Human v4 and Mouse v1 - Phalanx Biotech Group
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1. Human amp Mouse DNA Microarray User Guide www OneArray com Notice to the User Wee It is important that users read the entire manual before O commencing work Phalanx Biotech Group OneArray User Manual v3 3 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 seguence information of the Mouse OneArray product 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 satisfacti2. 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 18 OneArray User Manual v3 3 Phalanx Biotech Group Using OneArray k 50mm 1 96 9 5mm Probe Spotted Region 66 0 uwup cz ECBOLETL HO es v D 2 JI a o y m y a od m C p 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 42 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 OneArray User Manual v3 3 19 Using OneArray
3. Please refer to the respective company website for more information about the products listed below Table 8 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 Agilent Technology DNA Microarray Scanner G2565B 24 OneArray User Manual v3 3 Phalanx Biotech Group Using OneArray ci Ne 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 phalanxbiotech com Support Support html for more detailed information about the experimental controls on your OneArray product Alternatively SimpleMeasure is a small free Java based applet designed to analyze control probe data and generate easy to interpret graphs The program is included on your product CD or can be downloaded from http www phalanxbiotech com Support Downloads html Additional information about the control probes is included on the Product Support CD and on our Web site at www phalanxbiotech com Phalanx Biotech Group OneArray User Manual v3 3 25 Power of OneArray OneArray OneArray Human amp Mouse User Guide 2007 2008 Phalanx Biotech All rights rese
4. solution that meets your specific needs Stratagene Ambion Invitrogen and other reagent companies offer many different RNA isolation products For more information you can visit each company s Web site 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 Web site 10 OneArray User Manual v3 3 Phalanx Biotech Group Using OneArray EN Label the Target IMPORTANT For best results it is recommended that you use one of the 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 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 nor 4 it is recommended that you validate the method to test and determine its compatibility with the OneArray You may want to confirm the quality of the labeled target with the No Cuvettes Spectrophotometer from NanoDrop RNA
5. Figure 2 below provides an illustration of Step 4C 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 4C gt aRNA or cDNA 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 1 2 inch x 1 2 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 usascientific com 20 OneArray User Manual v3 3 Phalanx Biotech Group Using OneArray gt Wash the Hybridized Microarray Moutu Washed and dried microarrays should be scanned within a O couple of hours 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 1 Submerge the entire labeled target and microarray set up with the cover slide still intact into a large container filled with 42 C 2X SSPE 0 1 SDS solution 2 Carefully remove the cover slide from the glass by gent
6. Guide Product Support CD which contains the following e Sample Images e OneArray gal file e OneArray gene list and probe seguences e OneArray microarray layout e OneArray Control Probe list e OneArray User Guide electronic version e SimpleMeasure experimental control analysis program Phalanx Biotech Group OneArray User Manual v3 3 1 Getting Started Other Necessary Apparatus Not Supplied Apparatus Water bath heating block Powder free gloves Clean blunt forceps Micropipettors gt Sterilized and nuclease free pipet tips Sterilized and nuclease free microcentrifuge tubes High speed microcentrifuge Low speed tabletop microcentrifuge with slide holder attachment gt Vortex mixer Hybridization oven gt Hybridization accessories chamber cover slides etc Rectangular slide staining dish and slide rack for washing microarrays PCR polymerase chain reaction machine gt Microarray scanner for standard 1 x 3 format see Table 8 under OneArray Microarray Scanner Specifications for a list of compatible scanners Hybridization systems optional gt Automated hybridization station optional 2 OneArray User Manual v3 3 Phalanx Biotech Group Getting Started Other Necessary Reagents Not Supplied Reagents De ionized nuclease free water Cyanine 3 or 5 labeled amplified aRNA sample or Cyanine 3 or 5 labeled eDNA sample 20X SSPE stock solution ste
7. Sample Amounts Generally the amount needed of quality RNA is 10 20 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 30 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 loss of signal of many Phalanx Biotech Group OneArray User Manual v3 3 11 Using OneArray targets It is not recommended to perform hybridization with samples of low dye incorporation efficiency For aRNA Hybridization Follow the instructions provided by the reagent supplier Indirect labeling with NHS ester dye is recommended Table 3 below contains a list of products that have been tested for use with OneArray Table 3 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 gt 10 ug of quality aRNA is recommended Smaller
