Rice Microarray
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1. The Hinged 100 Place Storage amp Freezer Polypropylene Box from USA Scientific has been used to complete this step with frequent success The small approximately 1 2 inch x 12 inch chambers within the box are filled about 34 full of buffer then the microarrays are laid on top of the chambers The box is then closed and placed inside the oven For information about this product or other USA Scientific products access their Web site at www usasciesntific com 22 Rice OneArray User Manual v1 Phalanx Biotech Group C Hybridization Using the MAUI hybridization system Step 4Ca Prepare Hybridization Solution Using the OneArray Hybridization Buffer Included Tey For correct use of this buffer you must add a specific amount O of formamide and labeled target Please follow the instructions below carefully 1 Spin down the stock OneArray Hybridization Buffer 410 ulin each tube 2 Add 90 u 1 of deionized formamide 3 Warm the mixture to 42 C to completely dissolve the solution Mix thoroughly Yield 500 u l of 1 5X Hybridization Buffer solution 4 Make up X Hybridization Buffer by adding nuclease free H20 5 Aliquot the solution into individual tubes according to usage and store in darkness at 20 C Step 4Cb Prepare Target for Hybridization lt Hybridization Using Labeled Targets from aRNA Labeling Approaches 1 Mix 2 ug of your aRNA sample with nuclease free H5O to yield a final volume of 9 uL2. NOTE It is essential to use at least 2 ue of labeled target for each hybridization 2 Add 1 ul 10x Fragmentation Reagent and incubate at 70 C for 15 minutes 3 Add 1 ul Stop Solution and mix well Phalanx Biotech Group Rice OneArray User Manual v1 23 4 Mix with nuclease free H2O to yield a final volume of 14 2 uL 5 Keep on ice and in darkness until hybridization Step 4Dc Step 4Cc gt Complete the Hybridization 1 Ensure your work and experimentation area as well as the OneArray are clean before adding the Hybridization Buffer solution to the target array 2 Pre warm the Hybridization Buffer with formamide at 42 C for 10 minutes 3 Prepare the hybridization mix in a 1 5 ml Eppendorf tube according to the Table 5 below Table 5 Hybridization Mix Measurements For each slide 45 pu Component Final Volume 1 5X OneArray Hybridization 30 ul Buffer Sheared Salmon Sperm DNA 10 0 8 ul ng nb Target preparation plus nuclease 14 2 pl free ddH O Alternatives to Salmon Sperm DNA Blocking Mixtures Ambion sheared Salmon Sperm DNA 10 ug ul or Invitrogen Cot 1 DNA 2 5 10 ug ul or Invitrogen Poly A 2 5 10 pg ul 4 Spin down the mixture for 5 minutes to eliminate potential debris 5 Transfer the mixture to a new tube 24 Rice OneArray User Manual v1 Phalanx Biotech Group 6 7 8 9 Heat the mixture to 95 C for 5 minutes thermocycler recommended Main
3. 105 experimental control probes e 3 features per probe 100 of Sanger miRBase v15 Human miRNAs gt H E Mouse amp Rat miRNA OneArray v2 e 785 unique miRNA probes e 105 experimental control probes e 3 features per probe e 100 of Sanger miRBase v15 miRNAs ym 32 Rice OneArray User Manual v1 Phalanx Biotech Group
4. 2 below contains a list of products that have been tested for use with OneArray Table 2 aRNA Preparation Products Manufacturer Product Name and Description Ambion Amino Allyl MessageAmp IITM aRNA Kit Ambion aRNA Fragmentation Reagent Epicentre Biotechnologies TargetAmp 1 Round Aminoallyl aRNA Amplification Kit For aRNA labeling 10 ug of guality aRNA is recommended Smaller volumes can lead to significant loss of sample and may increase the concentration of contaminants in the labeled aRNA sample leading to higher background signal It is best to use aRNA as soon as possible after labeling as exposure to air and light can reduce the signal of some dyes If it must be left overnight it is best to aliguot your labeled aRNA and store in the dark at 80 C Avoid thawing and re freezing aRNA if possible as freeze thaw cycles can damage the aRNA Finally aRNA fragmentation is best performed immediately prior to hybridization Step 4 10 Rice OneArray User Manual v1 Phalanx Biotech Group General Instructions SL OneArray requires a pre hybridization step prior to 8 hybridization of the labeled target The pre hybridization step reduces background signals and increases the performance of the microarray Complete the pre hybridization step by carefully following the instructions below 1 2 3 4 5 6 7 Phalanx Biotech Group Warm the pre hybridization solution 5X SSPE 0 1 SD
