Getting Started
Contents
1. ad Mouse OneAnay V2 1 iat User Guide www OneArmay com Notice to the User IMPORTANT It is important that users read the entire manual before commencing work Phalanx Biotech Group Mouse OneArray User Guide v2 1a 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 Mouse OneArray product and any other OneArray products Phalanx Biotech 1s founded on the mission to offer researchers high guality and user friendly solutions at an affordable price Your satisfaction in using our p2. Product Family ccccccccssssssssssssssssssscssssssssssssseeeees 3 Phalanx Biotech Group Mouse OneArray User Guide v2 1a vii Getting Started Please read the introductory information below to help familiarize yourself with Mouse OneArray before use Product Contents gt Mouse OneArray DNA Microarray gt OneArray Hybridization Buffer Tube e Each tube contains buffers sufficient for 50 microarray hybridization procedures Spare round cap tube gt Mouse OneArray User Guide gt Spotted Region Guide gt Product Support CD which contains the following e Sample Images e Mouse OneArray gal file e Mouse OneArray gene list and probe sequences e Mouse OneArray microarray layout e Mouse OneArray Control Probe list e Mouse OneArray User Guide electronic version Phalanx Biotech Group Mouse OneArray User Guide v2 1a 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 fo
3. 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 1hr of completing the pre hybridization process Mouse OneArray User Manual v2 1a Phalanx Biotech Group gt Complete the Hybridization Protocol Once you have completed the pre hybridization step using the method 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 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 A Using the Phalanx hybridization system Step 4Aa gt Prepare Hybridization Solution Using the OneArray Hybridization Buffer Included IMPORTANT 6 1 2 3 4 5 Phalanx Biotech Group For correct use of this buffer you must add a specific amount of formamide and labeled target Please follow the instruc
4. DD DD DD Label the Target ms For best results it is recommended that you use one of the commercially available labeling kits that has been tested for use with the Mouse OneArray microarray please refer to Tables 2 below General Guidelines for Target Labeling There are many commercially available 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 2 it is recommended that you validate the method to test and determine its compatibility with the Mouse OneArray You may want to confirm the quality of the labeled target with the No Cuvettes Spectrophotometer from NanoDrop RNA Sample Amounts Generally the amount needed of guality 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 1000 bases 17 33 bases dye molecule yield the most usable data Rates below 20 dyes per 1000 bases 50 bases dye are very low and may lead to a
5. Guide v2 1a 3 Important Notes on Microarray Handling and Storage Storage Conditions gt OneArray chip can be stored at room temperature for several weeks For Long term storage please keep 1t at 2 8 C 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 mel Please read this section carefully and follow the instructions gt Polynucleotide probes are printed on the side of the slide with the barcode gt 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 Opened arrays should be used within a week 4 Mouse OneArray User Manual v2 1a Phalanx Biotech Group EE EE EN ES EU EU O EN ED EN EN ED NE EN ED ED ED EU ED ED EI EN ED ED EU EN EN EN EN EN ED EU EU EN EN ED EN ED ED EE DA ED ED ED ED ED EN DA ED ED ED ED EG ED EN ED ED ED EU EN ED EN EN ED ED EE EN DA ED ED ED EE EA EN ED EN ED ED EN EN EN ED EN ED EU EN EE EN EN ED ED EN EN EN ED ED ED ED ED EN DA ED ED ED ED ED EN DA ED ED ED EU EE EN EN ED ED EU EE EN EN ED ED EU EN ED EN DA ED ED EU EN DA EN EN ED ED EU EE EN ED ED ED DA EG EN EN ED ED ED ED EG ED EN ED ED ED ED ED EN DA ED ED ED EE EN DA ED ED ED EU EG ED DA EN ED ED E
