User Manual
Contents
1. precursor miRNA as well as mature miRNA The content also includes probes for positive controls e g 5S rRNA tRNAs U6 as well as negative controls Customizable Chip Formats GenoExplorer chip products come in several formats Check our website for updates on the species specific chips available Orders of other species are also available upon request 3 D Gel Matrix for Optimal Hybridization Environment The surface of the GenoExplorer chip is composed of a 3 D gel matrix which provides a more optimal hybridization environment by extending the probe into the solution and increasing its access to the target GenoExplorer microRNA Biochips Kit Kit Components and Storage Conditions GenoExplorer miRNA Full Kit 20 reactions Components Amount Storage Array Chip 5x4chips 4 C 2x Hyb Buffer 5 x 280 ul 4 C Array Layout CD 1 User s Manual 1 Shipping and Storage GenoExplorer biochips and hybridization reagents are shipped at room temperature Components should be stored at temperatures shown in the above table At proper storage conditions components are stable for 1 year from the date received Expiration dates are also noted on the tube labels of each component Safety Warnings and Precautions For research use only Not recommended or intended for the diagnosis of disease in humans or animals Do not use internally or externally in humans or animals Consider all chemicals a2. G GenoSensor Corporation GenoExplorer miRNA Biochips Kit Catalog s 1201 99 Version B January 2006 GenoExplorer microRNA Biochips Kit Table of Contents Prod ct OV CEVICW otiacaninnacuubawiiidnn cane haw ddenatwobansinabatdabaweiienedwnbauneampacdebnasaneibabes 2 Kit Components and Storage Condition 0 cece cece eee ene eee eee e eee ene eee e eee 3 Additional Required Materials 5s ssid sess oachekessntdveusdsaruesddeceeveaseiacs ura danendaeciieens 4 Related Products from GenoSensor seis scasaniverancceianddexesxseutdeucarcatahivexesexerasados 4 GenoExplorer miRNA Biochip Hybridization Assay Guidelines cseeceeeeereeeeeeeees 5 PUT ciessc esses E EE E E A E EE 8 TROUBLES NO OTIS sssrini E EA EEE EEE REES 8 Technical Service soniers aeres AT E N EERTE EEEE ETE 10 Literature Citation When describing a procedure for publication using this product we would appreciate that you refer to it as the GenoExplorer miRNA Biochips Kit Patents and Trademarks GenoExplorer is a trademark of GenoSensor The GenoExplorer miRNA Biochips Kit is covered by patents pending GenoExplorer microRNA Biochips Kit Product Overview GenoExplorer Biochips Comprehensive Probe Content GenoExplorer chips consist of probes for all miRNAs as registered and annotated in the miRBase at The Wellcome Trust Sanger Institute A key differentiator of the GenoExplorer chip s content is the inclusion of probes for
3. epend on the total hybridization volume which is determined by the type of hyb chamber or coverslip that is being used Table 2 shows the volumes of each component required for different total hybridization volumes Table 2 Hybridization Reaction Mix Ww Nn Hybridation Labeled 2x Hyb Added Volume ul RNA ul Buffer ul H20 uD 40 20 20 0 60 20 30 10 100 20 50 30 120 20 60 40 Incubate the sample at 90 95 C for 5 min Cool sample by spinning down for 2 min Load the labeled RNA on to the array Consult with the slide chamber manufacturer s recommended protocol for further information on assembling the slide in its chamber Run the hybridization at 42 C for 16 hours or longer For fluorescently labeled RNA target after hybridization the arrays can undergo post hybridization washing and drying GenoExplorer labeled wash buffers are strongly recommended for the post hybridization wash processing These buffers have been optimized to enhance signal and reduce background For detailed instructions on the use of these buffers consult the user manual for the GenoExplorer Full Kit For GenoExplorer labeled miRNA containing biotin labels please refer to the user manual of the GenoExplorer Full Kit for further instructions on the post hybridizaton process Scanning and Detection Consult the scanner manufacturer instructions for laser and PMT settings GenoExplorer
4. ing slides Hold the slide at its edges and avoid touching the side of the slide containing the probe array The probes are on the side of the slide which faces you when reading the slide label correctly refer to figure 1 Prepare the chips for hybridization promptly to minimize possible accumulation of dust from the air on to the surface of the arrays even if some dust can be washed away after post hybridization washing procedures Use assembly methods as recommended by hybstation or hybchamber manufacturers For automated hybridization using a hybstation orient the chips such that the probe side is up in the chamber and the slide end opposite the label where the arrays are located are next to the sample injection port Once the hybridization solution is introduced to the chip avoid any drying of the chips until after the final wash step GenoExplorer microRNA Biochips Kit Assay Guidelines The GenoExplorer miRNA Biochips Kit has been optimized for microarray experiments using GenoExplorer reagents for hybridization assay Below are general guidelines that can be used for running the hybridization assay on GenoExplorer BioChips For a detailed protocol on running GenoExplorer labeled miRNA on GenoExplorer BioChips please consult the user manual for the GenoExplorer full kit 1 Prepare the labeled RNA sample for hybridization by combining with 2x Hyb buffer and water The volumes of each component to be added will d
5. is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about a GenoSensor product or service please contact our Technical Service at tech_service genosensorcorp com GenoSensor warrants that all of its products will perform according to the specifications stated on the certificate of analysis This warranty limits GenoSensor Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions GenoSensor reserves the right to select the method s used to analyze a product unless GenoSensor agrees to a specified method in writing prior to acceptance of the order GenoSensor makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore GenoSensor makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service GenoSensor assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or imp