5. cun ne Y nd RG NU d dwn neu a Product Contents 22 022220 2 222 1 Other Necessary Apparatus Not Supplied 2 Other Necessary Reagents Not Supplied 3 Important Notes on Microarray Handling and Storage 4 Product Descriptions and Overview 5 Using OneArray ss ss 7 Step 1 Prepare the RNA Sample 8 Step 2 Label the Target 9 Step 3 Pre Hybridize the Microarray 11 Step 4 Complete the Hybridization Protocol 13 Step 5 Wash the Hybridized Microarray 26 Step 6 Scan and Extract Gene Expression Results 28 Step 7 Check the Control Probe Data 31 OneArray Product Family 32 Phalanx Biotech Group Rice OneArray User Manual v1 vii Getting Started Please read the introductory information below to help familiarize yourself with OneArray before use Product Contents gt Rice OneArray DNA Microarray gt OneArray Hybridization Buffer Tube e Each tube contains buffers suff
6. filtered o 3 0 M Sodium chloride o 0 3 M Sodium phosphate pH 7 0 gt 20X SSPE stock solution sterile filtered o 3 6 M Sodium chloride o 0 2 M Sodium phosphate pH 7 7 o 20mM EDTA gt Wash Solutions sterile filtered four types approximately 250 mL of each is required per experiment o 2 X SSC 0 2 SDS o 2X SSC o 0 2 X SSC NOTE SDS must be molecular biology grade gt Pre hybridization Buffer prepared and sterile filtered immediately prior to pre hybridization o 5X SSPE 0 1 SDS 1 BSA NOTE BSA must be molecular biology grade gt Deionized formamide to be added to the OneArray Hybridization Buffer prior to use see Step 4 gt RNA Fragmentation Reagent and Stop Solution for hybridization using aRNA DNA Blocking Mixture Phalanx Biotech Group Rice OneArray User Manual v1 3 o Ambion sheared Salmon Sperm DNA 10 ug ul or Invitrogen Cot 1 DNA 2 5 10 ug ul or Invitrogen Poly A 2 5 10 ug ul Important Notes on Microarray Handling and Storage Storage Conditions gt Store unopened OneArray product at room temperature gt Store opened OneArray product at room temperature gt Store OneArray Hybridization Buffer at room temperature NOTE If the product is received with an open bag please contact Phalanx Biotech Customer Service for an immediate replacement Handling Microarrays mn Please read this section carefully and follow the Oo instructions g
7. labeling kits for microarray analysis Select a labeling kit or labeling method that is most suitable for your specific needs If you use a labeling kit that is not listed in Tables 3 it is recommended that you validate the method to test and determine its compatibility with the OneArray You may want to confirm the guality of the labeled target with the No Cuvettes Spectrophotometer from NanoDrop RNA Sample Amounts Generally the amount needed of quality RNA is 10 ug for each labeling reaction If you have an ample supply of RNA samples you have the choice of using a protocol that either amplifies or does not amplify the RNA sample If you have a limited amount of RNA samples it is recommended that you use a protocol that includes a linear amplification of the RNA samples Dye Incorporation Efficiency Good dye incorporation rates are important for yielding the best data from microarray hybridization Incorporation rates of 20 60 dye molecules per 1 000 bases 20 33 bases dye molecule yield the most usable data Rates below 20 dyes per 1 000 bases 50 bases dye are very low and may lead to a loss of signal of many targets It is not recommended to perform hybridization with samples of low dye incorporation efficiency Phalanx Biotech Group Rice OneArray User Manual v1 9 For aRNA Hybridization Follow the instructions provided by the reagent supplier Indirect labeling with NHS ester dye is recommended Table