6. are welcome V Mouse OneArray User Guide v2 1a Phalanx Biotech Group Trademarks and Copyrights amp Mouse 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 CyDye and Cy are trademarks of GE Healthcare AlphaScan is a trademark of Alpha Innotech Inc Array WoRx Biochip Reader is a registered trademark of Applied Precision Inc GenePix is a trademark of Molecular Devices GeneTAC M is a trademark of Genomic Solutions _ Inc ScanArray 5000 is a trademark of Perkin Elmer Inc msSerries 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 Nov 2012 2005 2012Phalanx Biotech Group All rights reserved Phalanx Biotech Group Mouse OneArray User Guide v2 1a V Thank You Phalanx Biotech Group would like to extend special thanks to our customers who have provided feedback that enabled us to improve the Mouse OneArray User Guide Vi Mo
7. loss of signal of many targets It is not recommended to perform hybridization with samples of low dye incorporation efficiency Phalanx Biotech Group Mouse OneArray User Guide v2 1a 9 For aRNA Hybridization Follow the instructions provided by the reagent supplier Indirect labeling with NHS ester dye is recommended Table 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 II aRNA Kit Ambion aRNA Fragmentation Reagent Epicentre Biotechnologies TargetAmp 1 Round Aminoallyl aRNA Amplification Kit For aRNA labeling 10 ug of quality 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 aliquot 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 Mouse OneArray User Manual v2 1a Phalanx Biotech Group General Instructions POZZ Mouse OneArray requires a pre hybridization step prior to O hybr
8. of the assembly during hybridization has been shown to increase the signal intensity Vill Prepare the first wash solution of 2x SSC 0 2 SDS and warm to 42 C More details can be found in the OneArrayTM Microarray User Guide 1X 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 X Wash the array in the solution and proceed to follow the remaining steps according to the step 5 22 Mouse OneArray User Manual v2 1a Phalanx Biotech Group C Hybridization Using the Glass Cover Slide Method Step 4Ca gt Prepare Hybridization Solution Using the OneArray Hybridization Buffer Included For correct use of this buffer you must add a specific amount of formamide and labeled target Please follow the instructions below carefully IMPORTANT 1 Spin down the stock OneArray Hybridization Buffer 5 5 ml in each tube 2 Add 4 5 ml of deionized formamide 3 Warm the mixture to 42 C to completely dissolve the solution Mix thoroughly Yield 10 ml of 1 5x Hybridization Buffer solution 4 Make up 1x Hybridization Buffer by adding nuclease free H O 5 Aliquot the solution into individual tubes according to usage and store in darknes
9. 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 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 24 Mouse OneArray User Manual v2 1a Phalanx Biotech Group Table 4 Hybridization Mix Measurements For each slide 55 uL Component Final Volume 1 5x OneArray Hybridization Buffer 37 uL Sheared Salmon Sperm DNA 10 ug uL 1 pL Target preparation plus nuclease freeddH O 17 uL Alternatives to Salmon Sperm DNA Blocking Mixtures Ambion sheared Salmon Sperm DNA 10 ug uL or Invitrogen Cot 1 DNA 2 5 ug uL or Invitrogen Poly A 2 5 ug uL 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 8 Place the OneArray slide bar code up atop the Probe Printed Region Guide included see Figure 1 It may be helpful to set a Denature program in the thermocycler as follows 95 C 5 minutes 60 C Hold Phalanx Biotech Group Mouse OneArray User Guid
10. U EN EN EN EN EN ED EU EG EN EA EN EN ED EU EN DA ED ED ED ED EU ED DA ED ED ED ED DE ED ED ED ED ED ED ED ED ED ED ED EU ED ED ED EN ED ED ED EU ED ED ED ED Mouse 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 25mm x 75 mm standard format microarray glass slide Updated information of genome content from public domains is used to design approximately 27 000 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 Mouse OneArray v2 1 Genome Content Each microarray contains 27 307 oligonucleotides 26 423 mouse genome probes and 884 experimental control probes Mouse oligonucleotide probe is designed to hybridize to a specific target gene described in the current public domain contents such as NCBI Reference Sequence RefSeq Database and Ensembl database Table 1 below provides an example of the contents of a mouse genome that can be studied using the Mouse OneArray v2 1 Table 1 Mouse OneArray v2 1 probe Content Probe Type Number of Probes MOA 2 1 probes 27 307 total New Probes Design based on 26 423 RefSeg release 42 Ensembl release 59 Control Probes 884 Mouse OneArray is guar