6. lied including any warranty of merchantability or fitness for a particular purpose 10
7. microRNA Biochips Kit Using the Grid Files The enclosed CD provides an array list in txt file and gal file formats Four arrays were printed on each chip The name and location of each chemical probe is listed and labeled in the software Each chemical was printed in triplicate Arrays include known microRNA probes as well as both positive and negative controls Load the gal files or convert the text file to the correct format that your scanner requires Since the first chemicals on each array are positive controls you should see them on the upper left corner of each array and they are in triplicate Move the array grid until it aligns with the positive controls in the upper left corner Figure 2 Proceed with data computation after gridding Figure 2 Grid and Align the Arrays oo0000 OO0O00000 GenoExplorer microRNA Biochips Kit Appendix Troubleshooting Guide Poor Hybridization Signal Poor quality e Use higher quality RNA samples RNA samples e Use proper laboratory techniques when handling RNA samples e Label and hybridize more sample to microarray Suboptimal e Label and hybridize more sample to amount of microarray sample applied to microarray Improper e Verify that detection instrumentation is detection compatible with detection reagents strategy e Adjust detection settings Signal lost by e Minimize exposure to light exposure to light environmental conditions Poor biotin e E
8. nsure that the samples have been SA S dye purified and quantified properly before detection labeling e Ensure that the kit components have been stored properly e Ensure that the detection solution was prepared and stored properly Hybridization e Ensure correct temperatures are used signal for each of the process steps stripped from microarray Suboptimal e Extend hybridization time hybridization time GenoExplorer microRNA Biochips Kit High Background Excess e Do not allow slides to dry out during detection hybridization protocol reagents e Do not touch microarray directly or remaining forcibly remove coverslip at any time on microarray Excess e Quantify the amount of labeled sample sample and use less in hybridization applied to microarray Ink or e Avoid using markers or ink to identify marker used to identify slide use a diamond scribe pen microarray Dust Dust on e Avoid dust from environment microarray e Minimize exposure of the microarray to the air e Use filtered distilled water for final wash GenoExplorer microRNA Biochips Kit Technical Service For more information or technical assistance please call write fax or email GenoSensor Corporation 4665 S Ash Avenue Suite G 18 Tempe Arizona 85282 Tel 1 480 598 5378 Fax 1 480 755 3319 Email tech_service genosensorcorp com Web www genosensorcorp com Limited Warranty GenoSensor
9. s potentially hazardous Only persons trained in laboratory techniques and familiar with the principles of good laboratory practice should handle these products Wear suitable protective clothing such as laboratory overalls safety glasses and gloves Exercise caution to avoid contact with skin or eyes if contact should occur wash immediately with water Material Safety Data Sheet for products is available upon request GenoExplorer microRNA Biochips Kit Additional Required Materials Recommended GenoExplorer miRNA labeling kit Recommended GenoExplorer reagents for hybridization assay Total RNA containing the small RNA RNase free water Adjustable pipettors RNase free tips RNase free polypropylene microcentrifuge tubes 0 2 0 5 or 1 5 ml Graduated cylinder Microcentrifuge Incubator set at 37 C Incubator or heating block set at 75 Heating block at 95 C Hybridization Station optional for automated hybridization e g Genomic Solutions Tecan or Coverslips for manual hybridization e g Erie LifterSlips Product 22x30I 2 4374 Slide Chamber for manual hybridization e g Corning Microarray Hybridization Chamber Product 2551 Incubator or water bath set at 42 C for manual hybridization 50 ml screw top tubes for manual post hybridization and post stain washing Bottles for storing diluted Wash Buffers Centrifuge or air blower Microarray scanner and Image Processing Software The GenoE
10. xplorer Biochips kit is open to a variety of equipment The examples given are only suggestions rather than specific recommendations Please contact technical support if you have specific questions Related Products from GenoSensor GenoExplorer microRNA Full Kit GenoExplorer microRNA Labeling kit GenoExplorer microRNA Probe Set GenoExplorer Reagents for Hybridization Assay GenoExplorer microRNA Biochips Kit GenoExplorer miRNA Biochips Hybridization Assay Guidelines General Description GenoExplorer biochips are built on a 1 x 3 25 mm x 75 mm slide The chips are coated with patented polymers used for DNA probe attachment The biochips covalently immobilize amine modified DNA oligonucleotide probes for use in microarray analysis The chips demonstrate high probe attachment efficiency and accessibility of nucleic acid targets for hybridization Figure 1 shows the positions of the label and probe arrays on the slide The four arrays are positioned lengthwise 15 mm from the end of the slide opposite the label and centered widthwise 10 mm from the side of the slide The arrays area covers a length of 18 mm and a width of 4 6 mm Figure 1 Array positions on the chips 1 15 mm 18 mm 10 mm s pA z uy Biochip Handling and Assembly Biochips are stored in a chip container It is recommended to wear powder free gloves when handling the Biochips Open the container carefully to avoid dropp
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