User Manual
Contents
1. Limited Warranty GenoSensor 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 warrant2. molar ratio thus providing for the reliability of hybridization signals The streptavidin conjugated dye stain SA S and the staining buffer Buffer S reagents are also included in this kit Their preparation and use in the post hybridization staining procedure is described below in the hybridization assay section Handling RNA Samples When working with RNA always use proper microbiological aseptic techniques Use RNase and DNase free reagents water glassware and plasticware Use non powdered gloves during all steps of sample labeling chip hybridization washing detection and scanning RNA Preparation The GenoExplorer miRNA Labeling Kit patent pending provides an easy and quick way to label microRNAs and other small RNAs Total RNA isolation not provided using traditional methods such as Trizol is recommended Some commercial kits can be used Users should be aware of the harvest efficiency for small RNAs when choosing them Checking with manufacturers is highly recommended A total RNA starting amount of 5 to 10 micrograms is recommended High quality and sufficient amounts of RNA samples is crucial for experiments with microarrays RNA quality can be evaluated by visualizing the RNA on a gel as well as by calculating the A A ratio On a denaturing gel or on an ordinary agarose gel in denaturing buffer the RNA should appear as two bright distinct bands that represent the 28S and 18S ribosomal species The 28S band s
3. G GenoSensor Corporation GenoExplorer miRNA Labeling Kit Catalog 1301 Version B January 2006 GenoExplorer microRNA Labeling Kit Table of Contents Prod ct OV CIVIC W atiennnaewubaniteingcaxeboudcenavotaustanbandabantucswedinbauntanbacdebpasanieiactas 2 Kit Components and Storage Condition 0 ccc cece eee ce eee ee ence cnet nee een ee ee 3 Additional Reg ired Materials sas xc ddcosss eaciacedasnosadcensnl rasi aE EE ER Gees 4 Related Products from iemO SIS OF anseia catexpeaeaieraia iaaeaeanieea ae d a Eaa 4 GenoExplorer miRNA Labeling Protocol s usussiscnscesvssissnisiwnasssavabeasebicnsaieresontsossvetadedsboes 5 GenoExplorer labeled miRNA Hybridization Assay Guidelines ssesessseesseeseeseeeseeseeee 7 PRI 555 sca sap sedis eee iE Ee EAEE EAEE EEE AE E AREA ERE EE 8 Troubleshooting mersininin anaa a a ace REKE Y E dene aa 8 Techmic l SErViCE cas isvesereunen sensnseasaetsussionscauinmwrsaanraunsuallievcaasannanastaneansdsnaundienmiatseiusnassuaness 10 Literature Citation When describing a procedure for publication using these products we would appreciate that you refer to them as the GenoExplorer miRNA Labeling Kit and the GenoExplorer miRNA Biochips Kit Patents and Trademarks GenoExplorer is a trademark of GenoSensor The GenoExplorer miRNA Labeling Kit and the GenoExplorer miRNA Biochips Kit are covered by patents pending 1 GenoExplorer microRNA Labeling Kit
4. Kit reagents are shipped on dry ice 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 as 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 Labeling Kit Additional Required Materials Recommended GenoExplorer miRNA Biochips kit Recommended GenoExplorer miRNA probe set 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 aut
5. Product Overview The GenoExplorer miRNA Labeling Kit Selective and Uniform Labeling GenoExplorer s labeling system employs a 5 end ligation approach This offers greater selectivity in the labeling of RNA compared with more traditional 3 approaches since RNA molecules with capped 5 ends e g intact mRNA will not be labeled The ligation reaction also ensures that the labeling is uniform in that a single label is attached to each RNA molecule with an uncapped 5 end GenoExplorer currently offers a biotin label Simplified Sample Prep Protocol With GenoExplorer s more selective labeling system RNA samples do not require the additional miRNA isolation step as some labeling protocols recommend Users can begin their labeling reaction with total RNA It is important however to ensure that small RNA is retained during the initial RNA extraction procedure Streptavidin Dye Staining The GenoExplorer full kit also includes a streptavidin dye conjugate which is used for the post hybridization dye staining step This step is required for RNA labeled with biotin GenoExplorer microRNA Labeling Kit Kit Components and Storage Conditions GenoExplorer miRNA Full Kit 20 reactions Components Amount Storage Buffer L 140 ul 20 C Enzyme L 10 ul 20 C SA S Dye 10 ul 20 C Buffer S 2x 1 4ml 20 C User s Manual 1 Shipping and Storage GenoExplorer miRNA Labeling
6. ditions Poor biotin e Ensure 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 Labeling Kit High Background Excess e Do not allow slides to dry out during marker used to identify 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 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 Labeling 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