8. the OneArray Buffer Table 3 Compatible Glass Cover Slide Products Manufacturer Product Name BioRad Laboratories SLS 6001 24x60 mm Erie Scientific Company mSeries LifterSlip 25x601 M 5439 Corning Cover Glass 24 X 60 mm 1 Ensure your work and experimentation area as well as the OneArray are clean before adding the Hybridization Buffer solution to the target array Phalanx Biotech Group Rice OneArray User Manual v1 19 2 Pre warm the Hybridization Buffer with formamide at 42 C for 10 minutes 3 Prepare the hybridization mix in a 1 5 ml Eppendorf tube according to the Table 4 below Table 4 Hybridization Mix Measurements For each slide 55 pu Component Final Volume 1 5X OneArray9 Hybridization 37 pl Buffer Sheared Salmon Sperm DNA 10 1 pl yg pl Target preparation plus nuclease 17 pl free ddH O Alternatives to Salmon Sperm DNA Blocking Mixtures Ambion sheared Salmon Sperm DNA 10 ug ul or Invitrogen Cot 1 DNA 2 5 10 ug ul or Invitrogen Poly A 2 5 10 ug l 4 Spin down the mixture for 5 minutes to eliminate potential debris 5 Transfer the mixture to a new tube 6 Heat the mixture to 95 C for 5 minutes thermocycler recommended 7 Maintain the mixture at a temperature of 60 C until pipetting onto the array thermocycler recommended It may be helpful to set a Denature program in the thermocycler as follows 95 C 5 minutes 60 C Hold
9. 20 Rice OneArray User Manual v1 Phalanx Biotech Group 8 Place the OneArray slide bar code up atop the Probe Printed Region Guide included see Figure 1 5 50mm 1 96 9 5mm Probe Spotted Region EZ6OLEZLYO 66 0 WW GZ U Y V V lt vV O o F m 4 O m m 75mm 2 97 Figure 1 OneArray Microarray Glass Slide with Probe Printed Region Guide Plastic Underlay 9 Pipette the hybridization mixture onto the spotted region of OneArray DNA Microarray Avoid creating any bubbles 10 Carefully place the glass cover slide over the spotted area in an even manner 11 Place the entire labeled target plus the microarray set up into a closable chambered box that is humidified by 2X SSPE buffer in the 50 C oven for 14 to 16 hours A sealed chamber ensures that the appropriate humidity level is maintained during incubation See Figure 2 Phalanx Biotech Group Rice OneArray User Manual v1 21 Figure 2 below provides an illustration of Step 4Bc where the hybridization protocol is completed using the glass cover slide method and specifically the OneArray DNA Microarray is placed into the chambered box Place the hybridized microarray slide on top of the filled chambers inside the box and close the box Figure 2 Step 4Bc gt aRNA Hybridization Glass Slide Inside Chamber Box
10. Make up X Hybridization Buffer by adding nuclease free H20 5 Aliquot the solution into individual tubes according to usage and store in darkness at 20 C Step 4Bb gt Prepare Target for Hybridization lt Hybridization Using Labeled Targets from aRNA Labeling Approaches 1 Mix 2 ug of your aRNA sample with nuclease free H5O to yield a final volume of 9 uL NOTE It is essential to use at least 2 ue of labeled target for each hybridization If you are performing a dual dye experiment use at least 2 ug of each labeled aRNA sample 18 Rice OneArray User Manual v1 Phalanx Biotech Group 2 Add 1 ul 10x Fragmentation Reagent and incubate at 70 C for 15 minutes 3 Add 1 ul Stop Solution and mix well 4 Mix with nuclease free H O to yield a final volume of 17 uL 5 Keep on ice and in darkness until hybridization Step 4Bc Step 4Bc 2Complete the Hybridization NOTE If you perform hybridization using methods other than the basic glass cover slide method it is recommended that you validate the protocol experimentally For example the phalanx hybridization system the MAUI System from BioMicro Systems or HS Series of Hybridization Stations from TECAN offer a higher throughput and more automated hybridization methods To complete this step you will need to select a type of glass cover slide Table 3 below contains a list of glass cover slides that have been tested and confirmed compatible for use with
11. Notice to the User IMPORTANT It is important that users read the entire manual before O commencing work Phalanx Biotech Group Rice OneArray User Manual v1 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 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 Rice 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 performi