8. Stared cnu ey iid DG L Product Contents 2 222 2022200 202 2 1 Other Necessary Apparatus and Reagents Not Supplied 2 Important Notes on Microarray Handling and Storage 4 Product Descriptions and Overview 5 Using ONGAN aY FHHHHHHHHHHHHH 9 Step 1 Prepare the RNA Sample 10 Step 2 Label the Target 11 Step 3 Pre Hybridize the Microarray 14 Step 4 Complete the Hybridization Protocol 15 Step 5 Wash the Hybridized Microarray 21 Step 6 Scan and Extract Gene Expression Results 22 Step 7 Check the Control Probe Data 25 Phalanx Biotech Group OneArray User Manual v3 3 vii Getting Started Getting Started Please read the introductory information below to help familiarize yourself with OneArray before use Product Contents gt Human or Mouse OneArray DNA Microarray gt OneArray Hybridization Buffer Tube e Each tube contains buffers sufficient for 5 to 10 microarray hybridization procedures gt Spare round cap tube gt OneArray User Guide Spotted Region
9. accompanied this product or at http www phalanxbiotech com OneArray User Manual v3 3 Phalanx Biotech Group Getting Started Human OneArray Genome Content Each microarray contains 32 050 oligonucleotides 30 968 human genome probes and 1082 experimental control probes Each oligonucleotide probe is designed to hybridize to a specific target gene described in the current public domain contents such as UniGene Cancer Genome Anatomy Project CGAP BioCarta Kyoto Encyclopedia of Genes and Genomes KEGG and validated by the Human Genome Seguencing Project HGSP Table 2 below provides an example of the contents of a human genome that can be studied using the Human OneArray Table 2 Human Genome Content Probe Type Number of Probes UniGene and RefSeg based 30 968 total UniGene build 175 based and or RefSeq based with Entrez 28 703 Gene ID including CGAP Cancer Genome Anatomy Project BioCarts and KEGG Kyoto Encyclopedia of Genes and Genomes pathways UniGene build 163 based with Gene ID and experimentally 2265 selected Human OneArray is guaranteed to contain gt 98 of the total probe content Phalanx Biotech Group OneArray User Manual v3 3 7 Getting Started Human OneArray Control Features There are 1 082 control probes built into the Human OneArray DNA microarray that monitor the sample quality and hybridization process These control probes provide valuable information to ensure experimen
10. e 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 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 25x601 M 5439 is a trademark of Erie Scientific Company Amino Allyl MessageAmp II aRNA is a trademark of Ambion ArrayControl is a trademark of Ambion Inc Amino Allyel cDNA Labeling Kit is a trademark of Ambion Superscript Indirect Labeling System is a trademark of Invitrogen Fairplay II Microarray Labeling Kit is a trademark of Strategene Mini Elute PCR purification is a trademark of Qiagen SpotReport is a registered trademark of Strategene Inc Last updated July 2008 2005 2008 Phalanx Biotech Group All rights reserved Phalanx Biotech Group OneArray User Manual v3 3 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 OneArray User Guide vi OneArray User Manual v3 3 Phalanx Biotech Group Table of Contents Getting
11. halanx Biotech Group OneArray User Manual v3 3 15 Using OneArray 3 Warm the mixture to 42C to completely dissolve the solution Mix thoroughly Yield 500 ul of 1 5X Hybridization Buffer solution 4 Aliquot the solution into individual tubes according to usage and store in darkness at 20 C Step 4B Prepare Target for Hybridization Hybridization Using Labeled Targets from aRNA or cDNA Labeling Approaches For aRNA Hybridization 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 ug 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 3 Add 1 ul Stop Solution and mix well 4 Mix with nuclease free H20 to yield a final volume of 17 uL 5 Keep on ice and in darkness until hybridization Step 4C For cDNA Hybridization 1 Mix 15 ug of your cDNA sample with nuclease free H2O to yield a final volume of 17 uL NOTE When using cDNA as your target fragmentation is not necessary 2 Keep on ice and in darkness until hybridization Step 4C 16 OneArray User Manual v3 3 Phalanx Biotech Group Using OneArray Step 4C 2Complete the Hybridization Using the Glass Cover Slide Method NOTE If you perform hybridization using methods other than the basic glass cover slide method it is reco
12. ides more than once in the pre hybridization buffer OneArray User Manual v3 3 Phalanx Biotech Group Using OneArray 8 After 2 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 completing the pre hybridization process EZZ Complete the Hybridization Protocol 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 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 Step 4A Prepare Hybridization Solution Using the OneArray Hybridization Buffer Included sau For correct use of this buffer you must add a specific O amount of formamide and labeled target Please follow the instructions below carefully 1 Spin down the stock OneArray Hybridization Buffer 410 ul in each tube 2 Add 90 ul of deionized formamide P