11. 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 28 Mouse OneArray User Manual v2 1a Phalanx Biotech Group EN EN EN EU EN EN EN EN DG EN EN EN EN EN EN EM EN EN EN EN EN EM EN EN EU EU E2 EN EM EN EN EU EN EN EN EN EN EN EU EN EN EN EN EN EN EU EU EN EN EM D EN EN EU EN EN EN EN EN EU EN EN EN EN EN EN EA EN EN EN EN EN EN EN EN EN EN EN EN EN EN EI EN EN EM EN EN EU EN EN EN EN EN EN EU EU EN EN EN EN EN EN EN EN EN EM EN EN EN EN EU EN EN EN EN EN EN EN EN EN EN EN EN EN EN EN EN EN EN ED EN EU EN EM ED EN ED EN EN EN DA ED ED EN EU EN DA DA EN ED EN EU EN EN EN EN EN EU EN EE EN EE EN EN EN EN EN EN EN EU EN EN EN EN EN EN EU EU EN EM EN EN EN ED EN EE EN EN EN ED EU EN EN EE EN ED EU EN EN EN EN EN DN DU ED ED DD 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 5 below lists the setting for using the GenePix 4100 For a list of scanners that are compatible with the OneArray please refer to Table 8 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 p
12. anteed to contain gt 98 of the total probe content Phalanx Biotech Group Mouse OneArray User Guide v2 1a 5 Mouse OneArray v2 1 Control Features There are 884 control probes built into the Mouse 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 guality results for analysis NOTE Detailed control information gene lists gene annotations and probe seguences can be found on the Product Support CD that accompanied this product or at http www onearray com Mouse OneArray User Manual v2 1a Phalanx Biotech Group Using Mouse 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 Mouse OneArray microarray IMPORTANT Follow these detailed steps exactly to achieve the best experimentation results gt Step 1 Prepare the RNA Sample gt Step 2 gt Step 3 gt Step 4 gt Step 5 gt Step 6 gt Step 7 Phalanx Biotech Group Label the Target Pre Hybridize the Microarray Perform the Hybridization Protocol Wash the Hybridized Microarray Scan and Extract Gene Expression Results Check Control Probe Data Mouse OneArray User Guide v2 1a T Prepare the RNA Sample WANE High quality intact RNA is essential for all gene 6 expres
13. arameters 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 WWW oncarray CoOom Table 5 Scanner Settings Using GenePix 4100 from Molecular Devices Wavelength 635 nm 532 nm PMT 630 V 590 V Minimum diameter 90 80 Maximum diameter 90 150 CPI Threshold 100 NOTE For lower versions of GenePix software adjust the property parameter to 142 8 um manually to obtain best results Phalanx Biotech Group Mouse OneArray User Guide v2 1a 29 Figure 3 below provides a visual example of the OneArray glass slide with spotted probe region e 142 8um Figure 3 OneArray Glass Slide with Spotted Probe Region 30 Mouse OneArray User Manual v2 1a Phalanx Biotech Group OneArray Microarray Scanner Specifications 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 6 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 6 Compatible Microarray Scanners Manufacturer Produ
14. ct 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 Agilent Technology DNA Microarray Scanner G2565B Phalanx Biotech Group Mouse OneArray User Guide v2 1a 31 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 tech_support support_tab html 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 WWWw oncarTay com 32 Mouse OneArray User Manual v2 1a Phalanx Biotech Group OneArray Product Family Human OneArray v6 32 679 human genome probes 938 experimental control probes Composition RefSeq release 51 and Ensembl release 65 Mouse OneArray v2 1 26 423 mouse genome probes i 884 experimental control probes ot a Composition RefSeq release 42 and Ensembl release 59 Rat OneArray vi N 24 358 rat genome probes y ka 980 experimental control probes SI Composition RefSeq release 42 and Ensembl release 59 Rice OneArray vi 22 003 rice genome