12. Protocol Step 5 Wash the Hybridized Microarray Step 6 Scan and Extract Gene Expression Results gt Step 7 Check Control Probe Data Phalanx Biotech Group Rice OneArray User Manual v1 7 Prepare the RNA Sample Lee Nil High quality intact RNA is essential for all gene expression microarray experiments There are many different RNA isolation protocols and commercially available RNA isolation kits You should choose a solution that meets your specific needs Qiagen Ambion Invitrogen and other reagent companies offer many different RNA isolation products For more information you can visit each company s website Once the RNA samples are isolated you must confirm the quantity and quality of the samples Similarly many different protocols are available and you should choose a solution that is suitable for your needs For faster and more automated RNA analysis you may want to consider the No Cuvettes Spectrophotometer from NanoDrop or the 2100 Bioanalyzer from Agilent Technologies For more information visit each company s website 8 Rice OneArray User Manual v1 Phalanx Biotech Group ESE Label the Target mu For best results it is recommended that you use one of the O commercially available labeling kits that has been tested for use with the OneArray microarray please refer to Tables 3 and 4 below General Guidelines for Target Labeling There are many commercially available
13. S and 1 BSA to 42 C Pour 25 ml room temperature 100 ethanol into the Spare array tube Preheat the OneArray s in the round cap tube at 60 C for 10 min hybridization oven recommended Remove the OneArray s from the round cap tube place in the two outermost slots inside the tube containing 100 ethanol close the cap and let sit for approximately 15 sec Shake the round cap tube for 20 sec Remove and thoroughly rinse each array with deionized water to remove any residual ethanol Carefully and slowly fully submerge the OneArray in an abundant amount of pre hybridization solution for 1 hr at 42 C 35 ml is sufficient if using a round cap tube Rice OneArray User Manual v1 11 Avoid inserting and removing the slides more than once in the Try to insert the slides into the correct position the first time K pre hybridization buffer Replace the slides in the same location in which they were removed 1 Remove the glass slide s then pour in the pre hybridization buffer 2 Replace the slide s in the plastic tube in the same place from which itwas removed Try to get them in the correct location the first time 8 After 1 hr transfer the slide s to room temperature distilled water and wash gently for 2 min 9 Spin dry the slide s for 2 min Store in a dry dark place until hybridization It is recommended that you use the slides in the hybridization protocol within 1 hr of co
14. icient for 50 microarray hybridization procedures gt Spare round cap tube gt OneArray User Guide gt Spotted Region Guide Product Support CD which contains the following e Sample Images e OneArray gal file e OneArray gene list and probe sequences e OneArray microarray layout e OneArray Control Probe list e OneArray User Guide electronic version Phalanx Biotech Group Rice OneArray User Manual v1 1 Other Necessary Apparatus Not Supplied Apparatus e Water bath heating block e Powder free gloves e Clean blunt forceps e Micropipettors e Sterilized and nuclease free pipet tips e Sterilized and nuclease free microcentrifuge tubes e High speed microcentrifuge e Low speed tabletop microcentrifuge with slide holder attachment e Vortex mixer e Hybridization oven e Hybridization accessories chamber cover slides etc e Rectangular slide staining dish and slide rack for washing microarrays e PCR polymerase chain reaction machine e Microarray scanner for standard 1 x 3 format see Table 6 under OneArray Microarray Scanner Specifications for a list of compatible scanners e Hybridization systems optional e Automated hybridization station optional 2 Rice OneArray User Manual v1 Phalanx Biotech Group Other Necessary Reagents Not Supplied Reagents De ionized nuclease free water Cyanine 3 or 5 labeled amplified aRNA sample gt 20X SSC stock solution sterile