13. ly 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 3 Wash the slide s in the rectangular slide staining dish and slide rack with the excess amount of pre warmed 2X SSPE 0 1 SDS solution for 5 min at 42 C 4 Transfer the slide rack to a second slide staining dish that contains 0 1X SSPE 0 1 SDS solution and wash for 5 min at room temperature 5 Transfer the slide rack to a third slide staining dish that contains 0 1X SSPE and wash for 5 min at room temperature 6 Rinse each array carefully with 0 1X SSPE using a squeeze bottle 7 Spin dry with a centrifuge for at least one minute 8 Keep the microarray dry and in the dark until ready to scan Phalanx Biotech Group OneArray User Manual v3 3 21 Using OneArray Ew 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 7 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 en
14. mmended that you validate the protocol experimentally For example 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 5 below contains a list of glass cover slides that have been tested and confirmed compatible for use with the OneArray Buffer Table 5 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 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 6 below Phalanx Biotech Group OneArray User Manual v3 3 17 Using OneArray Table 6 Hybridization Mix Measurements For each slide 55 ul Component Final Volume 1 5X OneArray Hybridization 37 ul Buffer Sheared Salmon Sperm DNA 1l 10 pg pl Target preparation plus 17 ul nuclease 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 l or Invitrogen Poly A 2 5 10 ug ul 4
15. ng reaction often contributes to increased background noise The Qiagen Mini Elute PCR has been found to consistently provide more reliable results Phalanx Biotech Group OneArray User Manual v3 3 13 Using OneArray gt Pre Hybridize the Microarray General Instructions IMPORTANT 6 IMPORTANT 14 OneArray requires a pre hybridization step prior to 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 Warm the pre hybridization solution SX SSPE 0 1 SDS and 1 BSA to 42 C 2 Pour 25 ml room temperature 100 ethanol into the spare array tube 3 Preheat the OneArray s in the round cap tube at 60 C for 10 min hybridization oven recommended 4 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 5 Shake the round cap tube for 20 sec 6 Remove and thoroughly rinse each array with deionized water to remove any residual ethanol 7 Carefully and slowly fully submerge the OneArray in an abundant amount of pre hybridization solution for 2 hr at 42 C 35 ml is sufficient if using a round cap tube Try to insert the slides into the correct position the first time Avoid inserting and removing the sl
16. on 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 ii OneArray User Manual v3 3 Phalanx Biotech Group Contact Information U S Office Asia Office Phalanx Biotech Group 1400 Page Mill Road Bldg B Palo Alto CA 94304 1124 Ph 650 320 8669 FAX 650 320 8488 E mail info phalanxbiotech com Web site www phalanxbiotech com www onearray com Phalanx Biotech Group 6 Technology Road 5 6th Floor Hsinchu Science Park Hsinchu 30077 Taiwan Ph 886 3 5781168 FAX 886 3 5785099 E mail info phalanxbiotech com Web site www phalanxbiotech com Phalanx Biotech Group OneArray User Manual v3 3 iii User Guide and Technical Support Electronic version of this manual is available on the enclosed Product Support CD and online at www phalanxbiotech com To reach technical support by telephone call Within the US 1 650 320 8669 Outside the US 886 3 5781168 Feedback We welcome your feedback regarding our products and this manual Please contact us at feedback phalanxbiotech com All comments are welcome iv OneArray User Manual v3 3 Phalanx Biotech Group Trademarks and Copyrights Mouse OneArray Human OneArray and OneArray ar
17. rile filtered o 3 6 M Sodium chloride o 0 2M Sodium phosphate pH 7 7 o 20mM EDTA Wash Solutions sterile filtered four types approximately 250 mL of each is reguired per experiment 2X SSPE 0 1 SDS 2X SSPE 0 1X SSPE 0 1 SDS 0 1X SSPE o NOTE SDS must be molecular biology grade O O O O Pre hybridization Buffer prepared and sterile filtered immediately prior to pre hybridization o 5X SSPE 0 1 SDS 1 BSA o NOTE BSA must be molecular biology grade gt Deionized formamide to be added to the OneArray Hybridization Buffer prior to use see Step 4A RNA Fragmentation Reagent and Stop Solution for hybridization using aRNA DNA Blocking Mixture o Ambion sheared Salmon Sperm DNA 10 ug ul or o Invitrogen Cot 1 DNA 2 5 10 ug ul or o Invitrogen Poly A 2 5 10 ug ul Phalanx Biotech Group OneArray User Manual v3 3 3 Getting Started Storage Conditions Store unopened OneArray product at room temperature gt Store opened OneArray product at 4 C 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 Wei Please read this section carefully and follow the 6 instructions Polynucleotide probes are printed on the side of the slide with the barcode To avoid irreparable damage of the printing area do not touch