15. e v2 1a 25 50mm 1 96 9 5mm Probe Spotted Region cc60l clP0 66 0 WWE s iz Uu _ D Y BW O E A y o 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 Mouse OneArray User Manual v2 1a Phalanx Biotech Group 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 2 aRNA Hybridization Glass Slide Inside Chamber Box 2 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 2 inch x inch chambers within the box are filled ab
16. idization 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 SDS and 1 BSA to 42 C Pour 25 ml room temperature 100 ethanol into the spare array tube Preheat the Mouse OneArray s in the round cap tube at 60 C for 10 min hybridization oven recommended Remove the Mouse 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 Mouse OneArray in a sufficient amount of pre hybridization solution for 1 hr at 42 C 35 ml is sufficient if using a round cap tube Mouse OneArray User Guide v2 1a 11 IMPORTANT 12 Try to insert the slides into the correct position the first time Avoid inserting and removing the slides more than once in the 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
17. ix from the previous step in a PCR machine at 95 C for 5 minutes and hold at 60 C Assembling Process ll lll 1V VI 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 1f 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 ensure a secure adhesion Figure 4 Mouse OneArray User Guide v2 1a 21 VII Keep the chamber microarray assembly at 50 C for 14 16 hrs Rotation
18. osed hybridization system If the bubble does not appear or appears to be much smaller please try to tap on the slide slightly to disturb the mixture into forming a bubble Also make certain that all the air bubbles in the mixture eventually mix into one big bubble X Place the hybridization assembly into a 50 C OneArray Cover Slide oven and set it on a rotator Remember to rotate Phalanx the assembly at 2 rotations per minute RPM for overnight Note Please check to see that the bubble rotates and move TII freely in the solution to aid in the mixing effect during hybridization Mouse OneArray User Manual v2 1a Phalanx Biotech Group B Using OneArray Full Length Chamber Step 4Ba gt Prepare Hybridization Solution Using the OneArray Hybridization Buffer Included For correct use of this buffer you must add a specific amount of formamide and labeled target Please follow the instructions below carefully IMPORTANT 1 Spin down the stock OneArray Hybridization Buffer 5 5 ml in each tube 2 Add 4 5 ml of deionized formamide 3 Warm the mixture to 42 C to completely dissolve the solution Mix thoroughly Yield 10 ml of 1 5x Hybridization Buffer solution 4 Make up 1x Hybridization Buffer by adding nuclease free HO 5 Aliquot the solution into individual tubes according to usage and store in darkness at 20 C Step 4Bb gt Prepare Target for Hybridization l
19. out 3 4 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 Phalanx Biotech Group Mouse OneArray User Guide v2 1a 27 Wash the Hybridized Microarray Washed and dried microarrays should be scanned within a sau couple of hours 1 2 3 4 5 6 7 8 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 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 Transfer the slide rack to a third slide staining dish that contains 2x SSC
20. probes 824 experimental control probes Composition RGAP v6 1 and BGI 2008 Yeast OneArray v1 3 6 946 yeast genome probes 695 experimental control probes Composition AROS v1 1 and YBOX v1 0 Human miRNA OneArray v4 1 087 unique miRNA probes 3 105 experimental control probes 3 features per probe DA 100 of Sanger miRBase v18 Human miRNAs Mouse amp Rat miRNA OneArray v5 1 506 unique miRNA probes 48 experimental control probes 3 repeats per probe 100 of Sanger miRBase v19 Rodent miRNAs Phalanx Biotech Group Mouse OneArray User Guide v2 1a 33 Model Plant miRNA OneArray v3 ee 3 171 unique miRNA probes F 117 experimental control probes 3 features per probe 100 of Sanger miRBase v17 Plant miRNAs 34 Mouse OneArray User Manual v2 1a Phalanx Biotech Group Power of OneArray OneArray Mouse OneArray UserGuide 2013 Phalanx Biotech All rights reserved www OneArmay com
