrigerator for this incubation because the temperature will fluctuate too much Note You may want to preheat the Nuclease free Water for use in step II E 4 during this incubation 3 Place reactions on ice briefly or freeze immediately After the 2 hr incubation at 16 C place the reactions on ice and proceed to section E cDNA Purification below or immediately freeze reactions at 209C Do not leave the reactions on ice for more than hr Stopping Point This is a potential overnight stopping point at 20 C but it is better to complete the cDNA purification next section before stopping E cDNA Purification Important All centrifugations in this purification procedure should be done at 10 000 x g typically 10 000 rpm at
50. ser Guide 1 Figure 2 Amino Allyl MessageAmp ll aRNA Amplification Procedure Sa total RNA TTTT T7 l Mix RNA and I yl T7 Oli Primer and adjust volume to 12 pl TERETE TTTT T7 2 Incubate 10 min at 70 G then place on ice Sip 3 Add 8 ul of Reverse Transcription Master Mix and place at 42 C H 4 Incubate for 2 hr at 42 C 57 AAA 3 111T T75 cDNA i Saal I Add 80 pl Second Strand Master Mix to each sample s Y amra 2 Incubate for 2 hr at 16 C 3 TTTT T7 5 enzymes i sals aga 20min I Add 250 ul cDNA Binding Buffer to each sample RNAs 2 Pass the mixture through a cDNA Filter Cartridge SXCESS pomer 3 Wash with 500 ul Wash Buffer 5 AAAA T7 3 4 Elute cDNA with 2 x 9 pl 50 55 C Nuclease free Water 3 TTT T dsDNA template 4 l4hr 3 wv 5 1 Add 26 ul of IVT Master Mix to each sample and mix al gm 2 Incubate for 4 14 hr at 37 C jee UUUU 5 3 Add 60 ul Nuclease free Water to each sample e amino allyl aRNA enzymes i 1 Add 350 ul aRNA Binding Buffer to each sample salts Ki 20 min 2 Add 250 ul 100 ethanol and pipet 3 times to mix unused NTPs i 3 Pass samples through an aRNA Filter Cartridge s i 4 Wash with 650 ul Wash Buffer s y 5 Elute aRNA with 100 ul preheated Nuclease free Water 34 5 H ARNA Quantitation and Expected Yield ate UUUU 5 te 2 l Resuspend one CyDye vial in 11 pl of DMSO prepared san 3 Aona 2 Quantitate the aRNA and vacuum dry 5 20 ug NHS ester dye 3 Resuspend dried
51. te 2 hr at 16 C Incubate 2 hr in a 16 C thermal cycler If the lid temperature cannot be adjusted to match the 16 C block temperature cover the reactions with the heated lid turned off or if the lid cannot be turned off do not cover the reactions Do not use a water bath or a heat block in a 4 C refrigerator for this incubation because the temperature will fluctuate too much Stopping Point This is a potential overnight stopping point but it is better to complete the cDNA purification section I E before stopping 5 Continue with the procedure at section II E After the 2 hr incubation at 16 C place the reactions on ice and proceed to section I E cDNA Purification or immediately freeze reactions at 20 C Do not leave the reactions on ice for long periods of time Complete the rest of the second round amplification start at section I E cDNA Purification and continue through the remainder of section II Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide 19 Vi Troubleshooting A Positive Control Reaction Control RNA amplification instructions To establish if the kit is working properly Control RNA consisting of 1 mg mL HeLa cell total RNA is provided Use 1 uL of the Control RNA in a single round OneArray Amino Allyl aRNA Amplification reaction follow the protocol starting at step IL C 1 At step ILF 2 use a 14 hr incubation for the IVT reaction Continue with the procedure for making amino allyl aRNA
52. ter Mix to each sample Mix thoroughly by pipetting up and down 2 3 times then flicking the tube 3 4 times and centrifuge briefly to collect the reaction in the bottom of the tube Once assembled place the tubes at 37 C Phalanx Biotech OneArray Amino Allyl aRNA Amplification Kit User Guide Il 2 Incubate for 4 14 hr at 37 C The minimum recommended incubation time is 4 hr the maximum is 14 hr There are data and a detailed discussion of the length of the IVT incubation in section Z A Input RNA quantity and IVT reaction incubation time It is important to maintain a constant 37 C incubation temperature We recommend incubating in a hybridization oven to minimize condensation inside the tubes 3 Add 60 pL Nuclease free Water to each sample Stop the reaction by adding 60 uL Nuclease free Water to each aRNA sample to bring the final volume to 100 uL Mix thoroughly by gentle vortexing Proceed to the aRNA purification step below or store at 20 C Stopping Point The aRNA can be stored overnight at 20 C at this point if desired G aRNA Purification This purification removes unincorporated aaUTP and Tris from IVT reactions that would otherwise compete with the aRNA for dye coupling it also removes enzymes salts and other unincorporated nucleotides Important All centrifugations in this purification procedure should be done at 10 000 x g typically 10 000 rpm at room temp aRNA Filter Cartridges should not be subjected
53. vailable A aRNA Dye Coupling Reaction 1 Resuspend one CyDye vial in 11 pL of DMSO Prepare dye immediately before starting the dye coupling procedure add 11 uL of DMSO to one tube of Cy3 or Cy5 reactive dye and vortex to mix thoroughly Keep the resuspended dye in the dark at room temperature for up to 1 hr until you are ready to use it Recommendations for solubilizing several other commercially available NHS ester dyes are shown in section VILA Important The preparation and storage of solubilized dye is important for the efficient labeling of amino allyl modified aRNA It is imperative that the dye compounds remain dry both before and after dissolving in DMSO because any water that is introduced will cause hydrolysis of the NHS esters lowering the efficiency of coupling 2 Quantitate the aRNA and vacuum dry 5 20 yg See section III A for instructions on determining the aRNA concentration e Place 5 20 ug of amino allyl modified aRNA in a nuclease free microfuge tube and vacuum dry on medium or low heat until no liguid remains Check the progress of drying every 5 10 min and remove the sample from the concentrator as soon as it is dry do not overdry 3 Resuspend dried aRNA in 9 pL Coupling Buffer To a tube containing the dried amino allyl aRNA 5 20 ug add 9 uL Coupling Buffer and resuspend thoroughly by vortexing gently 4 Add 11 pL prepared dye to the aRNA and mix well Add 11 uL of prepared dye from step 1 to the aRN
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