Getting Started - Phalanx Biotech Group
Contents
1. 2 Other Necessary Reagents Not Supplied 3 Important Notes on Microarray Handling and Storage 4 Product Description and Overview 5 Using MIRNA ONneArray ssssssssseeeeeeeeeecceesssssseeeseeeeeeeseensO Step 1 Prepare the RNA Sample 7 Step 2 Label the Target 8 Step 3 Pre Hybridize the Microarray 9 Step 4 Complete the Hybridization Protocol 10 Step 5 Wash the Hybridized Microarray 15 Step 6 Scan and Extract Gene Expression Results 16 Step 7 Check the Control Probe Data 18 Phalanx Biotech Group miRNA OneArray User Manual v1 0 vii Getting Started Please read the introductory information below to help familiarize yourself with OneArray before use Product Contents gt miRNA OneArray DNA Microarrays gt OneArray 1 5X Hybridization Buffer I gt miRNA OneArray Hybridization Buffer II gt Spare round cap tube gt miRNA OneArray User Guide Spotted Region Guide Product Support CD which contains the following e Sample Images e miRNA OneArray gal file2. MIRNA OneArray User Guide www OneArray com Notice to the User Wet is important that users read the entire manual before commencing work Phalanx Biotech Group miRNA OneArray User Manual v1 0 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 all OneArray products Phalanx Biotech is founded on the mission to offer researchers high guality and user friendly solutions at an affordable price Your satisfaction in using our products is very important to us Therefore if any of our p
3. 0 2 SDS e Wash II 2X SSC e Wash III 0 2X SSC e NOTE SDS must be molecular biology grade gt 100 Ethanol Pre hybridization Buffer prepared and sterile filtered immediately prior to pre hybridization e 5X SSPE 0 1 SDS 1 BSA e NOTE BSA must be molecular biology grade Deionized formamide to be added to the OneArray 1 5X Hybridization Buffer prior to use see Step 4A Phalanx Biotech Group miRNA OneArray User Manual v1 0 3 Storage Conditions Store unopened miRNA OneArray product at room temperature gt Store opened miRNA OneArray product at 4 C gt Store OneArray 1 5X Hybridization Buffer I at room temperature gt Store miRNA OneArray Hybridization Buffer II at 20 C temperature NOTE If the product is received with an open bag please contact Phalanx Biotech Customer Service for an immediate replacement Handling Microarrays Ie i Please read this section carefully and follow the instructions Q 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 gt Whenever possible handle microarrays with clean blunt forceps to avoid contamination IMPORTANT O Open arrays should be used within a week 4 miRNA OneArray User Manual v1 0 Phalanx Biotech Group Product Description and Overview miRNA OneArray microarrays are
4. Figure 1 1825mm 95mm 3mm 10 55mm NOJS u20101g xuejeud Figure 1 miRNA OneArray Microarray Glass Slide with Probe Printed Region Guide Plastic Underlay 8 Pipette the hybridization mixture onto the spotted region of miRNA OneArray Microarray Avoid creating any bubbles 9 Carefully place the glass cover slide over the spotted area in an even manner 10 Place the entire labeled target plus the microarray set up into a closable chambered box that is humidified by 2X SSC buffer in the 37 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 miRNA OneArray User Manual v1 0 13 Figure 2 below provides an illustration of Step 4C where the hybridization protocol is completed using the glass cover slide method and specifically the miRNA OneArray 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 4B gt 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 o
5. made of polydeoxynucleotide 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 Each probe is spotted onto the array in a highly consistent manner using a proprietary non contact spotting technology adapted for microarray manufacturing Phalanx Biotech Group miRNA OneArray User Manual v1 0 5 Using miRNA 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 miRNA OneArray microarray Wei Follow these detailed steps exactly to achieve the best 6 experimentation results Step 1 Prepare the RNA Sample Step 2 Label the Target Step 3 Pre Hybridize the Microarray Step 4 Perform the Hybridization Protocol Step 5 Wash the Hybridized Microarray Step 6 Scan and Extract Gene Expression Results gt Step 7 Check Control Probe Data 6 miRNA OneArray User Manual v1 0 Phalanx Biotech Group Stepi Prepare the RNA Sample IMPORTANT 6 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 Kreatech Stratagene Ambion Invitrogen and other reagent companies offer many different RNA isolation products For