18. rved Phalanx Biotech Group www OneArray com
19. s the following Web sites http arrays ucsf edu archive meebo html http genome www5 stanford edu Special thanks to Dr John Coller for his helpful discussion in the selection of probe sets for reduction Phalanx Biotech Group OneArray User Manual v3 3 5 Getting Started Table 1 below provides an example of the contents of a mouse genome that can be studied using the Mouse OneArray Table 1 Mouse Genome Content Probe Type Number of Probes MEEBO abridged version 29 922 total Group Code MC 24 858 Mouse constitutive exonic Rockefeller MouSDB3 Constitutive exons and locuslink2ucsf constitutive exons Group Code MR 5 064 Mouse mRNAs mRNA derived 70 mer probes which may span gt 1 exon Mouse OneArray is guaranteed to contain gt 98 of the total probe content Mouse OneArray Control Features There are 1 880 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 quality results for analysis SimpleMeasure is a small free Java based applet designed to analyze control probe data and generate easy to interpret graphs The program can be downloaded from http www phalanxbiotech com Support Downloads html NOTE Detailed control information gene lists gene annotations and probe sequences can be found on the Product Support CD that
20. sure 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 Web site at www phalanxbiotech com Table 7 Scanner Settings Using GenePix 4100 from Molecular Devices Wavelength 635 nm 532 nm PMT 630 V 590 V Minimum diameter 80 Maximum diameter 180 CPI Threshold 100 NOTE For lower versions of GenePix software adjust the property parameter to 142 8 um manually to obtain best results 22 OneArray User Manual v3 3 Phalanx Biotech Group Using OneArray Figure 3 below provides a visual example of the OneArray glass slide with spotted probe region 50 10 um Cj 142 8um i k 142 8um Figure 3 OneArray Glass Slide with Spotted Probe Region Phalanx Biotech Group OneArray User Manual v3 3 23 Using OneArray 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 8 below contains a partial list of microarray scanner products that are compatible for use with the OneArray microarray
21. the surface with bare hands or with any other objects Whenever possible handle microarrays with clean blunt forceps to avoid contamination IMPORTANT O Open arrays should be used within a week 4 OneArray User Manual v3 3 Phalanx Biotech Group Getting Started 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 30 000 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 Genome Content Each microarray contains 31 802 oligonucleotides 29 922 mouse genome probes and 1880 experimental control probes Mouse OneArray content is based on an abridged version of the Mouse Exonic Evidence Based Oligonucleotide MEEBO MEEBO is a new set of 70 mer probes specifically for DNA microarrays that was created by a team of researchers from the University of California San Francisco and elsewhere and was headed by Dr Ahs Alizadah MEEBO has been made available to the public For more information about MEEBO acces
22. ts are done correctly resulting in higher guality results for analysis SimpleMeasure is a small free Java based applet designed to analyze control probe data and generate easy to interpret graphs The program can be downloaded from http www phalanxbiotech com Support Downloads html 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 phalanxbiotech com 8 OneArray User Manual v3 3 Phalanx Biotech Group Using OneArray 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 Wide Follow these detailed steps exactly to achieve the best experimentation results gt Step 1 Prepare the RNA Sample Step 2 Label the Target Step 3 Pre Hybridize the Microarray gt Step 4 Perform the Hybridization Protocol Step 5 Wash the Hybridized Microarray Step 6 Scan and Extract Gene Expression Results Step 7 Check Control Probe Data Phalanx Biotech Group OneArray User Manual v3 3 9 Using OneArray EE Prepare the RNA Sale su High guality intact RNA is essential for all gene expression 6 microarray experiments There are many different RNA isolation protocols and commercially available RNA isolation kits You should choose a
23. 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 4B 12 OneArray User Manual v3 3 Phalanx Biotech Group Using OneArray For cDNA Hybridization Follow the instructions provided by the reagent supplier Indirect labeling with NHS ester dye is recommended Table 4 below contains a list of products that have been tested for use with OneArray Table 4 cDNA Preparation Products Manufacturer Product Name and Description Ambion Amino Allyl MessageAmp II cDNA Kit Invitrogen Superscript Indirect Labeling System Stratagene Fairplay ll Microarray Labeling Kit For cDNA labeling it is recommended that 15 pg of total nucleic acid be used as starting material For all cDNA preparation it is strongly recommended that you re purify the targets using the Qiagen Mini Elute PCR purification column prior to hybridization according to the manufacturer s protocol Incomplete removal of unincorporated dye from the labeli
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