15. mpleting the pre hybridization process 12 Rice OneArray User Manual v1 Phalanx Biotech Group Once you have completed the pre hybridization step using one of the methods outlined in the Step 3 Pre Hybridize the Microarray section you are ready to complete the hybridization protocol There are many different hybridization protocols apparatus and instruments available that may be compatible for use with the OneArray microarray Detailed instructions for using Phalanx hybridization system OneArray Full Length Chamber and the glass cover slide method are described below For best performance and consistent hybridization results it is recommended that you use the OneArray Hybridization Buffer included with this product to complete the hybridization process Phalanx Biotech Group Rice OneArray User Manual v1 13 A Using OneArray Full Length Chamber Step 4Aa Prepare Hybridization Solution Using the OneArray Hybridization Buffer Included me For correct use of this buffer you must add a specific amount O of formamide and labeled target Please follow the instructions below carefully 1 Spin down the stock OneArray Hybridization Buffer 410 ulin each tube 2 Add 90 u1 of deionized formamide 3 Warm the mixture to 42 C to completely dissolve the solution Mix thoroughly Yield 500 1 of 1 5X Hybridization Buffer solution 4 Make up X Hybridization Buffer by adding nuclease free H20 5 Aliquot
16. ng 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 Rice OneArray User Manual v1 Phalanx Biotech Group Contact Information lt gt Asia Office Phalanx Biotech Group 6 Technology Road 5 6 Floor Hsinchu Science Park Hsinchu 30078 Taiwan Ph 886 3 5781168 Toll Free 0800 777 988 FAX 886 3 5785099 E mail twsales Gphalanxbiotech com website www onearray com lt gt U S Office Phalanx Biotech Group 1301 Shoreway Road Suite 160 Belmont CA USA 94002 Ph 1 650 320 8669 Toll Free 877 730 3887 FAX 1 650 508 9889 E mail info phalanxbiotech com website www onearray com Phalanx Biotech Group Rice OneArray User Manual v1 iii lt User Guide and Technical Support Electronic version of this manual is available on the enclosed Product Support CD and online at www onearray com To reach technical support by telephone call Within the US 1 650 320 8669 Outside the US 886 3 5781168 lt gt Feedback We welcome your feedback regarding our products and this manual Please contact us at twsales phalanxbiotech com All comments are welcome iv Rice OneArray User Manual v1 Phalanx Biotech Group Trademarks and Copyrights Rice OneArray and OneArray are trademarks of Phalanx Biotech Group Inc in the United Sta
17. nsure a secure adhesion Figure 4 Rice OneArray User Manual v1 Phalanx Biotech Group Phalanx Biotech Group Vil viii ix Keep the chamber microarray assembly at 50 C for 14 16 hrs Rotation of the assembly during hybridization has been shown to increase the signal intensity Prepare the first wash solution of 2X SSC 0 2 SDS and warm to 42 C More details can be found in the OneArray Microarray User Guide Remove the chamber microarray assembly from the hybridization oven and completely submerge it under the wash solution Use forceps to slowly lift and remove the chamber starting from the tab end Use the holes in the tab for a better grip Be sure to keep the microarray under the wash solution during removal Figure 5 Wash the array in the solution and proceed to follow the remaining steps according to the step 5 Rice OneArray User Manual v1 17 B Hybridization Using the Glass Cover Slide Method Step 4Ba Prepare Hybridization Solution Using the OneArray Hybridization Buffer Included me For correct use of this buffer you must add a specific amount O of formamide and labeled target Please follow the instructions below carefully 1 Spin down the stock OneArray Hybridization Buffer 410 ulin each tube 2 Add 90 u 1 of deionized formamide 3 Warm the mixture to 42 C to completely dissolve the solution Mix thoroughly Yield 500 u l of 1 5X Hybridization Buffer solution 4