21. r Manual v2 1a Phalanx Biotech Group Final Total Volume of Target Hybridization Mix Labeled target mix 1 5x Working Hybridization Buffer Add RNAase free ddH5O 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 insufficient use distilled water to make up the volume 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 over Silde pane i Take cover slide out from the E package Phalanx Biotech Group Mouse OneArray User Guide v2 1a 15 WAE TE TUD Back sig PLAUKAI arcode LG hy ii Carefully laid the Mouse OneArray on top of the assembly of cover slide and spacer where the printed side of array should be facing toward the cover slide to form a hybridization assembly The printed side of array is on the side of the label i Insert the hybridization assembly into a 3 1 x 9cm heat shrank hybridization Hyb bag Sample loading side of the hybridization assembly should face the opening side of film bag Lower the assembly to the end of the bag Assembly viewed from the other side iv Clip the bag with a clipper securely from the opening end Immerse the assembly to the rim of the slide in 95 C hot water swiftly approximately 2 5 sec Do not dip the assembly too far into the wa
22. r 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 Mouse OneArray User Manual v2 1a Phalanx Biotech Group HE EE EL ES ED ED EU EE EN YD ES EYN EE ES Y ED YD IS DE ED DN YD EYN ES DS ES DS DS EDD ED EE DS ES DD DS DN ES EY EN EY E Reagents De ionized nuclease free water Cyanine 3 or 5 labeled amplified aRNA sample 20x SSC stock solution sterile 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 Wash Solutions sterile filtered approximately 250mL of each is required per experiment o 2x SSC 0 2 SDS o 2x SSC o 0 2 x SSC NOTE SDS must be molecular biology grade Pre hybridization Buffer prepared and sterile filtered 1mmediately 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 o Ambion sheared Salmon Sperm DNA 10 ug uL or Invitrogen Cot 1 DNA 2 5 ug uL or Invitrogen Poly A 2 5 ug uL Phalanx Biotech Group Mouse OneArray User
23. roducts 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 ii Mouse OneArray User Guide v2 1a Phalanx Biotech Group Contact Information Asia Office Phalanx Biotech Group 6 Technology Road 5 6 Floor Hsinchu Science Park Hsinchu 30078 Taiwan Tel 886 3 578 1168 Toll Free 0800 777 988 FAX 886 3 578 5099 E mail twinfo phalanxbiotech com website www onearray com China Office Phalanx Biotech Group E mail cninfo phalanxbiotech com website www onearray com cn Toll Free 4006 4008 60 U S Office Phalanx Biotech Group 1301 Shoreway Road Suite 160 Belmont CA USA 94002 Tel 650 320 8669 Toll Free 877 730 3887 FAX 650 508 9889 E mail info onearray com website www phalanxbiotech com www onearray com Phalanx Biotech Group Mouse OneArray User Guide v2 1a lil User Guide and Technical Support Electronic version of this manual is available on the enclosed Product Support CD and online at WWW OncarTay com To reach technical support by telephone call Within the US 650 320 8669 Outside the US 886 3 5781168 Feedback We welcome your feedback regarding our products and this manual Please contact us at twinfo phalanxbiotech com All comments
24. s at 20 C Step 4Cb 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 H2O 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 2 Add 1 uL 10x Fragmentation Reagent and incubate at 70 C for 15 minutes Phalanx Biotech Group Mouse OneArray User Guide v2 1a 23 3 Add 1 uL Stop Solution and mix well 4 Mix with nuclease free H2O to yield a final volume of 17 uL 5 Keep on ice and in darkness until hybridization Step 4Bc Step 4Cc gt Complete 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 the OneArray Buffer Table 3 Compatible Glass Cover Slide Products Manufacturer Product Name BioRad Laboratories SLS 6001 24x60 mm
25. sion 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 Mouse OneArray User Manual v2 1a Phalanx Biotech Group EN EN EN EN EN EN EN EN EN EN EN EN EU EN EN EN EN EN EN EU EN EN EN EN EN EU EU EN EN EN EN EN EN EU EN EN EN EN EN EU EU EN EN EN EN EN EN EU EN EN EN EN EN EN ED EN EN EN EN EN EN EN EN EN EN EN EN EN ED EN EN EN EN EN ED ED ES EN EN EN EN EN EN EN EN EN EN EN EN EU EN EN EN EN EN EU EU EN EN EN EN EN EU EU EN EN EN EN EN ED EDN EN EN EN EN ED EU EN DN EN EN EN ED EN EN EN EN EN EN EN EU EN EN EN EN EN EN EN ES EN EN EN EN EN UN EN DN EN EN EN EU UN EN EN EN EN EN EU EU EN EN EN EN EN EU EU EN EN EN EN EN EN EDEN EN EN EN EN ED EDN EN EN EN EN EN ED EN EN EN EN EN EN ES EN EN EN EN EN EU EN EY EN ED EN EN DU DN EN EN