7. e performed by adding 0 5 ul of the SA S to 249 5 ul of buffer S followed by gentle mixing and brief centrifugation Keep protected from light to minimize photobleaching of dye 7 Load the 1 500 diluted SA S dye stain solution on to the array and incubate at 25 C for 30 min Shield the slide from light to minimize effects of photobleaching of the dye 8 After completion of the post hybridization staining step slides should be washed and dried according to the optimized protocols established by the microarray and or probe set provider Scanning Detection and Image Analysis Consult the scanner manufacturer instructions for laser and PMT settings The SA S dye stain signal can be detected using the scanner s 635 nm wavelength Cy5 channel Consult the gal file provided by the microarray manufacturer for further data analysis GenoExplorer microRNA Labeling 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 con
8. eled miRNA Hybridization Assay Guidelines The GenoExplorer miRNA Labeling Kit has been optimized for microarray experiments using GenoExplorer microRNA chips and assay reagents If microarrays other than GenoExplorer microRNA chips are used consult the optimized protocols established by the microarray probe set provider However it is critical to also include the post hybridization staining step Below are general guidelines that can be used for running the hybridization assay using GenoExplorer miRNA labeled samples For a detailed protocol on running GenoExplorer labeled miRNA on GenoExplorer BioChips please consult the user manual for the GenoExplorer full kit 1 Prepare labeled RNA in hybridization buffer following the optimized protocol established by microarray probe set provider 2 Incubate the sample at 90 95 C for 5 min 3 Cool sample by spinning down for 2 min 4 Hybridize samples on microarrays following the optimized protocols established by the microarray and or probe set provider 5 After completion of the hybridization slides should be washed and dried according to optimized protocols established by the microarray and or probe set provider If hybridization is performed in an automated hybstation the drying step is probably not required 6 Within 15 minutes of beginning the post hybridization staining the SA S dye should be diluted 500 fold into Buffer S For a single sample this dilution should b
9. hould be brighter than the 18S band Tailing of these major bands down the gel or a background smear behind these bands that gets heavier at lower molecular weights can indicate degradation of the RNA Degraded RNA will produce high background and low signal intensity microarray results GenoExplorer microRNA Labeling Kit miRNA Labeling Procedure This procedure is used to attach a biotin label to the 5 ends of RNA molecules l D Place Buffer L on ice and thaw for 15 20 minutes Check for any precipitate If necessary warm the solution at 37 C and agitate to dissolve the precipitate completely Mix Buffer L by vortexing followed by brief centrifugation Combine reagents according to table 1 below Reagents should be combined in an RNAase free microcentrifuge tube and all reagents should be kept on ice during set up of the reaction For high accuracy pipet the viscous Buffer L slowly Table 1 miRNA Labeling Reaction Mix Reagents Volume ul Buffer L 7 0 RNA 5 10ug Adjustable RNAase free water Adjustable Enzyme L 0 5 Total 20 Mix reaction thoroughly by pipetting up and down or flicking tube gently several times Incubate at 37 C for 3 hours Incubate at 75 C for 20 min to inactivate the enzyme Store on ice until ready for the hybridization step The labeled sample can also be stored at 70 C and used later GenoExplorer microRNA Labeling Kit GenoExplorer Lab
10. omated 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 Related Products from GenoSensor GenoExplorer microRNA Full Kit GenoExplorer microRNA Biochips Kit GenoExplorer microRNA Probe Set GenoExplorer Reagents for Hybridization Assay GenoExplorer microRNA Labeling Kit GenoExplorer Labeling Protocol General Description The GenoExplorer miRNA Labeling Kit patent pending provides a direct end labeling method Biotin labels are ligated to the 5 ends of RNA molecules which do not contain a 5 capped structure These RNA molecules include rRNA tRNAs regulatory small RNAs such as microRNA siRNA snRNAs and other RNA transcripts of yet unknown function Due to labeling selectivity this method has resulted in low false positive hybridization signals that are usually caused by mRNA which are the most highly complex sequences of genome transcripts This protocol uses directly isolated RNA without RNA target amplification and ultimately reflects the cellular microRNA
11. y is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 10
Download Pdf Manuals
Related Search