18. nt Technology DNA Microarray Scanner G2565B Rice OneArray User Manual v1 Phalanx Biotech Group Check the Control Probe Data OneArray DNA Microarrays contains built in control probes for performance monitoring of the hybridization process They are used to confirm or deny whether the experiment was completed successfully Please visit http www phalanx com tw Support RiOA_CP php 1 for more detailed information about the experimental controls on your OneArray product Additional information about the control probes is included on the Product Support CD and on our website at Www onealiray cOom Phalanx Biotech Group Rice OneArray User Manual v1 31 OneArray Product Family E Human OneArray v5 e 29 187 human genome probes e 1 088 experimental control probes e Composition RefSeq release 38 and Ensembl release 56 E Mouse OneArray v2 e 26 423 mouse genome probes e 872 experimental control probes emay e Composition RefSeq release 42 and Ensembl release 59 E Rat OneArray vl N e 24 358 rat genome probes y e 980 experimental control probes N e Composition RefSeq release 42 and Ensembl release 59 E Yeast OneArray v1 e 6 958 yeast genome probes e 684 experimental control probes e Composition AROS v1 1 and YBOX v1 0 E Rice OneArray v1 e 22 003 rice genome probes e 824 experimental control probes e Composition RGAP v6 1 and BGI 2008 E Human miRNA OneArray v2 e 1 087 unique miRNA probes e
19. rack with the excess amount of pre warmed 2X SSC 0 2 SDS solution for 5 min at 42 C Transfer the slide rack to a second slide staining dish that contains 2X SSC wash for 5 min at 42 C 26 Rice OneArray User Manual v1 Phalanx Biotech Group Phalanx Biotech Group 5 6 7 8 Transfer the slide rack to a third slide staining dish that contains 2X SSC and wash for 5 min at room temperature Rinse each array carefully with 0 2X SSC using a squeeze bottle Spin dry with a centrifuge for at least one minute Keep the microarray dry and in the dark until ready to scan Rice OneArray User Manual v1 27 Scan and Extract Gene Expression Results There are many scanners available to extract signals from OneArray Data extraction using GenePix 4100 from Molecular Devices is described below Please refer to the respective company product instructions for appropriate use Table 6 below lists the setting for using the GenePix 4100 For a list of scanners that are compatible with the OneArray please refer to Table 6 below NOTE The performance of each scanner may differ Therefore to ensure best results it is recommended that the scanner be adjusted based on standard microarray calibration parameters Turn on and warm up the scanner for the duration according to manufacture instructions for the scanner Use the gal file and Gene List provided with this product or refer to our website at WWwW
20. release in 2008 Table 1 below provides an example of the contents of a Rice genome that can be studied using the Rice OneArray v1 Table 1 Rice OneArray v1 probe Content Probe Type Number of Probes RiOA 1 0 probes 22 003 total New Probes Design based on 21 179 RGAP v6 1 BGI 2008 Control Probes 824 Rice OneArray v1 is guaranteed to contain gt 98 of the total probe content Phalanx Biotech Group Rice OneArray User Manual v1 5 Rice OneArray v1 Control Features There are 824 control probes built into the Rice OneArray DNA microarray that monitor the sample quality and hybridization process These control probes provide valuable information to ensure experiments are done correctly to ensure higher quality results for analysis NOTE Detailed control information gene lists gene annotations and probe sequences can be found on the Product Support CD that accompanied this product or at http www onearray com Rice OneArray User Manual v1 Phalanx Biotech Group Using OneArray This section provides you with detailed information about how to perform the steps necessary to complete the hybridization process to study gene expressions using the OneArray microarray Follow these detailed steps exactly to achieve the best experimentation results Step 1 Prepare the RNA Sample Step 2 Label the Target Step 3 Pre Hybridize the Microarray Step 4 Perform the Hybridization