26. t Hybridization Using Labeled Targets from aRNA Labeling Approaches 1 Mix 10 ug of your aRNA sample with nuclease free H O to yield a final volume of 27 uL NOTE It is essential to use at least 10 wg of labeled target for each hybridization If you are performing a dual dye experiment use at least 10 ug of each labeled aRNA sample Phalanx Biotech Group Mouse OneArray User Guide v2 1a 19 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 H5O to yield a final volume of 67 uL 5 Keep on ice and in darkness until hybridization Step 4Bc Step 4Bc gt 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 1 e large beaker 3 Prepare Target Hybridization Mix Final Total Volume of Target Hybridization Mix Labeled target mix 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 use distilled water to make up the volume 20 Mouse OneArray User Manual v2 1a Phalanx Biotech Group 4 Phalanx Biotech Group Denature the Target Hybridization M
27. ter to avoid water leaking into the assembly The Hyb bag will shrink and tightly wrap around the assembly uana 00 Caution Hot water bath is gt 90 C Do not dip your hand into the water 16 Mouse OneArray User Manual v2 1a Phalanx Biotech Group V Remove the assembly from water and wipe off the water from the assembly Trim off the excess film from the top of the assembly Keep the assembly in a 50 C oven for at least 10 minutes EZIBDLEZLFMU riau UE ijj vi Load the entire target hybridization mix into the assembly through the sample loading opening Add 60 uL of 1x hybridization buffer and leave approximately 3mm 0 3cm of space from the top rim OneArra Cover SI Phala lae a x Caution Tilt the assembly slightly for easier loading cZeorcZtreo noi Ue 04 YS Bite Hb ieg GAH au AEL WU vii Cut the second Hyb Bag to approximately one la 8 half of its original length Phalanx Biotech Group Mouse OneArray User Guide v2 1a 1 18 Bupuag uu ied beg gAH Aeigaeuonp lw VIII Keep the assembly vertical and slip on the shortened Hyb bag from step viii all the way until the assembly reaches the end of the Hyb bag TPO czreotes DLHE 1 d IMI teas ee uj IX Use a pair of forceps to hold onto the top of the assembly Immerse the entire assembly into 95 C hot water with the assembly in upright position Caution Make sure a bubble forms in the cl
28. tions below carefully Spin down the stock OneArray Hybridization Buffer 5 5 ml in each tube Add 4 5 ml of deionized formamide Warm the mixture to 42 C to completely dissolve the solution Mix thoroughly Yield 10 ml of 1 5x Hybridization Buffer solution Make up 1x Hybridization Buffer by adding nuclease free H O Aliquot the solution into individual tubes according to usage and store in darkness at 20 C Mouse OneArray User Guide v2 1a 13 14 Step 4Ab gt Prepare Target for Hybridization lt Hybridization Using Labeled Targets from aRNA Labeling Approaches 1 Mix 10 ug of your aRNA sample with nuclease free H O to yield a final volume of 27 uL NOTE It is essential to use at least 10 ue of labeled target for each hybridization If you are performing a dual dye experiment use at least 10 ug of each labeled aRNA sample 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 HO to yield a final volume of 60 uL 5 Keep on ice and in darkness until hybridization Step 4Ac Step 4Ac gt Complete the Hybridization 1 Thaw and re suspend the 1 5x and lx 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 Mouse OneArray Use
29. use OneaArray User Manual v2 1a Phalanx Biotech Group Mouse OneArray User Guide Notice to he User scccecccdccsscocsccansececeschbacecececd acacececwsdcusdocssesesseeseeceeeel Thank Oll ee RR YN a aaa aaa VI Mouse OneATray a YY FYS US VIL Getting Started wascossssceseisscsssssscesvcsiestesesscscuscetenssdececessseeserssierseovesvenes dL Product Contents 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 Mouse OneArray 0 0 0 00 eeeneeneneenenenneneneens 7 Step 1 Prepare the RNA Sample 8 Step 2 Label the Target 9 Step 3 Pre Hybridize the Microarray 11 Complete the Hybridization Protocol 13 Step 5 Wash the Hybridized Microarray 28 Scan and Extract Gene Expression Results 29 Step 7 Check the Control Probe Data 32 OneArray
Download Pdf Manuals
Related Search