6. sample leading to higher background signal It is best to use RNA 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 RNA and store in the dark at 80 C Avoid thawing and re freezing RNA if possible as freeze thaw cycles can damage the RNA 8 miRNA OneArray User Manual v1 0 Phalanx Biotech Group EFE Pre Hybridize the Microarray General Instructions manwl miRNA OneArray requires a pre hybridization step prior to 6 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 5X 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 miRNA OneArray s in the round cap tube at 60 C for 10 min hybridization oven recommended 4 Remove the miRNA 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 miRNA OneArray in an abundant amount of pr
7. d 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 miRVana 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 December 2008 O 2005 2008 Phalanx Biotech Group All rights reserved Phalanx Biotech Group miRNA OneArray User Manual v1 0 V Thank You vi Phalanx Biotech Group would like to extend special thanks to our customers who have provided feedback that enabled us to improve the miRNA OneArray User Guide miRNA OneArray User Manual v1 0 Phalanx Biotech Group miRNA OneArray User Guide Getting Staned ne GN eek Product Contents 222220000002 22 2000 222 nn nL 1 Other Necessary Apparatus Not Supplied
8. e hybridization solution for 2 hr at 427C 35 ml is sufficient if using a round cap tube Lei Try to insert the slides into the correct position the first time 6 Avoid inserting and removing the slides more than once in the pre hybridization buffer Phalanx Biotech Group miRNA OneArray User Manual v1 0 9 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 Step4 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 miRNA 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 Buffers included with this product to complete the hybridization process Step 4A gt Prepare Hybridization Solution Using the OneArray 1 5X Hybridization Buffer I Included wuu For correct use of this buffer you must add a speci
9. fic O amount of formamide and labeled target Please follow the instructions below carefully 1 Spin down the stock OneArray 1 5X Hybridization Buffer I 410 ul in each tube 2 Add 90 ul of deionized formamide 10 miRNA OneArray User Manual v1 0 Phalanx Biotech Group 3 Warm the mixture to 42 C to completely dissolve the solution Mix thoroughly Yield 500 ul of OneArray 1 5X Hybridization Buffer I Step 4B Complete the Hybridization Using the Glass Cover Slide Method Hybridization Using Labeled Targets from Enriched miRNA Labeling Approaches NOTE The protocol that follows is for use with a single array Take care when using a coverslip that the solutions from neighboring arrays printed on the same slide do not mix NOTE If you perform hybridization using methods other than the basic glass cover slide method it is recommended that you validate the protocol experimentally To complete this step you will need to select a type of chamber or glass cover slide Table 5 below contains a list of products that have been tested and confirmed compatible for use with the OneArray Buffer Table 5 Compatible Glass Cover Slide Products Manufacturer Product Name Corning Cover Glass 22 X 22 mm Erie Scientific Company mSeries LifterSlip 22X251 M5226 BioMicro MAUI Mixer DC Phalanx Biotech Group OneArray Dual Hyb Chamber The MAUI DC Chamber requires the use of the MAUI Hybridization System Details ca
10. he miRNA OneArray microarray 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 Phalanx Biotech Group miRNA OneArray User Manual v1 0 17 E gt Check the Control Probe Data miRNA OneArray 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 miRNA OneArray product Additional information about the control probes is included on the Product Support CD and on our Web site at www phalanxbiotech com 18 miRNA OneArray User Manual v1 0 Phalanx Biotech Group Power of OneArray OneArray miRNA OneArray User Guide 2008 Phalanx Biotech All rights reserved Phalanx Biotech Group www OneArray com
11. ifuge for at least one minute 8 Keep the microarray dry and in the dark until ready to scan Phalanx Biotech Group miRNA OneArray User Manual v1 0 15 Step 6 Scan and Extract Gene Expression Results 16 There are many scanners available to extract signals from miRNA OneArray Data extraction using GenePix 4100 from Molecular Devices is described below Please refer to the respective company product instructions for appropriate use For a list of scanners that are compatible with the miRNA 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 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 miRNA OneArray User Manual v1 0 Phalanx Biotech Group miRNA 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 t