21. se distilled water to make up the volume Phalanx Biotech Group Rice OneArray User Manual v1 15 16 4 Denature the Target Hybridization Mix from the previous step in a PCR machine at 95 C for 5 minutes and hold at 60 C 5 Assembling Process ii iii iV yi Remove the clear liner on the back of the hybridization chamber Align the tab end of the chamber to the edge of the microarray opposite to the barcode It is easier to hold the long edges of the chamber in one hand and press down the tab with the other hand Figure 1 Use the applicator stick provided to press along the adhesive areas to ensure a secure seal Visually inspect the seal from underneath the microarray inconsistent patterns in the black adhesive may indicate an insecure seal Re use the applicator stick if needed Figure 2 Allow the adhesive to set for 30 minutes Pipette 200 uL of the labeled RNA solution through one port of the chamber while allowing air to escape through the other port Make sure there are no bubbles in the pipette tip If air bubbles form within the chamber light pressure may be applied to the surface to dislodge them Figure 3 Wipe excess solution from the ports Be careful not to draw solution from the chamber Cover ports with supplied circular seals Seals should be removed from the liner and applied using forceps The seals will adhere to most wet surfaces Apply pressure to both seals simultaneously to e
22. t Polynucleotide probes are printed on the side of the slide with the barcode To avoid irreparable damage of the printing area do not touch the surface with bare hands or with any other objects Whenever possible handle microarrays with clean blunt forceps to avoid contamination IMPORTANT Open arrays should be used within a week 4 Rice OneArray User Manual v1 Phalanx Biotech Group Product Descriptions and Overview OneArray Whole Genome DNA microarrays are made of sense strand polynucleotide probes spotted onto a proprietary chemical layer coated on top of a 1 x 3 25 mm x 75 mm standard format microarray glass slide Updated information of genome content from public domains is used to design approximately 22 000 highly sensitive long oligonucleotide probes for monitoring the expression level of corresponding protein coding genes Each probe is spotted onto the array in a highly consistent manner using a proprietary non contact spotting technology adapted for microarray manufacturing Rice OneArray v1 Genome Content Each microarray contains 22 003 oligonucleotides including 21 179 rice genome probes and 824 experimental control probes Based on One Gene One Probe design concept probe is designed to hybridize to a specific target gene described in the current public domain contents such as RGAP Rice Genome Annotation Project database v6 1 and BGI Beijing Genomics Institute database
23. tain the mixture at a temperature of 60 C until pipetting onto the array thermocycler recommended Load the MAUI SC Mixer as described in the MAUI Hybridization System User s Guide CDP 14 After sealing the fill and vent ports with port seals place the MAUI Mixer Slide Assembly on a pre warmed 50 C MAUI Hybridization System Close the MAUI Hybridization System lid and hybridize for 4 12 hours 4 Bay Mix Mode B 12 Bay Mix Mode A 10 Following hybridization disassemble the MAUI Phalanx Biotech Group Mixer Slide Assembly under pre warmed 42 C Wash Buffer 1 Rice OneArray User Manual v1 25 Washed and dried microarrays should be scanned within a O couple of hours 1 2 3 4 NOTE Do not allow the microarray s to be exposed to air for a significant amount of time otherwise an increased fluorescent background signal could appear Submerge the entire labeled target and microarray set up with the cover slide still intact into a large container filled with 42 C 2X SSC 0 2 SDS solution Carefully remove the cover slide from the glass by gently shaking the glass slide so that the cover slide is freed while the slide is submerged NOTE At this stage the microarray has the highest concentration of unhybridized target and dye Transfer the array quickly to the slide rack to minimize exposure to air Wash the slide s in the rectangular slide staining dish and slide