12. 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 Phalanx Biotech Group miRNA OneArray User Manual v1 0 7 EN Label the Target 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 You may want to confirm the guality of the labeled target with the No Cuvettes Spectrophotometer from NanoDrop 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 miRNA OneArray Table 3 miRNA Preparation Products Manufacturer Product Name and Description Ambion miRVana miRNA Isolation Kit Ambion miRVana miRNA Labeling Kit For RNA labeling 1 5 ug enriched miRNA is recommended Smaller volumes can lead to significant loss of sample and may increase the concentration of contaminants in the labeled RNA
13. n be found at the manufacturer s website http www biomicro com Phalanx Biotech Group miRNA OneArray User Manual v1 0 11 1 Ensure your work and experimentation area as well as the miRNA OneArray are clean before adding the Hybridization Buffers solution to the target array 2 Pre warm the OneArray 1 5X Hybridization Buffer I with formamide at 37 C for 10 minutes 3 Mix your labeled miRNA sample with nuclease free H20 to yield a final volume of 17 uL NOTE For enriched miRNA it is essential to use at least 1 ug of labeled miRNA If you are performing a dual dye experiment use at least 1 ug of each labeled miRNA sample 4 Prepare the hybridization mix in a 0 2 ml Eppendorf tube according to the Table 6 below Table 6 Hybridization Mix Measurements For each array 55 ul Component Final Volume 1 5X miRNA OneArray 37 ul Hybridization Buffer I miRNA OneArray 1 pl Hybridization Buffer ll Target preparation plus 17 ul nuclease free ddH O 5 Heat the mixture to 95 C for 2 minutes thermocycler recommended 6 Maintain the mixture at a temperature of 60 C until pipetting onto the array thermocycler recommended gt It may be helpful to set a Denature program in the thermocycler as follows 95 C 2 minutes 60 C Hold 12 miRNA OneArray User Manual v1 0 Phalanx Biotech Group 7 Place the miRNA OneArray slide bar code up atop the Probe Printed Region Guide included see
14. roducts 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 miRNA OneArray User Manual v1 0 Phalanx Biotech Group Contact Information U S Office Asia Office Phalanx Biotech Group 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 O phalanxbiotech com Web site www phalanxbiotech com www onearray com Phalanx Biotech Group 6 Technology Road 5 6 Floor Hsinchu Science Park Hsinchu 30077 Taiwan Ph 886 3 5781168 FAX 886 3 5785099 E mail info Ophalanxbiotech com Web site www phalanxbiotech com miRNA OneArray User Manual v1 0 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 O phalanxbiotech com All comments are welcome iv miRNA Onearray User Manual v1 0 Phalanx Biotech Group Trademarks and Copyrights OneArray is a trademark of Phalanx Biotech Group Inc in the United States and in other countries All trademarks an
15. s e miRNA OneArray probe sequences e miRNA OneArray annotation file e miRNA OneArray microarray layout e miRNA OneArray Control Probe list e miRNA OneArray User Guide electronic version Phalanx Biotech Group miRNA OneArray User Manual v1 0 Other Necessary Apparatus Not Supplied Apparatus Water bath heating block Powder free gloves Clean blunt forceps Micropipettors Sterilized and nuclease free pipet tips gt Sterilized and nuclease free microcentrifuge tubes High speed microcentrifuge Low speed tabletop microcentrifuge with slide holder attachment Vortex mixer Hybridization oven Hybridization accessories chamber cover slides etc Rectangular slide staining dish and slide rack for washing microarrays Thermocycler gt Microarray scanner for standard 1 x 3 format see Table 8 under miRNA OneArray Microarray Scanner Specifications for a list of compatible scanners Hybridization systems optional gt Automated hybridization station optional miRNA OneArray User Manual v1 0 Phalanx Biotech Group Other Necessary Reagents Not Supplied Reagents De ionized nuclease free water Cyanine 3 or 5 labeled miRNA sample 20X SSPE stock solution sterile filtered e 3 M Sodium chloride e 3M Sodium citrate Wash Solutions sterile filtered four types approximately 250 mL of each is required per experiment e Wash I 2X SSC
16. ven For information about this product or other USA Scientific products access their Web site at www usascientific com 14 miRNA OneArray User Manual v1 0 Phalanx Biotech Group EEB 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 37 C 2X SSC 0 2 SDS solution 2 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 3 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 37 C 4 Transfer the slide rack to a second slide staining dish that contains 2X SSC solution and wash for 5 min at room temperature 5 Transfer the slide rack to a third slide staining dish that contains 2X SSC and wash for 5 min at room temperature 6 Rinse each array carefully with 0 2X SSC using a squeeze bottle 7 Spin dry with a centr
Download Pdf Manuals
Related Search