24. tes 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 CyDye and Cy are trademarks of GE Healthcare AlphaScan is a trademark of Alpha Innotech Inc ArrayWoRx Biochip Reader is a registered trademark of Applied Precision Inc GenePix is a trademark of Molecular Devices GeneTAC is a trademark of Genomic Solutions Inc ScanArray 5000 is a trademark of Perkin Elmer Inc mSerries LifterSlip is a trademark of Erie Scientific Company Amino Allyl MessageAmp II aRNA is a trademark of Ambion ArrayControl is a trademark of Ambion Inc SpotReport is a registered trademark of Strategene Inc BioMicro and Maui are registered trademarks of BioMicro Systems Inc ULS is a trademark of Kreatech Diagnostics NanoDrop is a trademark of Thermo Fisher Scientific Last updated Jan 2011 2005 2011 Phalanx Biotech Group All rights reserved Phalanx Biotech Group Rice OneArray User Manual v1 Thank You Phalanx Biotech Group would like to extend special thanks to our customers who have provided feedback that enabled us to improve the OneArray User Guide vi Rice OneArray User Manual v1 Phalanx Biotech Group Rice OneArray User Guide Notice to the User ccssccssscessccssccssccsseenseenssenssenssecesesnssessensees Ban YOU cun Gn Cd He ne Caras VI Gettlng Stared
25. the solution into individual tubes according to usage and store in darkness at 20 C Step 4Ab Prepare Target for Hybridization lt Hybridization Using Labeled Targets from aRNA Labeling Approaches 1 Mix 10 ug of your aRNA sample with nuclease free H5O to yield a final volume of 27 uL NOTE It is essential to use at least 10 ug of labeled target for each hybridization If you are performing a dual dye experiment use at least 10 ug of each labeled aRNA sample 14 Rice OneArray User Manual v1 Phalanx Biotech Group 2 Add 3 ul 10x Fragmentation Reagent and incubate at 70 C for 15 minutes 3 Add 3 ul Stop Solution and mix well 4 Mix with nuclease free H O to yield a final volume of 63 uL 5 Keep on ice and in darkness until hybridization Step 4Bc Step 4Ac 2 Complete the Hybridization 1 Thaw and re suspend the 1 5X and 1X Working Hybridization buffer at 42 65 C for 10 minutes 2 Preheat water bath to 95 C The water batch should be sufficient to submerge a microarray slide vertically i e large beaker 3 Prepare Target Hybridization Mix Final Total Volume of Target Hybridization Mix Labeled target mix Sheared Salmon Sperm DNA 10 ug ul 1 5X Working Hybridization Buffer Add RNAase free ddH O to reach the final volume NOTE Different volumes of labeled target mix may be obtained due to different labeling protocols If the final volume of the labeling target mix is less u
26. w onearray com Table 6 Scanner Settings Using GenePix 4100 from Molecular Devices Wavelength 635 nm 532 nm PMT 630 V 590 V Minimum diameter 50 Maximum diameter 200 CPI Threshold 0 NOTE For lower versions of GenePix software adjust the property parameter to 142 8 um manually to obtain best results 28 Rice OneArray User Manual v1 Phalanx Biotech Group Figure 3 below provides a visual example of the OneArray glass slide with spotted probe region 142 8um cn k 142 8um Figure 3 OneArray Glass Slide with Spotted Probe Region Phalanx Biotech Group Rice OneArray User Manual v1 29 OneArray Microarray Scanner Specifications 30 Select and use a microarray scanner that meets the specifications below Microarray Scanner Specifications Format capabilities 1 x 3 one inch by three inch glass slide Molecular capabilities Able to accurately detect activate and read Cy3 and Cy5 fluorescent molecules Table 7 below contains a partial list of microarray scanner products that are compatible for use with the OneArray microarray Please refer to the respective company website for more information about the products listed below Table 7 Compatible Microarray Scanners Manufacturer Product Name and Description Molecular Devices Axon GenePix 4000 4100 and 4200 series Genomic Solutions Inc GeneTAC 2000 Perkin Elmer Inc ScanArray 5000 TECAN LS 200 300 400 Agile
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